Mice and experimental OA

All animal experiments were approved by The Third Affiliated Hospital of Southern Medical University and the Academy of Orthopedics in Guangdong province. All C57BL/6J and transgenic mice were housed in a specific-pathogen-free level animal room in an environment with a temperature of 20 °C–26 °C and a humidity of 50%–60%, and a 12-h light/dark cycle was maintained. We used Cre recombinase-mediated Fscn1 gene recombination to construct chondrocyte-specific Fscn1 knockout transgenic mice. Fscn1-flox (Strain #:T019490, purchased from GemPharmatech, Nanjing, China) homozygous females and Col2-CreER (Strain #:NM-KI-18029, purchased from Shanghai Model Organisms Center, Inc., Shanghai, China) or Col2-Cre transgenic males (Strain #:003554, purchased from Jackson Laboratories, Bar Harbor, ME, USA) were crossed to generate Col2-Cre: Fscn1flox/flox mice (FSCN1-cKO) or Col2-CreER: Fscn1flox/flox mice (FSCN1-iKO); Cre-negative Fscn1flox/flox mice (termed Fscn1flox/flox) served as controls. Tamoxifen (0.1 mg/g body weight) was administered to the 8-week-old mice via intraperitoneal injection (once per day for 5 consecutive days) to induce the Cre-recombinase-mediated deletion of Fscn1 in Col2-expressing cells. DNA isolated from mouse tails was used to perform PCR to identify the genotypes of transgenic mice with the primers for Col2-Cre, Col2-CreER, and Fscn1 loxp (Table S5). Mice were sacrificed at embryonic day 18.5 for histological analysis of the knee joint. Fscn1flox/flox mice (Cre-negative control) were injected with tamoxifen in the same way. All transgenic mice had been backcrossed on the C57BL/6JGpt strain (Strain #: N000013, purchased from GemPharmatech, Nanjing, China) for at least ten generations. We only used male mice because tamoxifen had been widely thought to have effects in female mice, including on cartilage, and male mice were more prone to OA. Post-traumatic OA was induced by destabilization of the medial meniscus (DMM) surgery in 12-week-old male mice.46 Sham-operated mice of the same sex and age were used as controls. Briefly, the meniscus ligament was located and cut in the DMM surgery group. However, in the sham group, no resection was performed after finding the meniscal tibial ligament. Mice were sacrificed by overdose anesthesia at 4, 6 or 10 weeks after DMM surgery. Knee joints were harvested for histological analysis according to each experimental design.

Collection of human tissue samples

Human OA cartilage specimens were obtained from eighteen OA patients undergoing total knee arthroplasty at the Third Affiliated Hospital of Southern Medical University (Guangzhou, China) for a diagnosis of end-stage OA, and were authorized by patients under the ethical approval of the hospital ethics committee. All patients provided complete written informed consent prior to total knee arthroplasty surgery. Patients’ information including patient ID, bed number, gender and age are recorded in Table S4. As drug treatment status is considered a key exclusion criterion for tissue explant culture study, we strictly used cartilage samples from OA patients who had no history of drug treatment for OA within the three years before total knee replacement surgery, including steroids or non-steroidal anti-inflammatory drugs (NSAIDs). It should be noted that we only used samples from female patients.

Human OA cartilage explant culture

The cartilage specimens were cut into 1 cm3 pieces and washed three times with PBS (pH 7.4, Boster, AR0030, Wuhan, China) containing 1% penicillin and streptomycin (Gibco/Life Technologies, Carlsbad, CA, USA, 15140122). Then the pieces of cartilage were immediately transferred into 12-well plates filled with Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, C11330500BT) containing 10% fetal bovine serum (Gibco, 10099141 C) and 1% antibiotics. The OOCHAS histopathology grading system and Collins’s tissue grading system were used to analyze the OA cartilage degeneration morphologically. To evaluate the effect of drugs on human OA, each compound was applied to 12-well plates containing cultured cartilage explants. Plates were treated with one of the following added to each well: 10 μmol/L NP (NP-G2-044, a Fscn1 inhibitor, Cat. No. #: HY-125506, MedChemExpress, Shanghai, China) or 2 μL DMSO (Cat. No. #: D8371, Solarbio, Beijing, China) as control, and cultured for 7 days in complete medium under a 5% CO2 atmosphere. The medium was replaced every other day. Each chemical treatment was performed in at least three replicates with cartilage specimens from different donors.

Intra-articular injections

Anesthetized mice were administered 10 μL adeno-associated virus 2/5 (AAV2/5) encoding mouse Fscn1 or negative control (AAV2/5-Fscn1 or AAV2/5-control, 2.5 × 1010 vg, created and packaged by Hanbio Biotechnology, Shanghai, China) by intra-articular injection 2 weeks before OA-inducing DMM surgery. Additionally, two supplementary AAV injections were performed 2 and 8 weeks after DMM operation. The mice were sacrificed 6 or 10 weeks after DMM surgery and the knee joints were harvested for histological analysis. For the drug treatments of OA in mice, 10 μL of drugs: 20 mmol/L LDN-193719 (Cat. No. #: HY-12274, MedChemExpress), 10 mmol/L NP, were injected once a week via intra-articular administration into the right knee of the mice, starting one week after DMM-inducing OA surgery. The drugs are dissolved in DMSO, but diluted 10-fold in saline before injection to minimize the toxicity. As described above, the mouse knee joints were collected at 6 or 10 weeks after OA surgery.

Safranin O staining and OA scoring

De-waxed and rehydrated tissue sections were soaked in PBS for 5 min, stained with a prepared 1% fast green solution (F7258-25G, Sigma-Aldrich, St Louis, MO, USA) for 60 s, rinsed with 3% acetic acid fixative solution for 3 s, stained with 0.5% safranin O solution (S8884-25G, Sigma–Aldrich) for 30 s, and then washed with deionized water to remove unbound stain. Finally, the sections were sealed with neutral gum after dehydrating and clearing. The histological scoring in the medial tibial plateau of the OA-model mice was quantified using the International Society for Osteoarthritis Research (OARSI) grading system (score 0–6), which required three experienced researchers, who had extensive experience in evaluating human and mouse OA, to blindly score three safranin O-stained sections for each specimen.47 Before grouped analysis, the mean score of the medial tibial plateau was computed for each animal. For synovitis scoring, sections were used to evaluate synovial changes using Krenn’s synovitis scoring system, where morphological parameters of synovitis including enlargement of the synovial lining layer, degree of inflammatory infiltration and activation of resident cells were graded separately (score, 0 to 3).47,48 Then individual scores were summed (score, 0 to 9) and classified as follows: no synovitis (score 0–1); slight synovitis (score 2–3); moderate synovitis (score 4–6) and strong synovitis (score 7–9).

Cell culture

Mouse primary articular chondrocytes, which were isolated from femoral condyles and tibial plateaus of 3-day-old C57BL/6 J mice, were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin and streptomycin in a humidified 37 °C and 5% CO2 atmosphere. When the cells reached 90% confluence, the P0 primary chondrocytes were digested with 0.25% Trypsin-EDTA (Gibco, 25200-072) and seeded into new dishes. P0 or P1 chondrocytes were stimulated with IL-1β (AF-211-11B, Pepro Tech, Rocky Hill, NJ, USA), strain loading or drug treatments. SW1353 cells (human chondrogenic cell line; ATCC, Manassas, VA, USA) were maintained in DMEM with 4.5 g glucose (Gibco, C11995) supplemented with 6% FBS and 1% antibiotics at 37 °C in a humidified atmosphere with 5% CO2. Primary chondrocytes were stimulated with 10 ng/mL IL-1β to establish a cell model of OA in vitro. Recent studies have shown that excessive mechanical stress leads to chondrocyte degeneration which also mimics an OA model in vitro.32 Consequently, we treated mouse primary chondrocytes with 0.5 Hz and 20% cyclic tensile strain loading via a Flexcell® FX-5000™ Tension System (Flexcell International Corp., Burlington, NC, USA) for 24 h. Cells were transfected with sh-FSCN1 (Hanbio Biotechnology), sh-DCN (Hanbio Biotechnology) or FSCN1-encoding plasmid (Hanbio Biotechnology) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the instruction. For primary culture of human articular chondrocytes, the cells were isolated from femoral condyles and tibial plateaus of OA donors. Human articular chondrocytes were maintained in DMEM/F-12 supplemented with 10% FBS, 1% antimycotics, and 1% antibiotics, at 37 °C in a humidified atmosphere with 5% CO2. Cells were treated with either: 10 μmol/L NP or 2 μL DMSO for 24 h under stimulation with 10 ng/mL IL-1β.

Actin-bundling assay

The in vitro F-actin bundling assays were performed according to the protocol provided with the Actin Binding Protein Biochem Kit™ (BK001, Cytoskeleton, Inc., Denver, CO, USA) containing G- or F-actin plus positive (α-actinin) and negative (bovine serum albumin; BSA) binding control proteins, which provided a way to obtain an answer concerning binding affinity for monomer (G-) or polymer (F-) actin. Briefly, a spin-down assay was used to measure F-actin binding, and centrifugation was performed to separate F-actin from G-actin by differential sedimentation where F-actin binding proteins co-sedimented with actin filaments and formed a pellet at the bottom of the tube. However, F-actin severing proteins, G-actin binding proteins, or non-actin binding proteins stay in the supernatant. The protein samples including pellets and supernatants were dissolved in an equivalent volume of 2× Laemmli reducing-sample buffer (Bio-Rad, 161-0737, Hercules, CA, USA). Finally, 20 μL of each sample was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subjected to electrophoresis until the dye front reached the bottom of the gel for immunoblotting.

Histology and immunohistochemistry

Histology is the gold standard for assessing OA in mice. Mouse knee joints were fixed with 4% paraformaldehyde (PFA, BL539A, Biosharp, Hefei, China) at 4°C for 24 h after removing skin and excess muscle, decalcified in 20% EDTA (pH 7.3) solution at room temperature (RT) for 2 weeks, then dehydrated in an automatic dehydrator (ASP300S, Leica Microsystems Ltd., Wetzlar, Germany) before embedding in paraffin. The joint specimens were cut into 4 μm thick sagittal slices and two sections were placed on every slide. About 10 tissue slides were harvested at approximately 80 μm intervals for histological and immunohistochemical staining according to standard protocols. The cultured human cartilage explants treated with the drugs mentioned above were harvested and fixed in 4% paraformaldehyde for 2 days, then 5 μm thick paraffin sections were cut and analyzed by immunohistochemical staining, as well as Safranin O & Fast Green staining. Mouse whole-mount skeleton staining of E18.5 embryos was performed with Alcian blue (A3157, Sigma–Aldrich) and Alizarin Red (A5533, Sigma-Aldrich). For immunohistochemistry (IHC), the following primary antibodies were used: FSCN1 (Abcam, ab126772, 1:200 dilution, Cambridge, UK), MMP3 (Abcam, ab52915, 1:50 dilution), and MMP13 (Abcam, ab39012, 1:100 dilution). For the quantification analysis of IHC-positivity in a standardized region of interest (ROI), the ratio of IHC-positive to total cell numbers was calculated with the number of IHC-positive cells and total number of cells counted in several ROI fields of articular cartilage for each mouse or human OA specimen in each group. Quantitation analysis of the density signal of IHC staining was performed by ImageJ (NIH, Bethesda, MD, USA) using the same signal threshold for all comparable IHC images in order to obtain non-biased results. A density value of 1 was given to the control group and all analyses were performed in at least three sections per animal from at least three animals.

Immunofluorescence

After routine dewaxing and hydration, paraffin sections were soaked in a TE 9.0 antigen retrieval solution in a 60 °C water bath for 3 h before blocking with ready-to-use goat serum (AR0009, Boster, Wuhan, China) for 1 h at RT. Primary mouse chondrocytes cells were fixed in 4% PFA at RT for 10 min, rinsed three times in PBS to wash off the PFA, and then blocked as before. Tissue sections or cell samples were incubated at 4 °C for 16 h with the primary antibodies indicated in Table S6. The cells and slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, 1:400) for 1 h at RT in the dark after carefully washing in PBS three times. F-actin was stained using Phalloidin Conjugates (Sigma-Aldrich, P5282, 100 µg/mL) for 15 min at RT in the dark. Finally, the nuclei were stained with DAPI (Sigma-Aldrich, F6057), and sections and cell samples were observed and captured at the corresponding excitation wavelength using a laser scanning confocal microscope (FV1000, Olympus, Tokyo, Japan). The immunofluorescence for G-actin staining was performed according to the manufacturer’s protocol of the Cell G-Actin Fluo Staining Kit (GMS102772, Genmed, Shanghai, China).

Immunoprecipitation

Immunoprecipitation (IP) was performed on the protein lysates isolated from cultured chondrocytes. Aliquots containing 2–5 × 107 cells were carefully washed three times with PBS (pH 7.4) and lysed in 1 mL ice-cold lysis buffer (40 mmol/L HEPES (pH 7.4), 2 mmol/L EDTA, 10 mmol/L pyrophosphate, 10 mmol/L glycerophosphate, 0.3% CHAPS and EDTA-free protease inhibitors (04693159001, Roche, Basel, Switzerland)) for 10 min at 4°C. Then, the lysates were centrifuged at a rcf of 10 000 × g at 4°C for 10 min and the supernatant was transferred to new centrifuge tubes. The supernatant samples were incubated with 5 μL IgG antibody (mouse anti-rabbit IgG LCS, IPKine™, A25022, Wuhan, China) and 20 μL pre-washed Protein G Sepharose™ 4 Fast Flow beads (17-0618-01, GE Healthcare, Little Chalfont, UK) under rotary agitation at 4°C for 30 min. Immediately after centrifuging samples at a rcf of 1 000 × g for 5 min, approximately 1 mL of the supernatant was pipetted into new 1.5 mL tubes. Then 5 μL primary antibody (Decorin or FSCN1, see Table S6) were added into the supernatant samples under rotary agitation at 4°C for 1 h and another 30 μL pre-washed Protein G Sepharose™ 4 Fast Flow beads were added as before and incubated under rotary agitation at 4°C for 4 h. After centrifugation at 1 000 × g for 5 min, the supernatant was discarded, and the pellet was washed 4 times with 1 mL of ice-cold PBS every time. After the last wash, the pellet was resuspended with 40 μL of 2 × SDS loading buffer, and denatured by heating on a metal plate at 100 °C for 3 min, and finally the samples were centrifuged at 10 000 × g for 5 min before SDS-PAGE electrophoresis.

Immunoblotting

Primary mouse chondrocytes were subjected to five consecutive passages, IL-1β stimulation, mechanical stress, or shFscn1 lentivirus transfection. Mouse joint tissue at 6 or 10 weeks after surgical induction of OA was lysed to isolate tissue proteins by homogenizing under liquid nitrogen. Chondrocytes were treated with IL-1β, drugs, shFscn1 lentivirus, or shDecorin-lentivirus transfection. In addition, primary chondrocytes from Fscn1-/- and Fscn1flox/flox mice were lysed to extract protein for immunoblotting analysis. Total proteins were extracted from tissues or cultured cells using 2 × SDS lysis buffer (1 mol/L Tris-HCl (pH 6.8), 10% SDS, glycerol, bromophenol blue and protease inhibitors) on ice for 10 min and analyzed by vertical electrophoresis. Then the separated proteins were transferred to 0.2 μm PVDF membranes (ISEQ00010, Merck-Millipore, Darmstadt, Germany). Next, the membranes were blocked with 5% non-fat dried milk dissolved in 1 × tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h at RT on a horizontal shaker and incubated at 4 °C overnight with primary antibodies (see Table S6). After removing unbound primary antibodies by washing three times with 1 × TBST, the PVDF membranes were incubated with secondary antibodies (see Table S6). Immunoreactive protein bands were detected by chemiluminescence. All immunoblotting experiments were repeated in at least three independent tests.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from chondrocytes treated with IL-1β and one of the five drugs, human chondrocytes treated with one of the five drugs, performed using Trizol Reagent (9109, TaKaRa Bio Inc., Osaka, Japan) according to the manufacturer’s protocols. cDNA was synthesized from total mRNA using a cDNA Synthesis kit (R333-01, Vazyme, Nanjing, China). The PCR primers for genes of interest are listed in Table S5 and real-time PCR or RT-PCR was performed with the LC96 system (Roche, Basel, Switzerland), using ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme) according to the manufacturer’s instructions. All reactions were run in triplicate, and the 2−ΔΔCt method was used for analysis of the expression of the respective genes.

RNA sequencing

Chondrocytes cells were treated with DMSO, 10 ng/mL IL-1β, 10 ng/mL IL-1β plus 10 μmol/L NP for 24 h. Three biological replicates were used to perform RNA sequencing for each experimental group. The extraction of total RNA, sequencing operation and quantification of the final libraries were performed by BGI Sequence Company (BGI Genomics Co., Ltd, Shenzhen, China). Hierarchical cluster analysis of differentially-expressed genes (DEGs), KEGG pathway enrichment analysis of DEGs, GSEA, and volcano plots were performed to find the expression changes of genes in the different groups and samples. The RNA-seq data in this study was deposited at Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession ID GSE232611.

ELISA for TGF-β1

Primary chondrocytes were plated in 6-well plates at an approximate confluency of 60%. Then the cells were treated with 10 ng/mL interleukin-1 beta (IL-1β). In parallel, the expression of FSCN1 was modulated by knockdown using shFSCN1 and by overexpression through plasmid-mediated transfection. Sixty hours post-treatment, the culture supernatants were harvested for the quantification of mouse TGF-β1 using an enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 (MEIMIAN, Cat#MM-0135M2, Jiangsu) according to the manufacturer’s instructions.

Chondrocyte Pellet culture

Primary chondrocytes were seeded in 6-well plates at an approximate confluency of 60%. The cells were subsequently treated with 10 ng/mL interleukin-1 beta (IL-1β). Concurrently, the expression of FSCN1 was modulated through knockdown using shFSCN1, and overexpression facilitated by plasmid-mediated transfection. Sixty hours post-treatment, 4 × 105 cells from each experimental group were centrifuged for 5 min at 150 × g in 1.5 mL polystyrene tubes. The culture medium was replenished every other day. After a period of 10 days, the resulting pellets were carefully collected from the tubes and fixed with 4% formaldehyde for 12 h at 4 °C, followed by dehydration in a graded sucrose series: 10% for 12 h, 20% for an additional 12 h, and finally, 30% for a further 12 h (totaling 48 h). The tissue samples were then embedded in OCT compound (Leica, Cat#3801480, Germany) and frozen sections of 5 μm thickness were prepared using a cryostat (Leica, Cat#CM3050S, Germany) according to a previously described protocol (PMID: 36696903). The sections were subsequently stained with Alcian blue or subjected to immunofluorescence analysis, and examined using a BX43 microscope (Olympus, Japan) or an Olympus FV1200 confocal microscope.

Immunoprecipitation-LC-MS/MS analysis

Mouse primary chondrocytes (1 ×107) were lysed in a buffer containing 25 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP-40, 5% glycerol, 1 mmol/L NaF, 1 mmol/L PMSF, and protease inhibitors. Clarified lysates (1 mg protein) underwent immunoprecipitation with anti-DCN antibody or control IgG antibodies coupled to Protein A/G Plus Agarose beads. The precipitated proteins were separated by SDS-PAGE, visualized by Coomassie staining, and subjected to in-gel trypsin digestion followed by liquid chromatography-tandem mass spectrometry analysis on a Q Exactive mass spectrometer coupled to UPLC, which was performed at the Fitgene Biotechnology Company (Guangzhou, China). Peptides were loaded onto a reversed-phase column (75 μm x 150 mm, C18) at 300 μL/min and ionized by nanospray. MS/MS was performed at 70 000 resolutions for intact peptides and 17 500 for fragments. Data-dependent acquisition involved one MS scan followed by 20 MS/MS scans of the top 20 precursors ( > 1E4 ions, 30 s exclusion). The AGC target captured ions up to 1E5 intensity within m/z 350–1 800. Protein identification used MASCOT to search the UniProt human database, allowing one missed cleavage, with carbamidomethylation as fixed and oxidation as variable modifications (20 × 106 peptide, 0.6 Da fragment tolerance). Monoisotopic mass calculation was used at P < 0.05 significance. Proteins from the IgG precipitate were considered contaminants and excluded.

RPLC-MS based DIA proteomics analysis

Proteomics analysis was performed on human osteoarthritic (OA) and control cartilage isolated from patients who underwent knee replacement surgery, as well as protein samples extracted from mouse chondrocytes with or without FSCN1 knockdown by shRNA and treated with IL-1β (n = 3 independent biological replicates per group). Samples were stored at −80 °C until shipment to the Fitgene Biotechnology Company (Guangzhou, China) for Reverse-Phase Liquid Chromatography-Mass Spectrometry based Data-Independent Acquisition (RPLC-MS DIA) proteomics analysis.

First, protein samples underwent quantification. A standard curve was prepared using 0, 1, 2, 4, 8, 12, 16, and 20 μg of BSA, with 2 μL of sample in duplicate. Following the instructions of the Beyotime BSA assay kit, samples were vortexed for 20 s to ensure thorough mixing, heated at 60 °C for 30 min, and absorbance was measured to construct the standard curve.

Next, protein samples were subjected to FASP digestion. After quantification, 30 μg of protein solution was transferred to a centrifuge tube, and 4 μL of TCEP Reducing Reagent was added, followed by incubation at 60 °C for 1 h. Subsequently, 2 μL of MMTS Cysteine-Blocking Reagent was added, and the mixture was incubated at room temperature for 30 min. The reduced and alkylated protein solution was then transferred to a 10 K ultrafiltration unit and centrifuged at 12 000 × g, 4 °C for 20 min. The flow-through in the collection tube was discarded. 8 mol/L urea (pH 8.5) (100 μL) was added, and the centrifugation step was repeated twice, discarding the flow-through each time. Next, 0.25 mol/L TEAB (pH 8.5) (100 μL) was added, and the centrifugation step was repeated three times, discarding the flow-through each time. A new collection tube was used, and 50 μL of 0.5 mol/L TEAB was added to the ultrafiltration unit, followed by the addition of trypsin (trypsin: protein ratio of 1:50). The mixture was incubated at 37 °C overnight. The next day, additional trypsin (trypsin: protein ratio of 1:100) was added, and the mixture was incubated at 37 °C for 4 h. After centrifugation at 12 000 × g for 20 min, the digested peptide solution was collected in the bottom of the collection tube. Subsequently, 50 μL of 0.5 mol/L TEAB was added to the ultrafiltration unit, and the centrifugation step was repeated at 12 000 × g, 4 °C for 20 min. The flow-through was combined with the previous collection, resulting in a total of 100 μL of the digested sample.

Next, the digested sample underwent proteomic analysis using second-dimension reverse-phase liquid chromatography-mass spectrometry (RPLC-MS). The mobile phase information was as follows: Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% acetonitrile. A home-made tip-column (150 μm × 250 mm, 3 μm-C18) was used for chromatographic separation. Peptides were dissolved in the sample solvent (0.1% formic acid), and an appropriate amount of iRT reagent was added. After thorough vortexing, samples were centrifuged at 13 500 r/min, 4 °C for 20 min, and the supernatant was transferred to an autosampler vial. 3 μg of sample was injected for high-performance liquid chromatography separation at a flow rate of 600 nL/min. The digested products, separated by high-performance liquid chromatography, were analyzed by an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) in positive ion mode for 120 min. The specific mass spectrometry parameters were as follows: Primary MS parameters: Resolution: 120 000; AGC target: 4e6; Maximum IT: 50 ms; Scan range: 350 to 1 250 m/z. DIA MS parameters: Resolution: 30 000; AGC target: 5e6; Maximum IT: 50 ms; NCE/stepped NCE: 31.

The fold change (FC) was computed as the ratio of the mean relative quantitative values of proteins between two sample groups. A T-test was performed on these values to assess the statistical significance of differences, yielding a P-value that acted as an index of significance. The significance threshold was predefined at a P-value of less than 0.05. To satisfy the normal distribution prerequisite for the T-test, the relative quantitative protein values were log2-transformed prior to analysis. From this differential analysis, a P-value less than 0.05 indicated a significant change: a differential expression level increase greater than 1.5-fold was considered significant upregulation, and a decrease below 1/1.5-fold was considered significant downregulation.

Statistical analysis

Statistical analysis was performed using SPSS version 20.0 software, and graphs were generated using GraphPad Prism 8.0. Data normality was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk tests. For comparisons between two independent groups, the unpaired, two-tailed Student’s t-test or Mann-Whitney rank-sum test was used, depending on data normality. One-way ANOVA with Tukey’s post hoc test or the Kruskal-Wallis test with Dunn’s multiple comparisons test was used for multiple comparisons, as appropriate. All statistical tests were two-sided, and a P-value < 0.05 was considered statistically significant. The sample size for each experiment was indicated in the figure legend and was determined to ensure adequate statistical power based on the observed effect sizes and available samples. All experiments were performed in triplicate, with at least three independent biological replicates, including cell, histology, and immunohistochemistry experiments.