Figure 9.

Cav-1 knockdown is sufficient to promote autophagy/mitophagy. (A) Acute Cav-1 downregulation activates the autophagy inducer NFκB. hTERT-fibroblasts treated with Cav-1 siRNA (right) or control siRNA (left) were fixed and immuno-stained with antibodies against phospho-NFκB (phospho-p65 at Ser 276, green). DAPI was used to visualize nuclei (blue). Note that Cav-1 knockdown is sufficient to induce phospho-NFκB nuclear localization and activation. White arrows point at the nuclear localization of phospho-NFκB in Cav-1 siRNA treated cells. Importantly, images were acquired using identical exposure settings. Original magnification, 40x. (B and C) Acute loss of Cav-1 increases the expression of autophagic markers. hTERT-fibroblasts were treated with Cav-1 siRNA or control (CTR) siRNA. (B) Western blot analysis. Cells were analyzed by western blot analysis using antibodies against the indicated autophagic markers. β-tubulin was used as equal loading control. (C) Immunofluorescence. Cells were fixed and immuno-stained with antibodies against beclin 1, BNIP3 and BNIP3L. DAPI was used to visualize nuclei (blue). Importantly, paired images were acquired using identical exposure settings. Original magnification, 40x. Note that acute Cav-1 knockdown is sufficient to greatly increase the expression levels of all the autophagy/mitophagy markers we examined.