Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Cell lines and culturing conditions

U-2 OS osteosarcoma (HTB-96), HEK293T embryonic kidney epithelial (293T; CRL-3216), and KG-1 acute myelogenous leukemia (AML; CCL-246) cells were obtained from the American Type Culture Collection (ATCC, Manassass, VA, USA). HCT116 and HCT116 3-6 colon carcinoma cells were originally obtained from Dr. Bert Vogelstein (Johns Hopkins University). U-2 OS and HEK293T cells were cultured in DMEM high glucose, GlutaMAX medium (Thermo Fisher Scientific), HCT116 and HCT116 3-6 cells were cultured in McCoy’s 5a, GlutaMAX medium (Thermo Fisher Scientific), and KG-1 cells were cultured in IMDM (Thermo Fisher Scientific). For in vitro luciferase read-outs, FluoroBrite DMEM or phenol red-free DMEM (Thermo Fisher Scientific) supplemented with GlutaMAX was used. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; except for KG-1, which had 20%) and penicillin/streptomycin. Cell cultures were maintained at 37 °C with 5% CO₂ in a humidified incubator. Purchased cell lines were authenticated by the ATCC (STR profiling), and no further authentication was performed. The cells were routinely screened for mycoplasma using the MycoAlert kit (Lonza Bioscience) and none were listed as misidentified on ICLAC or known to be cross-contaminated.

Antibodies and chemicals

anti-HA probe (mouse, clone F-7, cat. #sc7392, lot #L1281), anti-GFP (rabbit, cat. #sc8334, lot #D1907), anti-GFP (mouse, clone B-2, cat. #sc9996, lot #H2018), anti-PARP1 (mouse, clone F-2, cat. #sc8007, lot #D3019), and anti-SOD1 (mouse, clone G-11, cat. #sc17767, lot #G3119) were obtained from Santa Cruz Biotechnology. anti-CHK1 (mouse, clone 2G1D5, cat. #2360S, lot #8), anti-p-CHK1 Ser345 (rabbit polyclonal, cat. #2341S, lot #8), anti-p-CHK1 Ser345 (rabbit, clone 133D3, cat. #2348S, lot #18), anti-p-Histone H2A.X Ser139 (γH2A.X, rabbit, cat. #2577S, lot #12), and anti-vinculin (rabbit, cat. #4650S, lot #5) were obtained from Cell Signaling. anti-NUDT15 (rabbit, cat. #GTX32759, lot #822105550) was purchased from GeneTex. anti-V5 tag (mouse, clone SV5-Pk1, cat. #46-0705, lot #2735895) and anti-V5 tag (mouse, clone E10/V4RR, cat. #MA5-15253, lot #XI358694) was purchased from Invitrogen (now Thermo Fisher Scientific). anti-NUDT5 (rabbit polyclonal) was generated in-house as previously described⁷³. anti-MTH1 (NUDT1, rabbit, cat. #NB100-109, lot #F-2) was obtained from Novus Biologicals. anti-p-Histone H3 Ser10 (rabbit, cat. #ab5176, lot #GR3396345-3), anti-β-actin (mouse, clone AC-15, cat. #ab6276, lot #0000182472), anti-α-tubulin (mouse, clone DM1A, cat. #ab7291, lot #GR3341361-15), and anti-OGG1 (rabbit recombinant, clone EPR4664(2), cat. #ab124741) were purchased from Abcam. anti-p-Histone H2A.X Ser139 (γH2A.X, mouse, clone JBW301, cat. #05-636, lot #3313712), and pan-ADP-ribose binding reagent (rabbit Fc tag, cat. #MABE1016, lot #2901597) were obtained from Millipore. anti-DHFR (rabbit, cat. #15194-1-AP, lot #00102546), anti-MTH1 (mouse, clone 2D7G4, cat. #67443-1-Ig, lot #10011993), and anti-PARP2 (rabbit, cat. #55149-1-AP, lot #00073384) were purchased from ProteinTech. Donkey anti-mouse IgG IRDye 680RD (cat. #925-68072, lot #D20803-13) and goat anti-rabbit IgG IRDye 800CW (cat. #925-32211, lot #D21109-25) were purchased from Li-Cor. anti-mCherry (rabbit, cat. # PA5-34974, lot #VB2946310D), donkey anti-mouse IgG Alexa Fluor 488 (cat. #A-21202, lot #1696430), donkey anti-mouse IgG Alexa Fluor 555 (cat. #A-31570, lot #2387458), donkey anti-rabbit IgG Alexa Fluor 568 (cat. #A-10042, lot #1020757), donkey anti-rabbit IgG Alexa Fluor 647 (cat. #A-31573, lot #2420695), goat anti-rabbit IgG Alexa Fluor 488 (cat. #A-11008, lot #913909), and donkey anti-mouse IgG Alexa Fluor 647 (cat. #A-31571, lot #1839633) were purchased from Thermo Fisher Scientific.

Doxycycline hydrochloride (Sigma-Aldrich) was dissolved in MilliQ water (2 mg/mL) and used at 1 μg/mL. MG-132 (Z-Leu-Leu-Leu-al, Sigma-Aldrich) was dissolved in DMSO (10 mM stock) and used at 5 µM. 6-methylthio-GDP, 6-methylthio-GMP, 6-thio-GMP, 6-thio-GDP, 6-thio-IMP, and 6-thio-IDP were purchased from Jena Bioscience and dissolved in MilliQ water to 10 mM. NSC56456 was obtained from the NCI Developmental Therapeutics Program and later re-synthesized⁴¹, while TH8228 and TH8234 were synthesized in-house (see Chemical synthesis and characterization section), but all were dissolved in DMSO. Methotrexate, raltitrexed, TH5487, SU0268, and EB-47 were purchased from MedChemExpress. TH588, AZ19, IACS-4759 and BAY-707 were obtained or synthesized in-house as described previously^{31,33,34,35,74}. Talazoparib, niraparib, olaparib, veliparib, and iniparib (SelleckChem) were dissolved in DMSO. 3-aminobenzamide (3-AB; Sigma-Aldrich) was dissolved in DMSO to a stock of 100 mM. All other inhibitors were dissolved at 10 mM. Furimazine was purchased as part of the Nano-Glo Assay kit (Promega), fluorofurimazine was obtained from Promega as a ready-to-use poloxamer-407 (P-407) desiccate that was reconstituted in sterile PBS⁶⁵, and akaLumine HCl (TokeOni; Sigma Aldrich) was dissolved in MQ water to 40 mM, aliquoted, and stored at -80 °C.

Chemical synthesis and characterization

Synthesis of TH008228 (9-cyclohexyl-6-(methylthio)-9H-purin-2-amine)

2-amino-9-cyclohexyl-3H-purine-6-thione (NSC56456, 10 mg, 0.040 mmol) was dissolved in 0.5 M NaOH (1 mL) and stirred for 10 min., after which time MeI (2 µL, 0.040 mmol) was added and stirred for 2 hours at RT. The product was purified by preparative HPLC to give 9-cyclohexyl-6-(methylthio)-9H-purin-2-amine (4.00 mg, 37.9% yield) as a white powder (Supplementary Fig. 7). 1H NMR (600 MHz, DMSO-d₆) δ 8.02 (s, 1H), 4.21–4.16 (m, 1H), 2.56 (s, 3H), 1.94 (d, J = 9.0 Hz, 2H), 1.82 (app. t, J = 12.5,4H), 1.68 (d, J = 12.5 Hz, 1H), 1.39-1.34 (m, 2H), 1.25–1.21 (m, 1H); 13 C NMR (150 MHz, DMSO-d₆) δ 159.7, 159.3, 150.2, 138.5, 124.3, 52.9, 32.2, 25.2, 24.7, 10.8; LCMS (m/z): [M + H]⁺ calcd. for C12H17N5S, 263.4; found, 264.2, Rt = 1.451 min., purity at 254 nm >95%.

Synthesis of TH008234 (2‐amino‐9‐(2‐phenylpropan‐2‐yl)‐3H‐purine‐6(9H)‐thione)

Step 1: N‐{2‐amino‐4‐chloro‐6‐[(2‐phenylpropan‐2‐yl)amino]pyrimidin‐5‐yl}formamide

α,α-dimethylbenzylamine (29.4 mg, 0.217 mmol) was added to a stirred solution of N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide (30 mg, 0.145 mmol) and NEt₃ (2 eq) in ⁱPrOH (3 mL) and heated at 85 °C for 18 h. The reaction was cooled to RT, and the crude amino pyrimidine (44 mg, 99.3% yield) was collected by filtration.

Step 2: 6‐chloro‐9‐(2‐phenylpropan‐2‐yl)‐9H‐purin‐2‐amine

The crude amino pyrimidine (47 mg, 0.154 mmol) was dissolved in triethyl orthoformate (1.5 mL), heated at 120 °C for 12 hrs, then cooled to RT. HCl (0.05 mL, 12 M) was added and the reaction mixture was stirred for 12 hrs before concentrating under reduced pressure to give the crude 6-chloropurine (44 mg, 99.5% yield).

Step 3: 2-amino-9-(2-phenylpropan-2-yl)-3H-purine-6(9H)-thione

The crude 6-chloropurine (44 mg, 0.153 mmol) was dissolved in EtOH (2 mL), and thiourea (46.6 mg, 0.612 mmol) and formic acid (1 drop) were successively added before heating at 80 °C for 2 hrs. The reaction mixture was purified directly by preparative HPLC (acidic method) to give 2-amino-9-(2-phenylpropan-2-yl)-3H-purine-6(9H)-thione (6 mg, 13.8% yield) as a white solid (Supplementary Fig. 8). 1H NMR analysis revealed a 58:42 ratio between the thioamide and iminothiol tautomeric forms. 1H NMR thioamide tautomer (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 8.17 (s, 1H), 7.33–7.28 (m, 2H), 7.26–7.21 (m, 1H), 7.08–7.05 (m, 2H), 6.60 (s, 2H), 2.03 (s, 6H); 13 C NMR both tautomers (125 MHz, DMSO-d₆) δ 174.3, 160.1, 150.1, 147.7, 147.2, 145.0, 139.2, 128.5, 128.3, 128.0, 127.0, 126.6, 126.0, 125.2, 124.8, 124.6, 62.0, 54.7, 30.7, 30.5, 29.3, 29.1; LCMS (m/z): [M + H]⁺ calcd. for C14H15N5S, 285.4; found, 286.2, Rt = 1.346 min., purity at 254 nm >95%.

General methods and equipment

All commercial reagents and solvents were used without further purification. Analytical thin-layer chromatography was performed on silica gel 60 F-254 plates (E. Merck) and visualized under a UV lamp. 1H NMR spectra were recorded on a Bruker DRX-400. Chemical shifts are expressed in parts per million (ppm) and referenced to the residual solvent peak. Analytical HPLC-MS was performed on an Agilent MSD mass spectrometer connected to an Agilent 1100 system with method B1090A: column ACE 3 C8 (50 × 3.0 mm); H₂O ( + 0.1% TFA) and MeCN were used as mobile phases at a flow rate of 1 mL/min, with a gradient time of 3.0 min; Preparative HPLC was performed on a Gilson HPLC system: column ACE 5 C8 (150 × 30 mm); H₂O (containing 0.1% TFA) and MeCN were used as mobile phases at a flow rate of 45 mL/min, with a gradient time of 9 min. For HPLC-MS, detection was made by UV using the 180 − 305 nM range and MS (ESI + ). For preparative HPLC, detection was made by UV at 254 or 220 nM. All intermediates and final compounds were assessed to be >95% pure by HPLC-MS analysis, unless stated otherwise.

Protein production

Full-length NUDT15 wild-type and R139C were cloned, expressed, and purified as described previously²⁰. Wild-type and L713F PARP1 catalytic domains were also expressed and purified as before²¹.

Differential scanning fluorimetry (DSF)

Protein unfolding was detected by differential scanning fluorimetry (DSF⁷⁵). For NUDT15 experiments, 4 µM NUDT15 wild-type or R139C protein were added to 5x SYPRO Orange (ThermoFisher Scientific) in assay buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM NaPO₄, 5 mM MgCl₂) in the presence of DMSO (1% final v/v), 50 µM NSC56456, or 50 µM TH8228. A CFX96 Touch Real-Time PCR Detection System (Bio-Rad) was used to increase the temperature from 25 °C to 95 °C in 1 °C/min increments, and fluorescence intensity was measured at each step. Data were acquired and melting temperature (T_m) calculated by CFX Maestro™ 1.0 Software (Bio-Rad, version 4.02325.0418) based on minima from the negative first derivative of the melt curve.

For PARP1 experiments, the wild-type catalytic domain (5 µM) or the L713F catalytic domain (5 µM) was incubated with PARPi (250 µM) or DMSO control in 25 mM HEPES, pH 8.0, 150 mM NaCl, 0.1 mM TCEP, 1 mM EDTA and 12.5% DMSO. The experiments were performed as previously described²¹ using 5x SYPRO Orange and a Lightcycler 480 (Lightcycler 480 Software, version 1.5.1.62; Roche). The melting temperature (T_m) was calculated based on the minima from the negative first derivative of the melt curve using Prism (GraphPad, version 10).

Enzyme-coupled malachite green assay

The enzyme-coupled malachite green assay for NUDT15 was performed as previously described³⁹. Compounds were dispensed by an Echo Acoustic Liquid Handler to generate final concentrations ranging from 1.69 nM to 100 µM. Purified, wild-type NUDT15 (8 nM) and pyrophosphatase (0.2 u/mL) were combined in assay buffer (100 mM Tris-Acetate, pH 8.0, 40 mM NaCl, 10 mM MgAc, 1 mM DTT, and 0.005% Tween-20) and incubated for 10 minutes at room temperature. Negative controls were samples incubated without NUDT15 protein. Then, 100 µM dGTP (NUDT15 substrate) was added prior to a 15-minute incubation. A malachite green working solution (3.2 mM malachite green carbinol hydrochloride [Sigma Aldrich] in 3 M H₂SO₄ complemented with a final concentration of 1.5% ammonium molybdate and 0.17% Tween-20) was then added and followed by an additional 15-minute incubation prior to measuring absorbance at 630 nm on a Hidex Sense microplate reader (Aurentia Solutions, software version 0.5.11.2).

NUDT15-NSC56456 co-crystallization and structure determination

Full length NUDT15 (15 mg/ml) was prepared in sample buffer containing 20 mM HEPES, pH 7.5, 300 mM NaCl, 10% Glycerol, and 2 mM TCEP in the presence of 5 mM NSC56456 dissolved in DMSO. Sitting drop vapor diffusion was performed at 4 °C and NUDT15 was mixed with reservoir solution (0.1 M Tris pH 8.5, 0.2 M sodium acetate, 30% PEG 4000) in a 1:3 protein/reservoir ratio. Diffraction-quality crystals appeared in the first week, were extracted quickly without additional cryoprotectant, and flash frozen in liquid nitrogen. Data collection was performed at beam line 14.1 at BESSY, Germany, at 100 K and wavelength 0.9184 Å. Data reduction and processing were carried out using DIALS (version 2.0) and AIMLESS (version 0.5.7) from the CCP4 software package^{76,77,78,79,80}. The structure was solved by molecular replacement of the template structure file with PDB ID 5BON using Phaser6 (version 2.8.2)⁸¹ followed by iterative building cycles using the Refine program in Phenix (version 1.14)⁸². TLS parameters were determined using the TLSMD webserver⁸³. The structure was further validated using PDB_REDO⁸⁴ and deposited under PDBID: 7NR6. Statistics are found in Supplementary Table 1.

Cloning

General subcloning procedures

3xHA-tagged wild-type and R139C NUDT15²⁰, as well as V5-tagged wild-type and G48E p18 MTH1¹⁹, lentiviral expression constructs were previously established in pINDUCER20 (a gift from Stephen Elledge (Addgene plasmid # 44012)⁸⁵. GFP-PARP1 was transferred into pINDUCER20 via SalI/NotI ligation into pENTR4-N-GFP from pEGFP-C3-PARP1⁸⁶. Similarly, PARP1 WT-GFP was made by subcloning into pENTR1a-C-GFP using SalI/NotI primers with the stop codon removed (non-stop, NS). PARP1 L713F was subcloned from pET28-PARP1 L713F²¹ into pENTR1a-C-GFP by flanking SalI/NotI restriction sites and subsequently transferred to pINDUCER20, pCW57.1 (a gift from David Root; Addgene plasmid # 41393), or pLenti CMV Blast DEST (706-1), which was a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17451)⁸⁷. To generate pENTR1a-C-nLuc, nanoLuc (gBlock, IDT DNA) was subcloned into pENTR1a by flanking XbaI/XhoI restriction sites. PARP1 L713F was then subcloned into pENTR1a-C-nLuc by flanking SalI/NotI restriction sites prior to transferring into pINDUCER20, pCW57.1, or pLenti CMV blast. mCherry was subcloned from H2B-mCherry (a gift from Robert Benezra; Addgene plasmid # 20972)⁸⁸ into pENTR1a by flanking XhoI/XbaI sites prior to transferring to pLenti CMV Blast. Codon-optimized akaLuc⁵⁵ was subcloned from pEX-A258-akaLuc (Eurofins) into pENTR4 by flanking NcoI/SalI sites and transferred to pLenti CMV Blast, as before. DHFR WT and P67L were subcloned into pENTR1a-C-V5 by SalI/NotI cleavage-ligation prior to transferring to pINDUCER20. PARP2 WT and L269A were subcloned from pET28 vectors²⁷ into pENTR1a-C-GFP with flanking SalI/NotI restriction sites prior to transfer to pLenti CMV Blast. All subcloning into entry vectors was validated by automated sequencing, while shuttling into destination vectors was performed with Gateway LR Clonase II (ThermoFisher Scientific) and positive clones were confirmed by colony PCR.

Site-directed mutagenesis (SDM)

Site-directed mutagenesis of OGG1 R229Q; PARP1 L698A, L701A, L765A, L768A, and L698A/L701A; and PARP2 L269A was performed based on the method reported by Zheng et al.⁸⁹ OGG1 R229Q mutagenesis primers were designed with Agilent QuikChange Primer Design. Successful mutagenesis was confirmed by automated sequencing. Mutagenesis primers are provided in Supplementary Data 2.

Plasmids, primers, and synthetic DNA

All primers and custom vectors were ordered from Eurofins Genomics. gBlock fragments were ordered from IDT. Other plasmids, unless developed in-house, were purchased from Addgene. The sources and sequences of all nucleic acids used in this study are summarized in Supplementary Data 2.

Lentivirus production and transduction

Lentiviral production was performed following transfection of third generation lentiviral packing vectors by calcium phosphate precipitation. pINDUCER20, pCW57.1, or pLenti CMV Blast lentiviral constructs were co-transfected with lentiviral packaging vectors (Gag-Pol, Rev, and VSV-G envelope) into subconfluent HEK293T cells. Viral particles were harvested at 48- and 72-hours post-transfection, and target cells were transduced at 1:1 dilution of lentivirus and fresh, complete medium in the presence of polybrene (8 µg/mL). Forty-eight hours post-transduction, target cells were re-plated at low density in the presence of G418/neomycin (Sigma-Aldrich, 400 µg/mL for six days; pINDUCER20), puromycin (Sigma-Aldrich, 1 µg/mL for three days; pCW57.1), or blasticidin (Sigma-Aldrich, 5 µg/mL for four days; pLenti CMV Blast) that was replenished at three-day intervals. HCT116 pCW57.1-PARP1 L713F-nLuc cells were selected with 10 µg/mL puromycin over four days to enrich for high transductants.

Reverse transcription quantitative PCR (RT-qPCR)

U-2 OS or HCT116 cells were plated at 40,000 cells/well in 12-well plates in the absence or presence of 1 µg/mL DOX. The following day, DMSO control or indicated inhibitor was added to the cells before harvesting with TRIzol (Thermo Fisher Scientific). RNA was purified with the Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions and quantified by NanoDrop (Thermo Fisher Scientific). cDNA was then generated with the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. qPCR was performed with 2.5 ng cDNA per sample and iTaq Universal SYBR Green Supermix (Bio-Rad) using a Bio-Rad CFX96 Real-Time PCR Detection System (CFX Maestro™ 1.0, version 4.0.2325.0418). Relative quantity of target genes was calculated using the ΔΔCt method via normalization to GAPDH, β-actin, and/or 18s. All qPCR primers are listed in Supplementary Data 2.

Western blotting

Cells were plated and treated as described. At the termination of the experiment, cells were harvested and lysed directly in 1x Laemmli buffer. Following heating for 5 minutes at 95 °C, the samples were homogenized by sonication and either directly loaded for electrophoresis or frozen at -80 °C for later use. Protein samples were separated on 4-20% gradient Mini-PROTEAN gels (Bio-Rad) or TruPAGE gels (Sigma) prior to transferring onto 0.2 µm nitrocellulose with a Trans-Blot Turbo Transfer System (Bio-Rad). After blocking with LI-COR Blocking Buffer (TBS; LI-COR) for 1 hour at room temperature, primary antibodies were added at the following concentrations in 1:1 LI-COR Blocking Buffer and TBS + 0.05% Tween-20 at 4 °C overnight: anti-HA probe (mouse monoclonal, 1:500), anti-GFP (rabbit polyclonal or mouse monoclonal, 1:500), anti-mCherry (rabbit polyclonal, 1:2000), anti-PARP1 (mouse monoclonal, 1:500), anti-SOD1 (mouse monoclonal, 1:500), anti-CHK1 (mouse monoclonal, 1:1000), anti-p-CHK1 Ser345 (rabbit polyclonal, 1:1000), anti-NUDT15 (rabbit polyclonal, 1:1000), anti-V5 tag (mouse monoclonal, 1:500), anti-NUDT5 (rabbit polyclonal, 1:1000), anti-MTH1 (rabbit polyclonal, 1:500 or mouse monoclonal, 1:2000), anti-p-Histone H3 Ser10 (rabbit polyclonal, 1:1000), anti-γH2A.X (mouse monoclonal, 1:1000 or rabbit polyclonal, 1:500), anti-vinculin (rabbit polyclonal, 1:1000), anti-OGG1 (rabbit, 1:1000), anti-α-tubulin (mouse, 1:5000), anti-PARP2 (rabbit, 1:2000). LI-COR secondary antibodies were diluted in 1:1 LI-COR Blocking Buffer (TBS) and TBS + 0.05% Tween-20 at (1:10,000) or HRP-conjugated goat anti-rabbit secondary antibody (1:5000 in 5% milk/TBS) prior to incubating at room temperature for 1 hour. Blots were imaged with a LI-COR Odyssey Fc and analyzed using Image Studio (LI-COR, version 5.2). All uncropped blots with crop marks denoted are available in the Source Data file.

Qualitative live cell fluorescence microscopy

To initially test GFP PARP1 biosensors in live cells, U-2 OS pINDUCER20-PARP1 L713F-GFP or HCT116 pLenti CMV Blast-PARP1 L713F-GFP/pLenti CMV Blast-mCherry cells were plated in the absence or presence of 1 µg/mL DOX. Where indicated, PARPi were added the following day for 24 to 48 hours. 5 µM MG-132, 100 nM bafilomycin A1, or a combination of both was added for 6 hours prior to imaging with either a ZOE Fluorescent Cell Imager (Bio-Rad, version 2.257) or EVOS FL Cell Imaging System (Thermo Fisher Scientific, version 1.4) at 10, 20, or 40x magnification.

High-content fluorescence microscopy

For NUDT15 R139C experiments in HCT116 3-6 cells, 750 cells were plated in black, clear bottom 96-well plates (BD Falcon) on day 0 in the absence or presence of 1 µg/mL DOX in a volume of 80 µL complete medium. The following day, the appropriate concentrations of inhibitors were added in 10 µL complete medium (3% DMSO v/v). After a 3-hour pre-incubation, either 6TG or equivalent volumes of DMSO were added to the cells in 10 µL complete medium for an additional 72 hours (final DMSO 0.3% v/v). Cells were then fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes, permeabilized with 0.3% Triton-X100 in PBS for 10 minutes, and then blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour. Anti-HA (1:500) and anti-γH2A.X (rabbit, 1:1000) antibodies were incubated overnight at 4 °C in 3% BSA/PBS. The next day, the cells were washed three times (PBS, PBS + 0.05% Tween-20, and then PBS again) before incubation with Alexa Fluor 647 donkey anti-mouse (1:1000) and Alexa Fluor 488 goat anti-rabbit (1:1000) secondary antibodies for 1 hour at room temperature. Following another round of washes, the cells were counterstained with Hoechst 33342 (1 µg/mL in PBS) for 10 minutes prior to imaging.

For PARP1 and PARP2 experiments with live U-2 OS cells, 1000 cells were plated in black, clear bottom 96-well plates (BD Falcon) on day 0 in the absence or presence of 1 µg/mL DOX in a volume of 90 µL complete medium. The following day inhibitors were added to their indicated final concentrations in 10 µL of complete medium (final DMSO 0.1% or 1% [3-AB] v/v). After 24 hours, cell-permeable Hoechst 33342 was added to a final concentration of 1 µg/mL for 20 minutes prior to imaging. In instances where cells were fixed, the same set-up and fixation protocol was used as above. Primary antibodies were anti-GFP (mouse monoclonal, 1:300) combined with anti-p-CHK1 Ser345 (rabbit monoclonal, 1:300) or anti-GFP (rabbit polyclonal, 1:300) with anti-γH2A.X (mouse monoclonal, 1:1000). Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647 (GFP mouse and p-CHK1 rabbit) or anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 647 (GFP rabbit and γH2A.X mouse) were all used at 1:1000 dilutions.

Imaging was performed on an ImageXpress Micro high-content microscope (Molecular Devices, version 5) or CELLCYTE X (CYTENA, CELLCYTE Studio version 2.7.4) at 10x magnification (20x for fixed cells stained with anti-γH2A.X). For live-cell imaging, the microscope temperature, humidity, and CO₂ environment controller module were used to maintain cell ambient conditions (ImageXpress), or, for the CELLCYTE X, the microscope was contained within the humidified incubator. Image analysis was then performed with CellProfiler software (Broad Institute, version 3.1.0) and data plotted with GraphPad Prism (version 10).

Confocal microscopy, FRAP, and microirradiation

Photobleaching and microirradiation microscopy experiments were carried out with a Zeiss LSM780 confocal laser scanning microscope, equipped with a UV-transmitting Plan-Apochromat 40x/1.30 Oil DIC M27 objective as previously described⁹⁰. U-2 OS cells stably expressing GFP-PARP1 WT or PARP1 L713F-GFP were incubated in phenol-red free media containing DMSO, 5 µM olaparib or 0.5 µM talazoparib for indicated time periods. Cells were transferred to the microscope and eGFP was excited with a 488 nm Ar laser. The microscope was equipped with a heated environmental chamber set to 37 °C.

Microirradiation was carried out using either the FRAP module of the ZEISS ZEN software (version 2.1) or the tile scanning mode. Cells were pre-sensitized before microirradiation by incubation in medium containing 10 µg/mL Hoechst 33342 for 10 min. For inducing DNA damage with the FRAP module, a 10-pixel diameter spot within the nucleus was irradiated with a 405 nm diode laser set to 100% power. Before and after microirradiation, confocal image series of one mid z-section were recorded at 2 second intervals (typically 6 pre-irradiation and 120 post-irradiation frames). For evaluation of the recruitment kinetics, fluorescence intensities at the irradiated region were corrected for background and for total nuclear loss of fluorescence over the time course and normalized to the pre-irradiation value. For the quantitative evaluation of microirradiation experiments, data of at least 20 nuclei from two independent experiments were averaged and the mean curve and the standard error of the mean calculated and displayed using Microsoft Excel 2010 and GraphPad Prism (version 10).

For DNA damage induction followed by immunofluorescence staining, cells were seeded on µ-Grid (35 mm with Grid, ibidi) dishes and sensitized with Hoechst 33342 (10 µg/mL, 10 minutes). Laser microirradiation was performed using the tile scan mode (3×3 tiles, image size 128×128, scan speed 177.32 µs, every 7^th line scanned, 405 nm laser set to 70%), as previously described⁹¹. After indicated time periods, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Unspecific binding was blocked by incubation in PBS/4% BSA before staining with respective primary antibodies (pan-ADP-ribose binding reagent, 1:1000; γH2A.X, 1:1000). Primary antibodies were detected using secondary antibodies (diluted 1:500-1:1000 in PBS/4% BSA) conjugated to Alexa Fluor 568 or 647 (Thermo Fisher Scientific). Cells were counterstained with DAPI and kept in PBS until images were taken with the LSM780 microscope.

For FRAP analysis, half of the nucleus was marked using the regions tool of the ZEN software (ZEISS, version 2.1) and photobleached with the 488 nm laser set to maximum power at 100% transmission using 5 iterations at scan speed 8 (5 µs). Before and after bleaching, confocal image series were recorded with the following settings: 500 ms time intervals (20 prebleach and 200 postbleach frames), frame size 256×256 pixels, 170 nm pixel size, bidirectional scanning and a pinhole setting of 2.52 airy units. Mean fluorescence intensities of the bleached region were corrected for background and for total nuclear loss of fluorescence over time. For the quantitative evaluation of photobleaching experiments, data of at least 45 nuclei from five independent experiments were averaged and the mean curve, the standard error of the mean (s.e.m.), halftime of recovery (t½) and mobile fraction (Mf) calculated and displayed using Microsoft Excel 2010 and GraphPad Prism (version 10).

Flow cytometry

MTH1 G48E studies

For MTH1 studies, 400,000 U-2 OS V5-MTH1 G48E clone #6 cells were plated in T25 flasks in the presence of 1 µg/mL DOX on day 0. The following morning (day 1), the cells were then treated with DMSO (0.01% v/v final concentration) or the indicated concentration of MTH1i for 24 hours. On the morning of day 2, the cells were harvested by trypsinization and pooling of culture medium, as well as PBS washes (to ensure collection of dead and mitotic cells). Following a wash with PBS, the cells were fixed with 4% PFA in PBS for 15 minutes, washed once with 1% BSA/PBS, then permeabilized with saponin buffer (0.1% saponin in 1% BSA/PBS) on ice for 30 minutes. The cells were then stained with anti-V5 (mouse, clone E10/V4RR, 1:300) and anti-p-HH3 Ser10 (rabbit polyclonal, 1:500) antibodies diluted in saponin buffer overnight at 4 °C. Next, the cells were washed twice with saponin buffer prior to incubation with donkey anti-rabbit Alexa Fluor 647 and donkey anti-mouse Alexa Fluor 488 antibodies (1:1000 in saponin buffer) for 30 minutes at 37 °C. Following two additional washes with saponin buffer, the cells were incubated with 0.1 mg/mL RNase A (Thermo Fisher Scientific) and 10 µg/mL Hoechst 33342 for 15 minutes at room temperature in 1% BSA/PBS. Control V5-MTH1 G48E cells were also used for singlet antibody controls (one for V5 and one for p-HH3 Ser10). The cells were then analyzed by flow cytometry on a BD Fortessa flow cytometer (Bectin Dickenson) using BD FACSDiva software (version 8.0.1) for acquisition (p-HH3 Ser 10-Alexa Fluor 647: R 670_30-A, V5-Alexa Fluor 488: B 530_30-A, Hoechst: V 450_50-A). Analysis (including cell cycle by the Watson Pragmatic method) and final gating of cell populations was performed with FlowJo software (Bectin Dickenson, version 10.7.1; see Supplementary Fig. 20a). FlowJo was also used to export raw, per-event values for subsequent plotting and analysis in GraphPad Prism (version 10).

PARP1 L713F studies

PARP1 experiments were performed with live U-2 OS pINDUCER20-PARP1 L713F-GFP #5/pLenti CMV Blast-mCherry or HCT116 pLenti CMV Blast-PARP1 L713F-GFP/pLenti CMV Blast-mCherry cells. Briefly, 200,000 cells were plated in T25 flasks (in the presence of 1 µg/mL DOX for U-2 OS cells). The following day, varying concentrations of veliparib/niraparib or an equivalent volume of DMSO was added to the cells prior to harvesting 24 hours later. Trypsinized cells were quenched with complete medium, pelleted at 400 x g for 5 minutes, washed by resuspending in sterile PBS/10% FBS, centrifuged again, and then transferred to 5 mL flow cytometry tubes via a 40 µm strainer cap (BD Falcon) in 500 µL sterile PBS/5% FBS. The samples were then analyzed on a BD Accuri C6 flow cytometer (Bectin Dickenson) with BD Accuri C6 software (version 1.0.264.21; GFP – PARP1 L713F stabilization, mCherry – fluorescence normalization). Unstained parental U-2 OS or U-2 OS pINDUCER20-L713F cells without DOX were used to establish final gating and compensation parameters with FlowJo (Bectin Dickenson, version 10.7.1; Supplementary Fig. 20b,c).

In vitro luciferase assays

nLuc assays

For nLuc luciferase assays with U-2 OS PARP1 pINDUCER20-PARP1 L713F-nLuc cells, 1,000 cells per well were plated in white 96-well plates (Greiner) in complete medium and in the absence or presence of 1 µg/mL DOX. The following day, the medium was changed to FluoroBrite DMEM phenol red-free medium (Thermo Fisher Scientific) in the presence of DMSO or PARP inhibitor (fresh DOX was also added where necessary), and the cells were incubated for another 24 hours. nanoLuc signal was assessed using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions and the signal was quantified on a Hidex Sense plate reader (software version 0.5.11.2) with 1-second reads and an open filter setting. Readings were made every 3 minutes for up to 30 minutes following the addition of the lysis reagent.

For sensitivity measurements, U-2 OS cells were plated at serial dilutions ranging from 12 to 1,500 cells in white 96-well plates (Greiner) in complete medium and the presence of 1 µg/mL DOX. The medium was changed to FluoroBrite DMEM + DOX the following day and either DMSO or 2 µM veliparib were added to the cells for an additional 24 hours, before assaying by Nano-Glo kit with a Hidex Sense plate reader (software version 0.5.11.2) and an open filter. Signal-to-background (S/B = mean signal/mean background – µ_S/µ_B) was also calculated⁵⁶.

For dose response experiments with HCT116 pCW57.1-PARP1 L713F-nLuc cells, 4000 cells were plated in white 96-well plates (Greiner) in the absence or presence of 1 µg/mL DOX. The following day the medium was changed to complete FluoroBrite DMEM containing talazoparib (max conc. 10 µM), 3-AB (max conc. 1 mM), or iniparib (max conc. 20 µM) for 24 hours. After 24 hours, the medium was changed to furimazine-containing FluoroBrite (1:200 dilution from Nano-Glo kit) and immediately read with a CLARIOStar microplate reader (BMG LABTECH; software version 5.40 R2) with an open filter followed by a nLuc filter (470 ± 40 nm), both with spiral averaging. Data were exported using CLARIOstar MARS (version 3.31, BMG LABTECH).

Combined nLuc and akaLuc assays

Luminescence spectral profiling for both nLuc and akaLuc was determined by pre-treatment of HCT116 pCW57.1-PARP1 L713F-nLuc/pLenti-CMV-blast-akaLuc cells with 1 µg/mL DOX for 24 hours and 1 µM veliparib for an additional 24 hours. nLuc spectral profiling was performed immediately after the addition of a 1:200 dilution of Nano-Glo substrate in FluoroBrite DMEM, while the akaLuc spectrum was determined with 200 µM akaLumine HCl in FluoroBrite DMEM. Spectral scanning was determined on a CLARIOstar microplate reader on the spiral averaging setting.

To determine akaLuc tolerance to detergent-mediated lytic conditions, HCT116 pCW57.1-PARP1 L713F-nLuc HI/pLenti-CMV-blast-akaLuc or U-2 OS pINDUCER20-PARP1 L713F-nLuc/pLenti-CMV-blast-akaLuc cells were prepared for detection with 200 µM akaLumine HCl diluted in Nano-Glo lysis reagent (according to the lysis conditions described by the manufacturer) or 100 or 200 µM akaLumine HCl diluted in complete FluoroBrite DMEM medium before luminescence detection on an open filter and spiral averaging feature.

For testing akaLuc luminescence and cross-reactivity with nLuc signal, HCT116 pCW57.1-PARP1 L713F-nLuc/pLenti-CMV-blast-akaLuc cells were plated at 4000 cells per well unless otherwise stated and subjected to several different conditions. While testing akaLuc alone, luminescence signals were first established by comparison to parental HCT116 cells. The day after plating, the medium was changed to complete FluoroBrite DMEM with 200 µM akaLumine HCl and immediately measured on a CLARIOStar microplate reader with an open filter and spiral averaging feature.

For experiments testing simultaneous detection of nLuc and akaLuc signals, the cells were plated in complete medium supplemented with 1 µg/mL DOX. The following day, the cells were supplemented with 2 µM veliparib for an additional 24 hours. Just prior to imaging, the medium was replaced with complete FluoroBrite DMEM medium supplemented with 200 µM akaLumine HCl and furimazine (1:200 dilution) and immediately detected sequentially on an nLuc filter (470 ± 40 nm) and an akaLuc filter setting (650 ± 40 nm) using the spiral averaging feature.

For sequential reading of akaLuc signal followed by nLuc signal, HCT116 pCW57.1-PARP1 L713F-nLuc/pLenti-CMV-blast-akaLuc cells were plated in complete medium in the absence or presence of 1 µg/mL DOX (or not at all for HCT116 pLenti-CMV-blast-PARP1 L713F-nLuc/pLenti-CMV-blast-akaLuc cells used for animal experiments) prior to incubation with indicated concentrations of PARPi for 24 hours. On the day of imaging, the medium was first changed to complete FluoroBrite DMEM with 200 µM akaLumine HCl and immediately imaged with an open filter and spiral averaging feature. Following a wash with FluoroBrite DMEM, the medium was changed again to FluoroBrite DMEM with a 1:200 dilution of furimazine and immediately imaged with an nLuc filter (470 ± 40 nm) and spiral averaging feature. nLuc signal was then normalized to akaLuc signal to obtain relative signal values and then normalized to DMSO-treated (in some cases, +DOX) controls to give fold change.

Time- and target concentration-dependence of L713F-nLuc stabilization

Time-dependent stabilization of PARP1 L713F-nLuc by PARPi was determined with HCT116 pCW57.1-PARP1 L713F-nLuc/pLenti CMV Blast-akaLuc cells plated at 4000 per well in the absence or presence of 1 µg/mL DOX. The following day, veliparib was added at different concentrations for 1, 8, or 24 hours prior to sequential akaLuc and nLuc detection as before. Fold nLuc signal was calculated by normalization of nLuc intensity to akaLuc intensity and relative to DMSO + DOX treatment. The influence of PARP1 L713F-nLuc target abundance on stabilization by PARPi was determined as above, but cells were pre-treated with 10, 100, 333, or 1 µg/mL DOX overnight followed by 24-hr treatment with veliparib at different concentrations. Luciferase activity signals were again detected sequentially and nLuc intensity was normalized to akaLuc intensity and set relative to DMSO + DOX readings.

Small molecule screen for PARP1 biophysical perturbagens

Complete screening details are summarized in Supplementary Table 2.

Composition, storage, and plating of screening library

The 1187 compound screening library consisted of the MedChemExpress Epigenetics and Selleck Nordic Oncology sets housed at the Science for Life Laboratory Compound Center, part of Chemical Biology Consortium Sweden (CBCS). The compounds are kept at -20 °C as 10 mM solutions in DMSO (some compound stocks were lower concentrations – see Supplementary Data 1 for more details) under low humidity using a REMP Small-Size Store system. Stocks were transferred to LabCyte 384 LDV plates (LP-0200) to enable dispensing into assay plates with an Echo 550™ acoustic liquid handler (LabCyte). 100 nL of compound stock solutions were dispensed into white CELLSTAR® 96-well plates with tissue culture-treated surface (Greiner, 655083). Similarly, 100 nL DMSO (negative control) was dispensed into the first column of each assay plate, while 100 nL 10 mM veliparib (positive control) was dispensed into the second column of each plate. After addition of 100 µL growth medium to each well, the final DMSO concentration was 0.1% (v/v) and compound concentrations were up to 10 µM.

Screen execution and data acquisition

HCT116 pCW57.1-PARP1 L713F-nLuc/pLenti CMV Blast-akaLuc cells were initially pre-treated with 1 µg/mL DOX for 24 hours to induce expression of the L713F transgene. Cells were then trypsinized and replated into drug-containing assay plates at a concentration of 2.5×10⁴ cells/100 µL and 1 µg/mL DOX using a Multidrop Combi liquid dispenser (Thermo Scientific). Cells were then incubated with drugs for 24 hours at 37 °C with 5% CO₂ in a humidified incubator. To minimize edge effects, the plates were placed in self-made humidity chambers that limited evaporation in the outer ring of wells. Luciferase signals were acquired sequentially on a CLARIOstar microplate reader as above using an open filter setting (akaLuc) followed by an nLuc-specific filter (470 ± 40 nm) with live cells.

Data analysis and confirmation

To eliminate high variations in nLuc/akaLuc ratios, an akaLuc cut-off was set at >4 standard deviations from the mean of control wells per plate, which improved stringency of the screening campaign and left 840 compounds eligible for analysis (Supplementary Fig. 18d and e). L713F-nLuc signals were first normalized to the akaLuc intensity, then set relative to the mean of the DMSO (negative) control to give relative fold change. Normality of the dataset was then improved by transformation to give log₂(fold change [FC]) (Supplementary Fig. 18f). Hits were defined as ±2 SDs from the mean (log₂[FC] for all valid test compounds), while ±3 SDs was added as an additional cut-off of significance. Complete screening data are supplied in Supplementary Data 1. Hit confirmations were performed with the same experimental set-up as the screen but with two independent sets of triplicate data points at 10 µM compound concentration.

In vivo target engagement in tumor xenografts

Animal husbandry and ethical statement

BALB/cAnNCrl nude mice (6–8-week-old; strain code: 194 [homozygous], Charles River Labs) were used for tumor xenograft experiments. The mice were housed in individually ventilated cages (type IVC, four per cage) under conditions of a 12-hour light/dark cycle and ambient temperature of 21  ±  4°C with 40–70% humidity. All work was performed in accordance with EU (European Union) and Swedish Ethical Review Authority regulations for animal experimentation under Karolinska Institutet permit no. 5718-2019. Tumors did not exceed 1000 mm3 in size in this study, as mandated by the Swedish Central Animal Research Ethics Committee (Centrala Djurförsöksetiska Nämnden).

Establishment of PARP1 L713F-GFP/mCherry tumor xenografts and treatment with PARPi

HCT116 pLenti CMV Blast-PARP1 L713F-GFP/pLenti CMV Blast-mCherry cells were injected subcutaneously into the flanks of BALB/cAnNCrl nude mice (2×10⁶ cells in 100 µL PBS). Once the tumors reached approximately 200 mm3 in size, mice were systemically administered vehicle, 15 mg/kg, or 60 mg/kg niraparib formulated in 0.5% methylcellulose (Sigma) by oral gavage once daily (qd) for two days. On day three, the tumors were harvested and sub-divided for downstream analysis as follows: one half was formaldehyde-fixed and paraffin-embedded for sectioning and immunofluorescence analysis, one quarter was snap frozen for western blot analysis, and one quarter was immediately dissociated for live cell flow cytometry analysis.

Tumor dissociation for flow cytometry

Dissociation of excised subcutaneous HCT116 L713F-GFP/mCherry tumors was performed similarly to previously reported methods⁹². Briefly, approximately 60-80 mm3 of tumor tissue was finely minced with a sterile scalpel and dissociated for 1 hour at 37 °C with shaking in 9 mL of an enzyme solution containing 1 mg/mL collagenase D (Sigma) and 100 ng/mL DNAse I ( ≥ 40 U/mL final; Sigma) in McCoy’s 5a GlutaMAX without additives. For the final 5 minutes of the incubation period, 1 mL TrypLE Express was added to each tube. Digestive enzymes were deactivated by the addition of 3 mL McCoy’s 5a medium containing 10% FBS and the cell suspension was sieved through a 40 µm strainer (Corning), followed by an additional rinse with 2 mL McCoy’s/10% FBS. The cells were pelleted by centrifugation at 400 x g for 5 minutes and washed by resuspension in sterile PBS/10% FBS. After centrifugation, the cell pellet was resuspended in 1 mL sterile PBS/5% FBS and transferred to a 5 mL flow cytometry tube via a 40 µm strainer cap (BD Falcon). Viability of the final samples was between 30-35% by trypan blue exclusion. Tumor suspensions were assayed on a BD Accuri C6 (Bectin Dickenson, software version 1.0.264.21) with identical gating strategy for cultured HCT116 GFP/mCherry cells (Supplementary Fig. 20b). Relative L713F-GFP was determined by normalizing median GFP intensity to median mCherry intensity.

Western blotting of tumor cells

Excised tumor samples were immediately snap frozen on dry ice and stored at −80 °C until use. In preparation for lysis, tumor masses were then pulverized with a mortar and pestle in liquid nitrogen prior to lysis in RIPA buffer and clarification of proteins by centrifugation. Following protein concentration measurements by BCA assay, equal concentrations of tumor lysate were prepared for western blotting, as above. Primary antibodies used were anti-GFP (mouse; 1:500), anti-mCherry (rabbit; 1:2000), and anti-β-actin (mouse; 1:5000).

Sectioning, mounting, and immunohistochemistry

Following excision, tumors were fixed in 4% PFA/PBS for one day and then submerged in 70% ethanol. Paraffinization, sectioning, and mounting of tumors was performed in the Pathology Core Facility at Karolinska Institutet Huddinge campus.

In preparation for immunofluorescence, the sections were deparaffinized and rehydrated with the following steps: 1. Xylene: 2 ×3 minutes, 2. Xylene/100% ethanol (1:1): 3 minutes, 3. 100% ethanol: 2 ×3 minutes, 4. 95% ethanol: 3 minutes, 5. 70% ethanol: 3 minutes, 6. 50% ethanol: 3 minutes, 7. Rinse with cold tap water. Following deparaffinization, antigen retrieval was carried out with citric acid buffer in a standard pressure cooker made for slides. The slides were then washed 2 ×5 minutes with TBS/0.025% Triton X-100 under gentle agitation, followed by blocking in 2% BSA/TBS for 2 hours at room temperature under high humidity. Anti-GFP primary antibody (1:100, mouse monoclonal, Santa Cruz) was then incubated overnight at 4 °C in 2% BSA/TBS. The next day, the slides were washed 3 ×5 minutes in TBS/0.025% Triton X-100 with gentle agitation. Following washing, donkey anti-mouse Alexa Fluor 555 secondary antibody (1:1000 in 2% BSA/TBS) was applied on the slides and incubated at room temperature for 1 hour. The slides were again washed under gentle agitation 3 ×5 minutes in TBS, followed by rinsing with distilled water. The sections were counterstained with DAPI (1:1000), rinsed once with distilled water, and mounted with ProLong Gold (Thermo Fisher). Images were taken using a 40x objective on a Zeiss LSM780 (ZEN software, version 2.1) confocal laser scanning microscope and processed in Fiji (ImageJ, version 2.1.0/1.53c).

Establishment of PARP1 L713F-nLuc/akaLuc tumor xenografts and treatment with PARPi

HCT116 pLenti CMV Blast-PARP1 L713F-nLuc/pLenti CMV Blast-akaLuc cells were injected subcutaneously and treated with vehicle control or 60 mg/kg niraparib as above.

Bioluminescence imaging of PARP1 L713F-nLuc/akaLuc tumors in live animals

akaLuc and nLuc bioluminescence signals were sequentially imaged on an IVIS Spectrum (Perkin Elmer). On day two, the animals were anesthetized by isoflurane gas and 1 mg (3 µmol) akaLumine HCl in 100 µL sterile saline was administered IP prior to immediately imaging to determine tumor akaLuc signals⁶⁵. akaLuc bioluminescence was measured every minute for 20 minutes with the instrument settings of open filter, medium binning, 60 second exposure time, and f-stop of 1. To ensure metabolic clearance of akaLumine HCl, the following day, 120 µL of fluorofurimazine/P-407 solution (1 µmol) was administered IP for detection of L713F-nLuc⁶⁵, and the mice were again immediately imaged on an IVIS Spectrum (Living Image software, version 4.7.2) with measurements every minute over 20 minutes. Instrument settings were the same as above. Results are presented as radiance (p/sec/cm2/sr) or total flux (photons/sec). Relative L713F-nLuc luminescence was calculated by normalization to akaLuc signal.

Ex vivo bioluminescence measurements

Following clearance of fluorofurimazine on day three of niraparib treatment, tumors were excised from the mice prior to sectioning with a sterile scalpel, and a quarter of each tumor was transferred to a well in a white, 96-well plate (Greiner). Tumor masses were gently flattened with sterile Dounce homogenizer plungers to spread tissue evenly in the well and submerged in 200 µL FluoroBrite DMEM containing 200 µM AkaLumine HCl. Immediately after addition of Akalumine, tumor akaLuc luminescence was measured with a CLARIOStar microplate reader (BMG LABTECH, software version 5.40 R2) on an open filter with the spiral averaging feature. The AkaLumine was then removed, and tumors were briefly washed with FluoroBrite DMEM prior to submerging in 200 µL furimazine-FluoroBrite (diluted 1:200 from the Promega Nano-Glo kit). L713F-nLuc signals were then immediately measured using nLuc-specific filter settings (470 ± 40 nm). To determine relative L713F-nLuc luminescence, signals were normalized to akaLuc readings and set relative to the vehicle control tumors.

Statistical analyses and data transformation

All graphing and statistical analyses were performed using GraphPad Prism, version 10. Saturation curve fitting was performed using the [agonist] vs response four parameter variable slope model in Prism. Specific post-hoc tests, variations, and statistical significances for relevant experiments are described within individual figure legends. In some instances, raw data were transformed to simplify graphical visualization. Specifically, fluorescence values in Fig. 2i, Fig. 7j and k, and Supplementary Fig. 14j were multiplied by a factor of 10. Data in Fig. 4h (γH2A.X) and j (γH2A.X); Supplementary Fig. 14a, b, e, and h; Fig. 5d, f (GFP), g (GFP); Supplementary Fig. 10d and g; Supplementary Fig. 11c; and Supplementary Fig. 15b, c, e (GFP, pCHK1) and 15g, h, and j (GFP, γH2A.X) were multiplied by a factor of 100. Data in Fig. 4j (HA); Supplementary Fig. 10c and f; and Supplementary Fig. 11k were multiplied by a factor of 1000. Data in Supplementary Fig. 13f were multiplied by a factor of 10,000.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54415-7