Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation – Nature Cardiovascular Research

Materials and standards

Antibodies

Rabbit anti-α-tubulin (Thermo Fisher Scientific, PA5-19489), rat anti-CD42b monoclonal (clone Xia.G7, Emfret, M042-1), rat FITC anti-mouse CD41 (BioLegend, 133904), rabbit anti-CSNK2A1 (Abcam, ab76040), mouse anti-CKIP-1 (Santa Cruz Biotechnology, sc-376355), mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), goat anti-rabbit secondary antibody (Life Technologies, A21069), goat anti-rat secondary antibody (Life Technologies, A11006), donkey anti-mouse secondary antibody (LI-COR, 926-32212), Alexa Fluor 594-conjugated anti-CD105 antibody (BioLegend, 120418), Alexa Fluor 546-conjugated anti-CD105 (self-generated, clone MJ7/18, ref. ⁶⁰), anti-CD42a (GPIX) Alexa Fluor 488 derivative (self-generated, p0p6, ref. ⁶¹).

Chemicals

DRAQ5 DNA stain (Thermo Fisher Scientific, 62251), Alexa Fluor 488 phalloidin (Life Technologies, A12379), antibody diluent (Zytomed, ZUC025-100), Roti-Load (Roth, K929.1), bovine serum albumin (BSA) (PanReac AppliChem, A1391,0500), triacsin C (Cayman Chemical, 10007448), FSG67 (Focus Biomolecules, 10-4577), mounting medium (Invitrogen, P36961), poly-L-lysine (Sigma-Aldrich, P8920-100ML, 0.1%), paraformaldehyde (PFA) (Otto Fischar GmbH & Co. KG, 27246), Cell Lysis Buffer (Cell Signaling Technology, 9803S), Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872S), FcR Blocking Reagent mouse (Miltenyi Biotec, 130-092-575), PureLink RNase A (Invitrogen, 12091-021), propidium iodide (Invitrogen, P1304MP), EZ-Link Sulfo-NHS-Biotin (SNB) (Thermo Fisher Scientific, 11851185), EZ-Link NHS-Biotin (NB) (Thermo Fisher Scientific, 10381394), L-lysine (Sigma-Aldrich, L5501), triethylamine (Sigma-Aldrich, 90335), medetomidine (Pfizer), midazolam (Roche), fentanyl (Janssen-Cilag), recombinant TPO (ImmunoTools, 12343615).

Chemicals specific for lipid analysis: formic acid (BioSolve, 6914143), ULC/MS-grade methanol (BioSolve, 13684102), ULC/MS-grade water (BioSolve, 23214102), ULC/MS-grade acetonitrile (ACN) (BioSolve, 1204102), methyl tert-butyl ether (MTBE) (VWR, 34875-1L), ammonium acetate (Merck, 73594-25G-F), ammonium formate (Sigma-Aldrich, 70221-25G-F), HPLC-grade phosphoric acid (Sigma-Aldrich, 79617-250ML, 85–90%), chloroform (Sigma-Aldrich, 650498-1L), isopropanol (IPA) (Sigma-Aldrich, 1010402500).

Chemicals specific for protein analysis: formic acid (VWR, 84865-180P), ULC/MS-grade methanol (VWR, 83638320), ULC/MS-grade water (Honeywell, 14263-2L), ULC/MS-grade ACN (Honeywell, 34967-2.5L), urea (Merck, 1084871000), triethylammonium bicarbonate (TEAB) (Sigma-Aldrich, 18597-100ML), sodium dodecyl sulfate (SDS) (GERBU, 1212), dithiotreitol (DTT) (Sigma-Aldrich, APOSBIMB1015-25G), iodoacetamide (IAA) (Sigma-Aldrich, I6125-25G), Trypsin/Lys-C Mix Mass Spec Grade (Promega, V5073), trifluoroacetic acid (TFA) (Sigma-Aldrich, T6508-100ML).

Peptide standards

Standard peptide [Glu¹]-Fribrinopeptide B (sequence EGVNDNEEGFFSAR, Sigma-Aldrich, F3261), standard peptide M48 (sequence TTPAVLDSDGSYFLYSK, PSL), standard peptide HK0 (sequence VLETKSLYVR, PSL), standard peptide HK1 (sequence VLETK(ε-AC)SLYVR, PSL).

Lipid standards

Mouse SPLASH LIPIDOMIX Mass Spec Standard (Avanti Polar Lipids, 330710X-1EA) consisting of PC 15:0-18:1(d7), PE 15:0-18:1(d7), PS 15:0-18:1(d7), PG 15:0-18:1(d7) (as internal standard for PG and CL), PI 15:0-18:1(d7), PA 15:0-18:1(d7), LPC 18:1(d7), LPE 18:1(d7) (as internal standard for all lysophospholipids except LPC), SE 18:1(d7), PC-ether (PCO-a) 18:0-18:1(d9), PE-ether (PEO-a) 18:0-18:1(d9), DG 15:0-18:1(d7), TG 15:0-18:1(d7)-15:0 and SM d18:1-18:1(d9); Ceramide/Sphingoid Internal Standard Mixture II (Avanti Polar Lipids LM6005-1EA) consisting of sphingosine (SPB) d17:1, sphinganine (SPB) d17:0, sphingosine-1-P (SPBP) d17:1, sphinganine-1-P (SPBP) d17:0, SM d18:1/12:0, Cer d18:1/12:0, glucosylceramide (GlcCer) d18:1/c12:0 (as internal standard for HexCer), lactosylceramide (LacCer) d18:1/12:0 (as internal standard for dihexosylceramide (Hex2Cer)) and ceramide-1-P (CerP) d18:1/12:0; cholesterol-d7 (Avanti Polar Lipids, 700041P); lysosphingomyelin (LSM)-d7 (Avanti Polar Lipids, 860639P); PS 14:0-14:0 (Avanti Polar Lipids, 840033P) (self-generated biotinylated internal standard for biotin-PS).

Animal models

Csnk2a1^lox/lox mice were generated elsewhere⁶². For MK-specific or platelet-specific deletion of CK2α, csnk2a1^lox/lox mice were crossed with Pf4-Cre transgenic mice (The Jackson Laboratory, 008535) and studied at the age of 12–14 weeks. All animal experiments were performed according to Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by local authorities (Regierungspräsidium Tübingen) following the ARRIVE guidelines (protocols M01/20G and M03/19M).

For in vivo treatment studies, 6-week-old C57BL6/J mice, were treated daily intraperitoneally with either 0.285 mg per kg (body weight) triacsin C, 5 mg per kg (body weight) FSG67 or dimethylsulfoxide (DMSO) over a period of 7 days. Concentrations were adapted according to refs. ^63,64.

Bone marrow isolation and MK differentiation

For the bone marrow isolation, a centrifugation protocol previously published by ref. ⁶⁵ was used. Briefly, 10–14-week-old, male C57BL/6J mice (The Jackson Laboratory) were anesthetized using isoflurane and killed by cervical dislocation following the institutional guidelines and the German law for the welfare of animals. Both femora were dissected and cleaned, cut open at the knee side, and placed with the cut side facing down in a 0.5-ml Eppendorf tube with a pre-pierced hole in the bottom. The tube was placed into a 1.5-ml tube, pre-filled with 100 µl of DMEM (supplemented with 1% penicillin/streptomycin and 10% FBS) and centrifuged for 1 min at 2,600 × g at room temperature (21°C (69.8°F)). Next, 1 ml of supplemented medium was added, and bone marrow cells were resuspended, then filtered through a pluriStrainer Mini (70 µm), and the strainer was rinsed with 1 ml of medium. Afterward, cells were centrifuged for 5 min at 300 × g at room temperature, and the supernatant was removed.

To induce MK differentiation, the freshly isolated bone marrow cells (pool of five individual animals) were cultivated in 10-cm cell culture dishes containing supplemented DMEM, and differentiation was initiated by adding (1%) recombinant TPO. Cells were cultivated at 37 °C, 5% CO₂ for different periods of time. On days 1, 3 and 7, cells were collected (1,000 r.p.m., 5 min) and resuspended in 950 µl of PBS. The cell suspension was carefully pipetted on a two-phase BSA gradient (bottom, 1.5 ml 3% BSA in PBS; top, 1.5 ml 1.5% BSA in PBS) to separate cells by weight. After 40 min, the supernatant was removed, and the cell pellet was washed three times with 500 µl of PBS. Cells were counted in a Neubauer chamber and adjusted to 200,000 cells per tube. Cell pellets were shock-frozen in liquid nitrogen and stored at −80 °C for later multiomics analysis.

Immunofluorescence microscopy

For immunofluorescence microscopy of MKs, cells were isolated and purified as described above and cultivated for 1, 3 and 7 days. After isolation via BSA gradient, 5,000 MKs were seeded on chamber slides pre-coated with 0.1% poly-L-lysine for 60 min at 37 °C and further incubated for 1 h. Cells were fixed for 15 min with 4% PFA at room temperature, washed three times for 3 min each with PBS, 10 min with PBS and 0.1% Triton X-100, and again three times for 3 min each with PBS. Cells were further incubated with 1% BSA in PBS to block the unspecific binding of antibodies. Cells were stained with either the primary antibodies Alexa Fluor 488 phalloidin (1:200 in antibody diluent) and α-tubulin (1:400 in antibody diluent) or CD42b (1:100 in antibody diluent), or CD42d (1:300 in antibody diluent). After overnight incubation at 4 °C and washing three times for 3 min each with PBS, secondary antibodies (anti-rabbit Alexa Fluor 568, 1:300 in PBS; anti-rat Alexa Fluor 488, 1:300 in PBS) were applied for 2 h at room temperature. After three washes for 3 min each with PBS, nuclei were stained for 15 min with DRAQ5 stain (1:1,000), washed again with PBS, and mounted using a mounting medium. An LSM510 confocal laser scanning microscope (Zeiss) and ZEN Blue software (Zeiss) were used for the analysis.

Protein and lipid extraction

Samples, consisting of 200,000 cells per tube, were used for lipid and protein extraction following the SIMPLEX protocol previously described by ref. ¹⁸. In brief, 225 µl of methanol and the internal standard mixture were added to all samples, and cell pellets were homogenized through 2–5 min of ultrasonication. Two blanks used as quality controls were processed in parallel, one with and the other without internal standards. Next, 750 µl of MTBE were added, and samples were incubated for 1 h at 950 r.p.m. at 4 °C. To induce phase separation, 188 µl of water (HPLC-grade) were added, and samples were incubated on ice for 5 min. After a 10-min centrifugation step at 10,000 × g at 4 °C, the upper organic phase (containing GPs, GLs, SPs and STs) was carefully removed and dried under a gentle nitrogen flow. The dried organic phase was reconstituted in 100 µl of IPA/methanol/CHCl₃ (4:2:1, v/v/v) containing 7.5 mM ammonium acetate for lipid analysis. To complete protein precipitation, 527 µl of methanol were added to the lower aqueous phase, and samples were stored for 2 h at −20 °C, following centrifugation for 30 min at 12,000 × g at 4 °C. The protein pellet was dried and further subjected to protein analysis.

Protein analysis

Proteomics sample preparation

Protein samples were diluted 1:2 in lysis buffer (8 M urea, 50 mM TEAB, 5% SDS), then heated at 90 °C for 5 min, and protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific). For enzymatic digestion, 20 µg of protein were used, and ProtiFi S-Trap technology was applied⁶⁶. In short, solubilized proteins were reduced and carbamidomethylated by adding 64 mM DTT and 48 mM IAA, respectively. Before loading the samples onto S-Trap mini cartridges (ProtiFi), trapping buffer (90% (v/v) methanol, 0.1 M TEAB) was added. Subsequently, samples were thoroughly washed and then digested using Trypsin/Lys-C Mix for 2 h at 37 °C. Finally, peptides were eluted, dried, and stored at −20 °C until LC–MS analysis.

Label-free proteomics

LC–MS/MS analysis was performed as described previously^67,68,69. In brief, reconstitution of dried peptide samples was achieved by adding 5 µl of 30% formic acid containing four synthetic standard peptides. Afterward, samples were diluted with 40 µl of loading solvent (97.9% H₂O, 2% ACN, 0.05% TFA), of which 5 µl were injected into the Dionex UltiMate 3000 nano high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific). A pre-column (2 cm × 75 µm, PepMap 100 C18, Thermo Fisher Scientific) run at a flow rate of 10 µl min⁻¹ using mobile phase A (99.9% H₂O, 0.1% formic acid) was used to pre-concentrate peptides before chromatographic separation. Peptides were then separated on an analytical column (25 cm × 75 µm, 25 cm, Aurora Series emitter column, IonOpticks) by applying a flow rate of 300 nl min⁻¹ and using a gradient of 8–40% mobile phase B (79.9% ACN, 20% H₂O, 0.1% formic acid) over 155 min, resulting in a total LC run time of 195 min including washing and equilibration steps. A timsTOF Pro mass spectrometer (Bruker) with a captive spray ion source run at 1,700 V was used for MS analysis. The timsTOF Pro was operated in parallel accumulation–serial fragmentation (PASEF) mode, and moderate MS data reduction was applied. Further parameters included a scan range (m/z) from 100 to 1,700 to record MS and MS/MS spectra and a 1/k0 scan range of 0.60–1.60 V.s/cm² resulting in a ramp time of 100 ms to achieve trapped ion mobility separation. All experiments were performed with ten PASEF MS/MS scans per cycle, leading to a total cycle time of 1.16 s. Furthermore, the collision energy was ramped as a function of increasing ion mobility from 20 eV to 59 eV, and the quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 700. All samples were analyzed in technical duplicates.

Label-free proteomics data analysis

The publicly available software package MaxQuant (v1.6.17.0) running the Andromeda search engine was used for protein identification and label-free quantification (LFQ)⁷⁰. Therefore, raw data were searched against the Swiss-Prot database Mus musculus (v220621 with 17,519 entries). Search parameters included an allowed peptide tolerance of 20 ppm, a maximum of two missed cleavages, carbamidomethylation on cysteines as fixed modification, methionine oxidation, and N-terminal protein acetylation as variable modification. A minimum of two peptides per protein, of which at least one has to be unique for each protein, was set as a search criterium for positive identifications. In addition, the ‘match between runs’ option was applied, using a 0.7-min match time window, a match ion mobility window of 0.05, a 20-min alignment time window, and an alignment ion mobility of 1. A false discovery rate (FDR) of ≤0.01 was set for all peptide and protein identification. LC–MS data evaluation and statistical analysis were accomplished using the Perseus software (v1.6.14.0)⁷¹. Identified proteins were first filtered for reversed sequences and common contaminants and annotated according to differentiation time points. Before statistical analysis, LFQ intensity values were log₂-transformed, means of technical duplicates were calculated, and proteins were additionally filtered for their number of independent identifications (a minimum of three identifications in at least one group). Two-sided t-tests and statistics for volcano plots were performed by applying an FDR cutoff of 0.05 and an S0 of 0.1, whereby S0 controls the relative importance of the t-test P value and difference between the means. Figure visualization was done using OriginPro (v2021), RStudio (v1.4.1106) and Instant Clue (v0.10.10)⁷².

Protein network and cluster analysis

For the generation of protein networks, we divided our proteomics data into two groups: kinases and lipid-binding proteins. All proteins that were significantly regulated on either day were used for network analysis using STRING. Networks generated were loaded in Cytoscape (v3.9.1). For protein clustering, the MCODE application inside the Cytoscape interface was used with the following conditions: network scoring including loops with a degree cutoff of 2, cluster finding with fluffing using a node density cutoff of 0.8 and node score cutoff of 0.24 with K-core of 2 and max depth of 100.

Lipid analysis

Shotgun lipidomics

A Q Exactive HF (Thermo Fisher Scientific) coupled to a TriVersa NanoMate ion source (Advion Biosciences) was used for direct infusion experiments. A total of 12 µl of the sample were delivered over 14 min with a backpressure of 0.95 psi. After 6 min, polarity switching from +1.25 kV to −1.25 kV was applied to acquire mass spectra in both positive and negative ion modes in one measurement. Full MS spectra covering the mass range of 350–1200 m/z in both positive and negative modes were acquired with a resolution of 240,000, an AGC target of 1e6, and a maximum IT of 105 ms. MS1 acquisition was followed by data-independent acquisition of precursor masses at an interval of 1,001 Da. The precursor isolation window was 1 Da, and normalized collision energy (nCE) was 21% and 26% for positive and negative modes, respectively. MS2 spectra were acquired with a resolution of 60,000, an AGC target of 1e5, and a maximum IT of 105 ms.

Targeted lipidomics

Analysis of SP and ST was performed as previously described by ref. ⁷³. Inclusion lists for targeted measurements were generated using LipidCreator (v1.2.0). For the reversed-phase LC, the UltiMate 3000 system was equipped with an Ascentis Express C18 main column (150 mm × 2.1 mm, 2.7 µm, Supelco) and fitted with a guard cartridge (50 mm × 2.1 mm, 2.7 µm, Supelco) in a column oven set to 60 °C. Solvent A was ACN/H₂O (3:2, v/v), solvent B was IPA/ACN (9:1, v/v), and both contained 0.1% formic acid, 10 mM ammonium formate and 5 µM phosphoric acid. The separation was carried out with a flow rate of 0.5 ml min⁻¹ with the following 25-min-long gradient: initial, 30% B; 0–2 min, hold 30% B; 2–3 min, 30–56.1% B; 3–4 min, 56.1–58.3% B; 4–5.5 min, 58.3–60.2% B; 5.5–7 min, 60.2–60.6% B; 7–8.5 min, 60.6–62.3% B; 8.5–10 min, 62.3–64% B; 10–11.5 min, 64–64.5% B; 11.5–13 min, 64.5–66.2% B; 13–14.5 min, 66.2–66.9% B; 14.5–15 min, 66.9–100% B; 15.0–19.0 min, hold 100% B; 19 min, 5% B; 19–22 min, hold 5% B; 22 min, 30% B; 22–25 min, hold 30% B. The injector needle was automatically washed with 30% B and 0.1% phosphoric acid, and a volume of 5 µl per sample was injected.

The LC was coupled to a QTRAP 6500+ (Applied Biosystems Sciex) with an electrospray ion source (Turbo V ion source). MS scans were acquired in positive ion mode with the following source settings: curtain gas, 30 arbitrary units; temperature, 250 °C; ion source gas I, 40 arbitrary units; ion source gas II, 65 arbitrary units; collision gas, medium; ion spray voltage, +5,500 V; declustering potential, +100 V/−100 V; entrance potential, +10 V; exit potential, +13 V. For the scheduled multiple reaction monitoring, Q1 and Q3 were set to unit resolution. The detection window was set to 2 min, and the cycle time was set to 0.5 s. Data were acquired with Analyst (v1.7.2, Applied Biosystems Sciex).

Lipid identification and quantification

All spectra from shotgun experiments were converted to centroid mode using msConvert (v3.0.20186-dd907d757) and analyzed using LipidXplorer (v1.2.8.1) under the following settings: MS1, mass tolerance of 5 ppm with an intensity threshold of 1e5; MS2, mass tolerance of 10 ppm with an intensity threshold of 5e4. For lipid identification, molecular fragmentation query language queries, based on the previous work from refs. ^74,75, were compiled to match precursor and fragment ions to recognize lipid species. The detection and quantification of GLs (DG and TG) were used in positive ion mode. GPs (cardiolipin (CL), LPA, LPI, LPG, LPC, LPE, PA, PG, PC, PCO, PE, PEO, PI and PS) were identified and quantified in negative ion mode. All signal intensities were normalized to the corresponding deuterated internal standard (Mouse SPLASH LIPIDOMIX Mass Spec Standard). Protein concentrations, determined by the Pierce BCA Protein Assay Kit, were used to quantify all lipid species. TGs and CLs were quantified based on precursor intensities (Supplementary Table 1).

SPs (Cer, HexCer, Hex2Cer, Sulfo-HexCer (SHexCer), LSM, SPBP, SPB and SM) and STs (ST and SE) were identified and quantified by LC–MS analysis (Supplementary Table 2). Integration of peaks from targeted measurements was performed using Skyline (v21.1.0.146). Lipid species abundance was calculated using peak areas and quantified to the respective internal standard (Ceramide/Sphingoid Internal Standard Mixture II; cholesterol-d7) and protein amount described above.

Generation of biotinylated PS standards

A biotinylated PS internal standard was generated for quantification of biotin-labeled PS species within the biological sample. The generation of biotinylated standards was performed according to the protocol of ref. ⁷⁶. In brief, 1 mg of PS 14:0_14:0 standard (Avanti Polar Lipids) was dissolved in 330 µl of chloroform/methanol (2:1, v/v), and 6 mg of NB were added. After vortexing, 3.3 µl of triethylamine (Sigma) were added and incubated for 30 min at room temperature. Excess NB was sedimented by centrifugation for 5 min at 500 × g at room temperature. The supernatant was transferred into a new glass vial. The sediment was washed once with 330 µl of 2:1 CHCl₃/methanol, vortexed and centrifuged, and the supernatant was combined from the previous step. After drying under nitrogen flow, the biotinylated standard was resuspended in 600 µl of methanol for HPLC purification. An Agilent 1200 Series LC system with a Discovery C18 column (250 mm × 4.6 mm, 5 µm) was used with the following conditions: temperature, 22 °C; flow rate, 1 ml min⁻¹; gradient elution profile, 50% B (A, water + 5 mM ammonium acetate; B, methanol + 5 mM ammonium acetate) to 100% B over 15 min, held at 100% B for 20 min, then re-equilibrated to 50% B. Ultraviolet absorbance was measured at 205 nm. Six times, 100 µl were injected and all fractions were manually collected, combined and dried using a Genevac and resuspended in 200 µl of methanol. The standard was transferred into a clean, pre-weighted glass vial, dried and weighted again. The standard concentration was adjusted to 100 ng µl⁻¹ in methanol and stored under nitrogen gas at −80 °C. The derivatization of the standard was validated by direct injection on an amaZon speed ion ETD trap instrument.

Surface labeling of externalized PS

Biotinylation of cell surface-exposed PS was performed based on the protocol from ref. ⁷⁶. A cell-impermeable reagent (SNB) was used to label PS on the outer leaflet, and a cell-permeable reagent (NB) was used for quantification of total PS content. In brief, MK cell suspensions containing 200,000 cells per sample were equally divided in two tubes (100,000 cells each) and treated with either 43 µl of 22 mM SNB in PBS or 20 µl of 20 mM NB in DMSO for 10 min at room temperature. To SNB-treated cells, 72 µl of 250 mM L-lysine in PBS were added and incubated for another 10 min at room temperature to quench excess SNB. To NB samples, 95 µl of LC-grade water were added to reach the final extraction volume of 315 µl. Samples were transferred into 5-ml polypropylene Eppendorf tubes and subjected to the SIMPLEX protocol as described in the section ‘Protein and lipid extraction’ using a tripled amount of all solvents. For normalization of lipid intensities, 10 µl of a self-generated biotinylated PS standard (biotin-PS 14:0_14:0, 10 ng µl⁻¹) and 5 µl of Mouse SPLASH mix (Avanti Polar Lipids) were added prior to extraction. Dried lipid extracts were resuspended in 25 µl of butanol solvent (1-butanol:IPA:H₂O, 8:23:69, v/v/v + 5 mM phosphoric acid), and lipids were identified using targeted LC–MS/MS.

Reversed-phase LC–MS/MS of biotinylated PS

Lipid extracts were separated by reversed-phase HPLC according to ref. ⁷⁷ with minor adaptions. For separation, an Ascentis Express C18 column (150 mm × 2.1 mm, 2.7 μm, Supelco) fitted with a guard cartridge (50 mm × 2.1 mm, 2.7 μm, Supelco) was used. Mobile phase A was ACN/H₂O (60:40, v/v), mobile phase B was IPA/ACN (90/10, v/v), and both contained 10 mM ammonium formate and 0.1% formic acid. The temperatures of the autosampler and the column oven were set to 10 °C and 60 °C, respectively. Separation was carried out with a flow rate of 0.5 ml min⁻¹ with the following 35-min-long gradient: initial, 30% B; 0.0–3.0 min, hold 30% B; 3.0–15.0 min, ramp to 75% B; 15.0–17.0 min, ramp to 100% B; 17.0–30.0 min, to 5% B; 30.1–35.0 min, to 30% B. The injector needle was automatically washed with 30% B, and a volume of 5 µl per sample were injected.

The LC was coupled to the Q Exactive HF instrument, and data were acquired in negative ion mode. The following electrospray ionization (ESI) source parameters were applied: spray voltage, 3.8 kV; capillary temperature, 270 °C; sheath gas flow rate, 50; auxiliary gas flow rate, 15; auxiliary gas heater temperature, 380 °C; S-lens RF level, 60. Full MS spectra from 500 to 1,200 m/z were acquired in negative mode with a resolution of 60,000, an AGC target of 106, and a maximum IT of 50 ms. For MS/MS, a resolution of 30,000, an AGC target of 105, a maximum IT of 115 ms and an nCE of 24 were applied.

Identification and quantification of biotinylated PS

Integration of peaks from targeted measurements was performed using Skyline (v21.1.0.146). The top two abundant PS species (PS 18:0_18:1 and PS 18:0_20:4) were monitored. For the identification of biotinylated PS, both FAs and the neutral loss of the biotinylated PS headgroup (m/z, 313) were used. Lipids were quantified on the MS1 level. Lipid species abundance was calculated using peak areas and quantified to the respective internal standard (biotinylated PS to B-PS 14:0_14:0, unlabeled PS to PS 15:0_18:1(d7)). To account for differences in PS total amount throughout the samples, the summed intensity of labeled and unlabeled PS within each sample were used to normalize the amount of labeled PS. A ratio was calculated of externalized:total PS. Day 1 was set to 1 and used as a reference to calculate relative changes during megakaryopoiesis.

Validation of shotgun lipidomics data by targeted LC–MS/MS

Trends of selected lipid species shown in Fig. 4c were validated by targeted LC–MS/MS to increase confidence in the data obtained by shotgun lipidomics. After extraction of lipids by SIMPLEX, the dried lipid phase was resuspended in 50 µl of butanol solvent (1-butanol:IPA:H₂O, 8:23:69, v/v/v + 5 mM phosphoric acid) and separated by reversed-phase LC–MS/MS. LC parameters are as described in the section ‘Reversed-phase LC–MS/MS of biotinylated PS.’ The LC was coupled to the Q Exactive HF instrument applying the following ESI source parameters: spray voltage, 4.0 kV and 3.8 kV in positive and negative modes, respectively; capillary temperature, 270 °C; sheath gas flow rate, 50; auxiliary gas flow rate, 15; auxiliary gas heater temperature, 380 °C; S-lens RF level, 60. GLs were analyzed in positive mode, and GPs were analyzed in negative mode. Except for TG and CL, all lipids were quantified on the MS2 level. High-resolution MS full scan and parallel reaction monitoring were performed in one measuring cycle (MS1: 0.0–35.0 min negative mode, resolution 60,000, 350–1500 m/z; 13.0–35.0 min positive mode, resolution 30,000, 400–900 m/z; MS2: 0.0–16.0 min negative mode, resolution 30,000, nCE 24; 13.0–16.0 min positive mode, resolution 30,000, nCE 21). An AGC target of 10⁶ and 10⁵ and a maximum IT of 50 ms and 115 ms were used in full scan and parallel reaction monitoring, respectively. A pooled sample was measured in both polarities separately to verify the identification and acquire MS2 data for TG and CL.

Visualization and network analysis

For further investigation of common patterns in the lipid profiles, we performed both similarity-based clustering and network analysis based on the analysis approach of ref. ¹². In the first step, we compared all lipids with each other pairwise. For each lipid, we computed the mean abundances for days 1, 3 and 7, which we denote as a lipid profile. For each lipid pair, we compared their lipid profiles using Pearson correlation. The result is a quadratic similarity matrix m. For the network analysis, we have drawn a graph in which we connected all lipids with each other that had a cosine similarity ≥99% in m. For the cluster analysis, we sorted m row-wise and column-wise equally. To do so, we applied hierarchical clustering on the columns of m with cosine similarity and unweighted average clustering. The result is a sorted matrix m. Since we were interested in lipid profiles even with anticorrelation, we worked only with the absolute values of m. Along the diagonal of the sorted matrix m, we searched for the biggest nonintersecting squared areas, where all values within the squares have a Pearson correlation value of ≥99%. The results are presented in Fig. 3e–h. Networks were generated using Cytoscape (v3.9.1).

Cell vitality assay

Cell vitality of control and inhibitor-treated MKs was determined by a Promega CellTiter-Glo 2.0 Assay, based on the quantification of ATP and indication of metabolically active cells, as done in ref. ⁷⁸. Experiments were performed according to the manufacturer’s protocol. Per 96 wells, 10,000 MKs were seeded in TPO-supplemented DMEM. Inhibitors of phospholipid synthesis (160 µM FSG67, 5 µM triacsin C), 1 µM ionomycin or DMSO were added to the cells in single wells and cultivated for 0, 3 and 7 days. Ionomycin was added to cells as a positive control for apoptotic cells. Plates were equilibrated to room temperature for approximately 30 min before the addition of CellTiter-Glo 2.0 reagent (equilibrated to 22 °C). The reagent was added to each well in a 1:1 ratio of reagent:cell culture medium, mixed and incubated for 2 min on an orbital shaker to induce cell lysis. After 10 min, luminescence signals were recorded using a GloMax-Multi Detection System (Promega, 9300-002).

Subcellular protein fractionation

Subcellular protein fractions were obtained using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, 78840) following the manufacturer’s instructions with some modifications. MKs were isolated after cultivation for 0 days and 7 days in TPO-supplemented DMEM and washed with ice-cold PBS. One hundred thousand cells were pelleted by centrifugation for 2 min at 500 × g. Cell pellets were dried, 100 µl of ice-cold CEB containing protease inhibitors (1:100) were added, and cell pellets were incubated for 30 min at 4 °C while mixing on an end-over-end shaker. After centrifugation for 5 min at 500 × g, the supernatant was collected, and to the remaining cell pellet, 100 µl of ice-cold MEB containing protease inhibitors (1:100) were added, vortexed for 5 s and incubated for 10 min at 4 °C while mixing. The supernatant (membrane fraction) was collected after 5 min of centrifugation at 3,000 × g and frozen at −80 °C until immunoblot analysis.

Immunoblot analysis

Immunoblot analysis was performed using the prepared membrane fraction of cultivated MKs (day 0 and day 7) in the absence or presence of the inhibitors. After centrifugation for 15 min at 20,000 × g at 4 °C, the supernatant was collected, and the protein concentration was measured using a Bradford assay from Bio-Rad. For immunoblotting, protein was loaded in 12% gels and electrotransferred onto a nitrocellulose membrane, followed by blocking with 5% nonfat milk or 5% BSA for 1 h at room temperature. Afterward, the membrane was incubated with the primary antibody against CSNK2A1 (1:1,000), PKHO1/CKIP-1 (1:200) or GAPDH (1:1,000) overnight at 4 °C. After washing with TBS-T, the blots were incubated with fluorochrome-conjugated secondary antibodies (1:15,000) for 1 h at room temperature. After washing, antibody binding was detected with an Odyssey infrared imaging system (LI-COR). Bands were quantified with ImageJ (National Institutes of Health)⁷⁹.

Functional assessment of megakaryopoiesis

To validate the importance of the observed lipidomic changes on megakaryopoiesis, inhibitors of two enzymes involved in phospholipid biosynthesis were used. We added 160 µM FSG67, 5 µM triacsin C or vehicle control (DMSO) to the cell suspensions 24 h after the start of cultivation.

Lipidomic analysis (see the section ‘Lipid analysis’) was performed on MKs isolated via BSA gradient on day 0 (before the addition of inhibitors and control) and after 7 days of cultivation.

Proplatelet formation assay

Proplatelet formation assays were performed in triplicates on 48-well plates. After isolation via BSA gradient after 3 days of cultivation, 15,000 cells per well were seeded. Proplatelet formation was examined every 6 h by microscopy (ECLIPSE Ti2, NIS-Elements imaging software, Nikon). Ratios of proplatelet-forming MKs compared with non-proplatelet-forming MKs were calculated.

MK polyploidization

Ploidy measurements were performed according to ref. ¹. In brief, the bone marrow of femora from B6 mice was flushed and homogenized. Cells were cultivated in 10-cm cell culture dishes containing DMEM (supplemented with 1% penicillin/streptomycin and 10% FBS), and differentiation was initiated by adding (1%) recombinant TPO. After 5 days of cultivation, the cell suspension was carefully pipetted on a two-phase BSA gradient (bottom, 1.5 ml 3% BSA in PBS; top, 1.5 ml 1.5% BSA in PBS) to separate cells by weight. After 40 min, the supernatant was removed, and the cell pellet was washed three times with 500 µl of PBS. Nonspecific bone marrow binding was blocked by incubation with 0.02 mg ml⁻¹ FcR Blocking Reagent. Afterward, MKs were stained using FITC-conjugated anti-CD41 antibody, and the cells were subsequently washed once with 2 mM EDTA in PBS. Then, cells were washed with PBS (5 min at 300 × g) and fixed in PBS containing 1% PFA/0.1% EDTA. Fixed cells were washed with PBS (10 min at 300 × g) and permeabilized in PBS containing 0.1% Triton X-100. Finally, DNA was stained using 50 μg ml⁻¹ propidium iodide staining solution containing 100 μg ml⁻¹ RNase A and 2 mM EDTA in PBS. Analysis was performed by flow cytometry (BD FACSCalibur, BD Biosciences) and FlowJo software (Tree Star, Inc.) (Supplementary Fig. 1).

2P-IVM

Mice were anesthetized by intraperitoneal injection of 0.5 mg per g (body weight) medetomidine, 5 mg per g (body weight) midazolam and 0.05 mg per g (body weight) fentanyl. A 1-cm midline incision was made to expose the frontoparietal skull, while carefully avoiding damage to the bone tissue. For immobilization of the head, the mice were placed on a custom-built metal stage equipped with a stereotactic holder. Bone marrow vasculature was visualized by injection of anti-CD105 Alexa Fluor 546 (0.6 µg per g (body weight)), and MKs and platelets were visualized by injection of anti-CD42a (GPIX) Alexa Fluor 488 derivative (0.8 µg per g (body weight)). Images were acquired on an upright two-photon fluorescence microscope (TCS SP8 MP, Leica Microsystems) equipped with a ×25 water objective with a numerical aperture of 1.0. A tunable broadband Ti:sapphire laser (Chameleon, Coherent) was used at 780 nm to capture Alexa Fluor 488 and 546 fluorescence. For each mouse, four to eight z-stacks with a step size of 0.51 µm were recorded from different positions in the bone marrow. Proplatelet-forming MKs were counted, and MK morphology was categorized as normal or fragmented by a blinded experimenter. ImageJ was used to generate movies (Supplementary Videos 1–3).

Immunofluorescence staining on femora cryosections

Femora of inhibitor-treated or csnk2α1^lox/lox and csnk2α1^{Pf4∆/Pf4∆} mice were fixed with 4% PFA in 5 mM sucrose solution (Sigma-Aldrich), transferred into 10% sucrose in PBS and dehydrated using a graded sucrose series (10%–20%–30%). Subsequently, femora were embedded in Cryo-Gel (Leica) and shock-frozen in liquid nitrogen. Cryosections with a thickness of 5 μm were generated using a CryoJane Tape Transfer System (Leica) and probed with a self-conjugated FITC-anti-CD41 antibody (1:100) for specific labeling of MKs and platelets, Alexa Fluor 647-conjugated anti-CD105 antibody (1:300) for endothelium detection, and DRAQ5 (1:1,000) for nuclei staining. Samples were visualized using a Leica Stellaris 5 (LMB R039a) confocal microscope.

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/s44161-023-00325-8