Fig. 10
Enhanced suppression of CAV2 using transcriptional repression by T0070907 promoted cell apoptosis, lipolysis, and mitochondrial dysfunction in OSCC. (A) CAV2 protein in SCC25 cells treated with T0070907 (25 μM, 48 h). Quantitative analysis of CAV2 protein expression was performed. (B) After treatment for SCC25 cells with T0070907 (25 μM, 48 h), dual luciferase assay using SCC25 cells after transfection with the indicated plasmids (RL-TK, pGL4-basic, pGL4-CAV2-promoter reporter). (C and D) Cell viability assay showing IC50 values of T0070907 (0, 1, 2, 5, 10, 25, 50, 100 μM) in OSCC cell lines expressing shRNA targeting CAV2 (KD) and scrambled control shRNA (NC) after culturing for 24 h, 48 h, and 72 h. Both KD and NC cells were labeled with GFP for green fluorescence tracking. (E) Colony formation assay in SCC25 and CAL27 cells expressing KD or NC, followed by treatment with T0070907 (25 μM, 48 h). Colonies were quantified after 7 days of treatment followed by 5 days of culture. The quantitative analysis compares T0070907-treated groups to untreated KD and NC groups. (F) Flow cytometry analysis of apoptosis in SCC25 and CAL27 cells expressing KD or NC, treated with T0070907 (25 μM, 48 h) or DMSO (control), using APC Annexin V/PI apoptosis detection. Quantitative apoptosis rate analysis was provided. (G) PLIN3 protein in SCC25 cells treated with T0070907 (25 μM, 48 h). Quantitative analysis of PLIN3 protein expression was performed. (H and I) Oil Red O staining of NC and KD OSCC cells treated with T0070907 (25 μM) for 48 h. Quantitative analysis of LD number per cell and average area per LD in OSCC cells. Scale bars = 20 μm. Seven visual fields per sample were randomly selected for statistical analysis. (J and K) The Mitotracker probe was used to measure the changes in mitochondrial staining intensity in the OSCC cell lines comprising CAL27 and SCC25 cells treated with shCAV2 or vehicle control treated with T0070907 (25 μM, 48 h) by the fluorescence microscope. Cells were labeled with GFP (green), and mitochondria were labeled with Mitotraker (red). Scale bars = 50 μm. Five visual fields per sample were randomly selected for statistical analysis. Normally distributed variables are presented as mean ± SD (n≥3). Significance was determined by Student’s t-test (A, G, J, and K), one-way ANOVA with Turkey’s multiple comparison tests (B, E, F, H, and I), and two-way ANOVA with Bonferroni post-test (C and D). *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance. CAV2: Caveolin2; OSCC: oral squamous cell carcinoma; KD: shRNA targeting CAV2, labeled with GFP for green fluorescence tracking; NC: scrambled control shRNA, labeled with GFP for green fluorescence tracking; PLIN3: Perilipin3; PPARγ: peroxisome proliferator-activated receptor gamma; DMSO: dimethyl sulfoxide; IQR: interquartile range