Fig. 7

Knockdown of CAV2 induced mitochondrial dysfunction in OSCC. (A) Measurement of ROS in CAL27 and SCC25 cells treated with CAV2 knockdown (KD-2) or negative control (NC-2). ROS levels were quantified using the DCFH-DA probe and observed under a fluorescence microscope. Cells expressed RFP (red), and ROS were labeled with DCFH-DA (green). Scale bars = 100 μm. Five visual fields per sample were randomly selected for statistical analysis. (B) Assessment of MMP in CAL27 and SCC25 cells treated with KD or NC using the JC-1 probe. Nuclei were stained with DAPI (blue), and MMP was visualized with JC-1 (red). The quantitative analysis of MMP was performed. Scale bars = 50 μm. (C and D) Seahorse analysis of mitochondrial respiration in CAL27 and SCC25 cells treated with KD or NC was conducted. Non-mitochondrial oxygen consumption showed no significant difference between the KD and NC groups. Results showed that KD significantly decreased basal respiration, maximal respiration, proton leak, and ATP production in both cell lines compared to NC. Spare respiratory capacity analysis revealed a significant reduction in SCC25 cells, whereas no significant change was observed in CAL27 cells. (E and F) Mitochondrial intensity in OSCC cells (CAL27 and SCC25) treated with NC or KD was measured using the Mitotracker probe. GFP (green) labeled cells and mitochondria were stained with Mitotracker (red). Scale bars = 50 μm. Quantitative analysis was based on five random fields per sample. (G and H) Mitochondrial morphology was assessed in OSCC cells using Mitotracker staining. In the control group (NC), mitochondria appeared disc-shaped, whereas in the CAV2 knockdown group (KD), mitochondria were elongated and fragmented. Scale bars = 10 μm. (I) KEGG pathway enrichment analysis based on RNA sequencing data identified lysosome- and mitophagy-related pathways as significantly enriched in KD cells compared to NC cells. (J and K) TEM images of CAL27 and SCC25 cells showed increased interactions between mitochondria and lysosomes in KD cells compared to NC cells, highlighting enhanced mitophagy. Pseudocolors were applied to distinguish mitochondria (blue) and lysosomes (purple-red). Scale bars = 1 μm. The frequency of mitochondria-lysosome contacts was quantified and compared between groups (NC: n=32; KD: n=31). All data were expressed as the mean ± SD (n≥3) without special instructions. The one-way ANOVA determined significance with Dunn’s multiple comparison tests (A and B) and the Student’s t-test (C-F, J, and K). *P<0.05, **P<0.01, ***P<0.001, ns = no significance. CAV2: CAVEOLIN2; KD: shRNA targeting CAV2, labeled with GFP for green fluorescence tracking) or negative control, 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. OSCC: oral squamous cell carcinoma; ROS: reactive oxygen species; DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate; DAPI: 4’,6-diamidino-2-phenylindole; MMP, mitochondrial membrane potential; TEM: transmission electron microscope; Mito: mitochondria; Lys: lysosomes