Fig. 3
CAV2 regulated LD dynamics in OSCC. (A) IF staining was performed to observe the expression of CAV2 protein (red) in SCC25 and CAL27 cells. The expression of CAV2 protein (red) was detected using anti-CAV2 antibodies, while LDs were stained with BODIPY™ 493/503 (green), and nuclei were counterstained with DAPI (blue). Confocal microscopy images revealed CAV2 localization in the cytoplasm and cell membrane, with co-localization between CAV2 and LDs observed as yellow regions in the merged images. Scale bar = 10 μm. (B) Co-localization of CAV2 with LDs was further demonstrated in representative high-magnification images. Co-localization was quantified using Pearson’s correlation coefficients (values between 0.5 and 1 indicate significant co-localization), calculated with ImageJ software. Seven randomly selected visual fields per sample were analyzed statistically. Scale bar = 4 μm. (C) Representative IF and BODIPY™ 493/503 staining images of SCC25 and CAL27 cells with CAV2 OE and respective CON groups. Nuclei were stained with DAPI (blue), LDs with BODIPY™ 493/503 (green), and CAV2 protein with anti-CAV2 antibodies (red). Scale bar = 10 μm. (D) High-magnification images of CAV2 and LD co-localization in CON and OE OSCC cells. Scale bar = 4 μm. Pearson’s correlation coefficients for co-localization were calculated for CON and OE cells, with seven visual fields per sample analyzed. (E and F) Representative images and quantitative analysis of LD number and area per cell in SCC25 and CAL27 cells subjected to CON, OE, KD, and NC treatments. LD staining was performed using Oil Red O assay. LD dynamics were quantified after 48 h of culture, with seven visual fields analyzed per sample. Scale bars = 20 μm. All data were expressed as the mean ± SD (n≥3). Significance was determined by the Student’s t-test (E-F). **P<0.01, ***P<0.001. CAV2: Caveolin2; LD: lipid droplet; OSCC: oral squamous cell carcinoma; IF: immunofluorescence; OE: CAV2 overexpression using lentiviral-mediated overexpression (no fluorescence labeling); CON: control using lentiviral-mediated empty vector (no fluorescence labeling); KD: shRNA targeting CAV2, labeled with GFP for green fluorescence tracking; NC: scrambled control shRNA, labeled with GFP for green fluorescence tracking; DAPI: 4’,6-diamidino-2-phenylindole