Fig. 3

Downregulation of PLA2G3 expression impairs PC formation in OC cells. Western blot analysis of acetylated α tubulin levels in (A) OVCAR5 NTC and sh35, sh33 KD cells and in (B) OVCAR8 SCG and KO cells was shown with PCNA as a loading control. Densitometric analysis indicates the fold change calculated using Image J software, normalized to PCNA and provided beneath the panel. (C) Primary cilia (PC) were detected by IF using fluorescently tagged-acetylated α-tubulin (red) in OVCAR5 NTC and sh35 KD cells. PLA2G3 expression (green) was also assessed in the same cells. Nuclei were stained with DAPI. Scale bar: 20 μm. Quantification, 100 cells per field were scored and the percent ciliated cell was plotted as mean ± SD and shown next to the IF images (*p < 0.05 vs. control). (D) Similar IF study was performed in the OVCAR8 SCG and PLA2G3 KO cells as represented, and percent ciliated cell was plotted (**p < 0.01 vs. control). Scale bar: 20 μm. (E) Immunoblot analysis of SREBP1c, PLA2G3 and acetylated α tubulin levels in the SREBP1c overexpressed FT257 cells with PCNA as a loading control was shown. Densitometric analysis using Image J software was calculated, normalized to PCNA, and the fold change was provided beneath the panel. (G) PC was detected by IF using fluorescently tagged-acetylated α-tubulin (red) in empty vector (EV)-transfected control, nSREBP1c overexpressed FT257 cells in combination with nontargeting siRNA (siNTC) and the siPLA2G3 and the percent ciliated cell was plotted as mean ± SD (EV vs. SREBP1c overexpressed *p < 0.05; siNTC vs. siPLA2G3 in the SREBP1c overexpressed cells #p < 0.05). (G) Immunoblot analysis for acetylated α tubulin was also performed in the similar treated cells and shown with PCNA as a loading control. Fold change calculated using Image J software was provided beneath the panel