Fig. 2

Downregulation of CAV2 in OSCC inhibited cell proliferation and promoted apoptosis. (A) Validation of CAV2 knockdown in SCC25 and CAL27 cells using shRNA targeting CAV2 (KD) or scrambled control shRNA (NC), both labeled with GFP for green fluorescence tracking. For additional validation, KD-2 and NC-2 groups were established using a second shRNA sequence targeting CAV2 (KD-2) and its respective scrambled control (NC-2), both labeled with RFP for red fluorescence tracking. mRNA expression was measured by RT-qPCR (A), and protein levels were assessed by Western blot (B). (C and D) Cell proliferation was measured by growth curve analysis over 72 h. Cells were seeded in 96-well plates and analyzed at 24 h intervals using a CCK-8 assay. (E and F) Colony formation assay showing reduced clone formation after CAV2 knockdown. Cells were seeded in 6-well plates and cultured for 14 days before staining with crystal violet. (G and H) EdU proliferation assay of SCC25 and CAL27 cells stably expressing KD or NC, respectively. Representative images of EdU labeling (red) and DAPI (blue) staining were shown. Quantification of the percentage of EdU-positive cells was calculated as follows: EdU-positive cell numbers (red dots)/total numbers (blue dots) ×100%. Scale bars of (G) = 20 μm, Scale bars of (H) = 50 μm. Five visual fields per sample were randomly selected for statistical analysis. (I and J) The apoptosis of SCC25 and CAL27 cells stably expressed KD or NC was measured by flow cytometry using APC Annexin V/PI apoptosis detection, respectively. Quantitative analysis of apoptosis rate. (K) KEGG pathway enrichment bubble charts displayed pathways involved in apoptosis and MAPK signaling. (I) Quantifying CAS9, JUN, BCL2, and CYTO C mRNA expression in CAL27 cells treated with shRNA targeting CAV2 (KD) or scrambled control shRNA (NC). (J and K) Protein expression and quantification of CAS9, CAS3, BCL2, CYTO C, p44/42 MAPK (Erk1/2), and c-JUN in SCC25 and CAL27 cells transduced with shRNA targeting CAV2 (KD) or scrambled control shRNA (NC). All data were expressed as the mean ± SD (n≥3). Significance was determined by the Student’s t-test (A-J, L, and N). *P<0.05, **P<0.01, ***P<0.001. CAV2: Caveolin2; OSCC: oral squamous cell carcinoma; RT-qPCR: real-time quantitative polymerase chain reaction; KD: shRNA targeting CAV2, labeled with GFP for green fluorescence tracking; NC: scrambled control shRNA, labeled with GFP for green fluorescence tracking; KD-2: shRNA targeting CAV2, labeled with RFP for red fluorescence tracking; NC-2: scrambled control shRNA, labeled with RFP for red fluorescence tracking; CCK8: Cell Counting Kit 8; EdU: 5-ethynyl-2’-deoxyuridine; DAPI: 4’,6-diamidino-2-phenylindole; CAS9: Caspase9; CAS3: Caspase3; CYTO C: Cytochrome C