1. Bioinformatics analyses
The DNA and protein sequences of TM9SF1 (Gene ID: 10548; NP_006396.2) and its homologs were retrieved from the public database NCBI (https://www.ncbi.nlm.nih.gov/). Subsequently, the sequences were subjected to homology analysis using BLAST and Clustal to identify conserved regions and potential functional domains (https://www.ebi.ac.uk/Tools/msa/clustalo/). Moreover, the topology of TM9SF1 was analyzed using topology prediction algorithms TMHMM, accessible at http://www.cbs.dtu.dk/services/TMHMM/. The results of these analyses were employed to identify the membrane-spanning regions of TM9SF1 and predict its cellular localization.
2. Plasmid construction
The gene sequences of tm9sf1 and gfp were obtained from the NCBI database. Subsequently, Polymerase Chain Reaction (PCR) amplifications were performed using cDNA extracted from 293T cells, and pcDNA3.1-gfp was constructed by Sangon Biotech Co., Ltd. (Shanghai, China). Primers, specific to these sequences, were designed, as shown in Supplementary Table. S1. The pcDNA3.1 vector was subjected to double digestion with BamHI and XhoI restriction enzymes to linearize it, while the pGAD vector was subjected to double digestion with EcoR1 and BamH1, and the pGBK vector double-digested with BamHI and NotI. The target gene, once amplified, was digested and ligated into the linearized vector using ClonExpress II One Step Cloning Kit (Vazyme, C112-01). Following ligation, the resulting product was transformed into competent E. coli (DH5α, Vazyme, C502) cells and plated on a solid culture medium after incubating in an LB liquid medium without ampicillin on a shaker for an hour. Subsequently, the cells were cultured in a liquid culture medium containing ampicillin and incubated at 37°C in a shaking incubator for 17 hours to select colonies that contained positive clones. Once a significant increase in cell growth was observed, plasmid extraction was performed using the TIANprep Mini Plasmid Kit (Tiangen, DP103-03) following the manufacturer’s instructions. Finally, the extracted target plasmid was sent for sequencing, and the sequencing results were compared to the target gene sequence to complete the entire process.
3. Cell culture
The DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin was used to culture 293T cells. The cells were then incubated at 37°C with 5% CO2 for 48–72 hours to facilitate colony formation. Once the primary cells reached 90% confluence, they were harvested using trypsin/ethylenediamine tetraacetic acid (EDTA) and subsequently expanded in a second passage.
4. Transfection Methods
For transfection experiments, 293T cells were seeded in 6-well plates at a concentration of 1.5 × 105 cells/mL. Transfection was performed using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) in Dulbecco's Modified Eagle's Medium (DMEM) medium. The transfection included plasmids (pcDNA3.1-gfp, pcDNA3.1-tm9sf11 − 606-gfp, pcDNA3.1-tm9sf1N1 − 236-gfp, pcDNA3.1-tm9sf1C237 − 606-gfp, pcDNA3.1-tm9sf11 − 606-stag, pcDNA3.1-tm9sf1N1 − 236-stag, pcDNA3.1-tm9sf1C237 − 606—stag) and mimics, inhibitors, scrambled oligonucleotides. The siRNA targeting TM9SF1 was synthesized using the sequences 5’-GGUUACGACCUGACGAGUUTT-3’ (RNAi#1) and 5’-AAUUAUCCAUACAGAUGUC-3’ (RNAi#2). Subsequently, the cells were collected for protein analysis after 48 hours of transfection. The siRNA was transfected at a concentration of 30 nM/mL. The oligonucleotides used in the current study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
5. Immunofluorescence
The 293T cells were cultured in the appropriate cell culture medium and seeded onto coverslips. At room temperature, the sample cells were fixed with 4% paraformaldehyde for 15 min. Subsequently, the fixed cells were treated with a Phosphate-buffered saline (PBS) solution, containing 10% goat serum, 0.3 M glycine, 1% bovine serum albumin (BSA), and 0.1% Tween for 1 h at room temperature to make the cells permeable and block non-specific protein-protein interactions. The cells were then incubated overnight at 4°C with the primary antibody at a dilution of 1:100 in the blocking solution, washed, and incubated again with the respective secondary antibody (Beyotime, A0423, A0428) at a dilution of 1:200 for 1 h in the dark at 37°C. Finally, the cell nuclei were stained with a 4,6-diamino-2-phenyl indole (DAPI) staining solution (Beyotime, C1006) and then washed. The following fluorescent dyes were used to label corresponding organelles following their respective manufacturers’ instructions: ER-Tracker Red (Beyotime, C1041S), Golgi-Tracker Red (Beyotime, C1043), Mito-Tracker Red CMXRos (Beyotime, C1049B), and Lyso-Tracker Red (Beyotime, C1046). The antibodies used in these experiments were as follows, Anti-TM9SF1 Rabbit mAb (Bioss, bs-10764R), FEN1 (7H8) Mouse mAb(zenbio, 201032), and pCDNA3.1-rfp-lc3 was constucted by HANBIO, Shanghai, China. The cells were visualized using a Leica SP8 confocal system equipped with appropriate filters to detect the fluorescence signals.
6. Western blotting
After transfection, the 293T cells were collected and prepared. Cell samples were loaded into the wells of a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and subjected to electrophoresis. Following electrophoresis, the proteins were transferred onto a PVDF membrane(Merck Millipore, ISEQ00010). The membrane was blocked using a 5% non-fat milk solution (prepared by mixing 5 g non-fat milk powder in 100 mL of 1X TBST buffer). Subsequently, the PVDF membrane was incubated with the diluted primary antibodies at room temperature on a rocking platform for 2 h. The membrane was then washed with 1X TBST buffer and incubated with diluted secondary antibodies (1:5,000) at room temperature on a rocking platform for 1.5 h. The membrane was then washed three times with 1X TBST buffer. Subsequently, the protein bands were visualized as previously described[40]. This substrate reacted with the enzyme conjugated to the secondary antibodies, resulting in colored precipitate formation at the sites where antibodies bound. The protein bands were then observed, and the expression levels were measured using the BCIP/NBT chromogenic substrate. The Image J software was used for Grayscale analysis of the bands, allowing for quantitative analysis of the protein expression levels. The primary antibodies and diluents used for Western blotting were as follows: Anti-TM9SF1 Rabbit mAb (Bioss, bs-10764R, 1:1000), Anti-FEN1 Rabbit mAb (proteintech, 14768-1-AP, 1:1000), FEN1 (7H8) Mouse mAb༈zenbio, 201032༉, β-Actin Mouse Monoclonal Antibody (Beyotime, AF0003, 1:1000), GAPDH Mouse Monoclonal Antibody (Beyotime, AF0006, 1:1000), Lamin B1 Monoclonal antibody (proteintech, 66095-1-Ig, 1:1000), anti-γ-H2AX antibody (Wuhan Fine Biotech Co., Ltd., FNab10441, 1:1000), P53 (Beyotime, AF7671, 1:1000), Chk1 Rabbit Polyclonal Antibody (Beyotime, AF6498, 1:1000), Chk2 (Beyotime AF6501, 1:1000), Phospho-Chk1(Ser345) (Beyotime, AC506 1:1000), and anti-Phospho-Chk2 Rabbit mAb (Thr68) (Zenbio, 340766, 1:1000). For detection, the previously described secondary antibodies were used[40].
7. Yeast two-hybrid (Y2H) assay
The Y2H assays were performed using a series of construct pairs to systematically identify protein-protein interactions between TM9SF1 and FEN1. Recombinant plasmids were constructed using Activation Domain (AD) fusion (using the pGADT7 vector) and Binding Domain (BD) fusion (using the pGBKT7 vector). Yeast-competent cells were prepared and co-transformed with these plasmids. After 3 days, four single colonies from the transformed plates were selected and transferred onto selective plates with a synthetic complete medium that lacked two amino acids (SD/-2). The plates were then incubated for 3 days at 30°C. Colonies from these plates were subsequently picked and transferred to a medium that lacked four amino acids, including SD/-4; lacking tryptophan, leucine, histidine, and adenine. The plates were incubated again at 30°C for 3 days, and the growth of colonies was then observed. The positive control (ha72 and p33), negative control (empty vectors), and the plasmids and yeast AH109 were obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences as described previously[41].
8. Co-IP assay
The immunoprecipitation assay was performed using Protein A + G Magnetic Bead Immunoprecipitation Kit (Beyotime, P2179S), following the manufacturer's instructions. The 293T cell samples were collected either after passaging or 48 hours post-transfection. In order to prepare the samples, cell lysis buffer with protease inhibitors (Biosharp Protease Cocktail, EDTA-free, BL630B) was added, and the cells were lysed on ice for 30 min. The lysate was then centrifuged at approximately 13,000 × g for 10 min to pellet cell debris. The supernatant was transferred to a new tube and protein concentration was determined. Equal amounts of the lysate were incubated with 1 µg of primary antibody [Anti-TM9SF1 Rabbit mAb (Bioss, bs-10764R), and Anti- FEN1 (7H8) Mouse mAb(zenbio, 201032)] overnight at 4°C with gentle shaking. Subsequently, unbound samples were removed, and magnetic beads were washed three times. The precipitated product was then eluted in the elution buffer. The pre-washed Protein A Sepharose beads (Beyotime, P2055-10 mL) were added to the cell lysate along with the overnight-incubated antibody and incubated at 4°C with gentle shaking for 2–4 hours, allowing the antibody to couple with Protein A Sepharose beads. After the completion of the immunoprecipitation reaction, the samples were centrifuged at 3000× g for 3 min at 4°C and the supernatant was removed carefully. The Sepharose beads were washed 3–4 times with 1 mL of lysis buffer. Subsequently, 15 µL of SDS-PAGE protein loading buffer (5X) (BioVision, P0015L) was added and boiled for 5 min. Western blot analysis was conducted to analyze the samples and determine the interaction between TM9SF1 and FEN1 by immunoprecipitation method.
9. CCK8 assay
Cells were seeded and cultured into 96-well microplates, at a density of 5 × 103 cells/well in 100 µL of medium. Subsequently, the cells were allowed to adhere and proliferate for a specific duration (typically 48 hours). Following the incubation period, 100 µL of CCK-8 (Beyotime C0038) reagent was added to each well and incubated again for 1–4 hours. The absorbance was measured at 450 nm using a microplate reader (SpectraMax i3x platform); the wells that lacked cells served as the control. Cell proliferation was determined based on the measured absorbance.
10. Construction of TM9SF1 gene knockout transgenic mice
The transgenic siRNA-TM9SF1, derived from the TM9SF1 gene knockout, was obtained from Sangon Biotech Co., Ltd., located in Shanghai, China[5]. Conditional TM9SF1 gene knockout mice and control mice were procured from Shanghai Model Organisms Center, Inc., China. Genomic DNA was isolated from the mouse tail and assessed using PCR to confirm gene knockout[5]. Mouse spleen cell proteins were isolated, and the expression of the target protein was determined using Western blot analysis.
11. Statistical analysis
GraphPad Prism software was used to analyze statistical significance. Unless otherwise specified, an unpaired t-test was employed, and the results were presented as mean ± SD. Differences were considered to indicate statistical significance at P < 0.05, *P < 0.05, and **P < 0.01.[40]