Stem Cell

Cytosolic N-terminal formyl-methionine deformylation derives cancer stem cell features and tumor progression – Scientific Reports

Time Stamp: June 27, 2024 8:00 PM
Source Node: 624061

Antibodies

The following primary antibodies were used: anti-flag (F3165; Sigma Aldrich, MO, USA), anti-tubulin (T5168; Sigma Aldrich), anti-TOM20 (sc-17764; Santa Cruz, CA, USA), and anti-SOX2 (ab97959; Abcam, CB, UK). The anti-fMet antibody was purified as previously described with slight modifications⁹. Briefly, anti-fMet sera were negatively selected using Affi-gel-10/15 resins (1536098; Bio-Rad, CA, USA) conjugated with extracts from EcPDF_3f-1 and EcPDF_3f-2 SW480 cells. After three rounds of negative selection with the resulting resins, the unbound sera were diluted in PBS-T (phosphate-buffered saline with 0.05% Tween 20, pH 7.4) at a 1:500 ratio and pre-incubated with polyvinylidene fluoride (PVDF; IPVH0010; Merck, NJ, USA), on which extracts from EcPDF_3f-1 SW480 cells were electro-transferred before use. The secondary antibodies were goat anti-rabbit IgG (170–6515; Bio-Rad) and anti-mouse IgG (170–6516; Bio-Rad).

Plasmid construction

The plasmids and primers used in this study are listed in Supplementary Tables 1 and 2. To construct pCH5542, EcPDF DNA was PCR-amplified from pCH5540 using the primer pair OCH5578/5579. Simultaneously, 3× FLAG DNA was PCR-amplified using the primer pair OCH5576/5577. Subsequently, both EcPDF and 3× FLAG PCR products were used as templates for overlap extension PCR using the primer pair OCH5578/OCH5577. The resulting PCR product was digested with KpnI/XhoI and subsequently ligated into KpnI/XhoI-digested pcDNA3(+).

Cell culture and transfection

SW480 cells were cultured in RPMI-1640 (11875-093; Cytiva, MA, USA) supplemented with 10% fetal bovine serum (FBS; SH30919.03; Cytiva) and transfected with either pcDNA3 or pCH5542 (pcDNA3-EcPDF_3f) using Lipofectamine 2000 (11668019; Thermo Fisher Scientific, MA, USA) for 24 h. The cells were subsequently treated with 1–2 mg/ml G418 (G-1033; AG Scientific, CA, USA) for two weeks. The cell medium was replaced with fresh medium supplemented with G418 every 3–4 days. Colonies were picked using cloning cylinders and cultured in RPMI-1640 supplemented with 10% FBS. EcPDF expression in EcPDF_3f stable cells was confirmed by immunoblotting using an anti-flag antibody.

HT29 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (SH30243.01; Cytiva) containing 10% FBS. For cell proliferation assays and flow cytometry, HT29 cells were seeded in a 12-well culture plate and transiently transfected with either pcDNA3 or pCH5542 (pcDNA3-EcPDF_3f) (1.5 µg) using 7.5 µl Lipofectamine LTX (15338030; Thermo Fisher Scientific) for 1–2 days before analysis. For immunoblotting, HT29 cells were seeded in a 12-well culture plate and transiently transfected with either OGS411 or pCH5540 (OGS411-EcPDF_3f) (1 µg) using 4 μl polyethylenimine solution (1 mg/ml) for 3 days. Additional information on the cell lines used in this study is provided in Supplementary Table 3.

Subcellular fractionation

Stably EcPDF_3f-expressing SW480 cells were fractionated using a Qproteome Mitochondria Isolation Kit (37612; Qiagen, Hilden, Germany). Subsequently, 2.5 μg of the mitochondrial and cytosolic fractions were subjected to immunoblotting with the indicated antibodies.

Immunoblotting analysis

Harvested cells were lysed with RIPA buffer (89900; Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (4693132001; Sigma Aldrich) and 20 µg/ml actinonin (HY-113952; MedChemExpress, NJ, USA). The cell lysates were incubated on ice for 20 min, briefly sonicated, and centrifuged at 12,000×g for 10 min at 4 °C. The collected supernatants were diluted to 2 μg/μl, mixed with 4× SDS sample buffer at a 3:1 ratio, and heated at 70 °C for 10 min. Subsequently, 10 μl of each sample was subjected to immunoblotting with the specified antibodies. For immunoblotting with multiple antibodies, the membrane was cut into several pieces before incubation with specific antibodies. Immunoblots were developed with ECL substrates in chemiluminescence mode using Amersham Imager AI680 (GE HealthCare, IL, USA). Immunoblotting band intensities were quantified using ImageJ software (https://imagej.nih.gov/ij/). The uncropped immunoblotting images of the main figures are provided in Supplementary Information.

Cell proliferation assay

Stably EcPDF_3f-expressing SW480 cells (1 × 10⁵) in RPMI-1640 containing 10% FBS were seeded into each well of a 24-well plate. Alternatively, transiently EcPDF_3f-expressing HT29 cells (2 × 10⁵) in DMEM containing 10% FBS were seeded into each well of a 12-well plate. On the specified day, the cells were harvested and counted using a TC20 automated cell counter (Bio-Rad).

Soft agar colony formation assay

A 1% agarose solution and RPMI-1640 supplemented with 10% FBS were mixed in a 1:1 ratio, and 2 ml of the mixture was poured into each well of a 6-well plate. Once the bottom layer solidified, the melted 0.7% agarose solution and RPMI-1640 supplemented with 10% FBS containing 2.5 × 10³ cells were mixed in a 1:1 ratio, and 2 ml of the mixture was gently poured on top of the solidified bottom layer. The plates were incubated at 37 °C in a 5% CO₂ incubator to allow the top layer to solidify. Throughout the colony formation period, the top layer was maintained in fresh medium every 2–3 days to ensure nutrient replenishment. After two weeks, the agar layers were stained with 0.5% crystal violet to visualize the colonies, and images were captured for analysis.

Phalloidin staining

Cultured cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and stained with phalloidin conjugated to Alexa Fluor 594 (A12381; Thermo Fisher Scientific) for 30 min. The phalloidin staining solution (0.2 µM phalloidin and 1% bovine serum albumin [BSA] in PBS) was used. Fluorescence was observed using a Zeiss Axio Scope-A1. Cell sizes were measured using the ImageJ software.

Spheroid assay

Non-confluent SW480 cells were trypsinized, dispersed into single-cell suspensions, and plated at a density of 500 cells per well in a 24-well ultra-low attachment plate (CLS3473; Corning, NY, USA) with DMEM/F-12 containing 10 ng/ml bFGF (F0291; Sigma-Aldrich) and 20 ng/ml EGF (AF-100–15; Thermo Fisher Scientific) for 10 days. The culture medium was replaced every 4 days. Spheres over 50 μm in size were enumerated using Olympus CKX53 microscope at × 20 magnification.

RT-qPCR

Total RNA was isolated from control and EcPDF_3f SW480 cells using the RNeasy Mini Kit (74104; Qiagen). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo Fisher Scientific). RT-qPCR was performed using the SYBR Green Master Mix (4367659; Applied Biosystems, MA, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems). Relative mRNA levels of target genes were normalized to β-actin using the ΔΔCt method. Primer sequences are listed in Supplementary Table 4.

Flow cytometry

Control vector- or EcPDF_3f-expressing SW480 and HT29 cells were harvested and resuspended in FACS buffer (PBS + 2% FBS) at 1 × 10⁷ cells/ml. Cell suspensions were incubated with anti-CD24-FITC (1:20 dilution; 11–0247-41; Thermo Fisher Scientific) or isotype control (11-4714-81; Thermo Fisher Scientific) on ice for 30 min. Subsequently, the cells were diluted in FACS buffer and analyzed using a FACSVerse flow cytometer (BD Biosciences, NJ, USA). A total of 25,000 events were acquired, and events with FSC-A values less than 50,000 were excluded from the analysis. The data were subsequently gated and plotted using FACSuite Software (BD Biosciences).

In vivo xenograft assay

BALB/c nude mice (5 weeks old; male) were obtained from Orient Bio (Seongnam, Gyeonggi, South Korea) and acclimated for 1 week. The mice were fed standard rodent chow and water ad libitum and maintained in a specific pathogen-free animal facility with a standard 12-h light/12-h dark cycle. For the xenograft tumor growth assay, vector alone- and EcPDF-expressing SW480 cells (5.0 × 10⁶ cells) in 50 µl culture medium were mixed with 50 µl Matrigel (CLS354234; Corning). The cell mixtures were subcutaneously injected into the posterior flank of 6-week-old male BALB/c nude mice. Tumor volume was measured every 2 days for a period of 26 days and calculated using the following formula: 1/2 × (largest diameter) × (smallest diameter)². On day 26, tumor tissues were extracted and lysed in RIPA buffer supplemented with a protease inhibitor cocktail for immunoblotting analyses.

TCGA database analysis

Gene expression data for normal and cancer cells (normalized RNA-seq FPKM-UQ) from patients with CRC were acquired from the TCGA database (TCGA-COAD, July 2023). The dataset comprises 41 normal and 473 cancer samples. Tumor stages were defined using the latest version of the American Joint Committee on cancer code at the time of diagnosis. Major tumor stages (I, II, III, or IV) were investigated for differences in gene expression. After normalization, the expression levels of HsPDF gene were compared. Statistical analyses and visualization were performed using GraphPad Prism 9 (GraphPad Software, CA, USA).

Statistical analysis

P values were determined using a two-tailed unpaired t-test and two-way analysis of variance (ANOVA) using GraphPad Prism 9/10. Statistical significance was set at P < 0.05.