Fig. 5.

PGAM5 dephosphorylates FUNDC1 to regulate mitochondrial fission and mitophagy in response to selenite. a HeLa and PGAM5 knockout HeLa cells were treated with selenite or vehicle for 2 h, lysed and analyzed by immunoblotting with the indicated antibodies. b HeLa cells were transfected with FLAG-BCL-xL, FLAG-FUNDC1 and PGAM5-MYC, treated with selenite for the indicated times, lysed and immunoprecipitated with anti-MYC antibody. The co-precipitated FLAG-BCL-xL and FLAG-FUNDC1 were detected by immunoblotting with anti-FLAG antibody. c Densitometric quantification of co-immunoprecipitated FLAG-BCL-xL or FLAG-FUNDC1 in (b). Normalized to the band from the untreated samples. Data are the mean ± SEM of three experiments. *p < 0.05; **p < 0.01. d HeLa and FUNDC1 or PGAM5 knockout HeLa cells were treated with selenite for 2 h, fixed and immunostained with a fluorescent dye-conjugated antibody against the mitochondrial protein cytochrome c (green). Confocal fluorescence images of the cells are shown (scale bar, 10 μm.) e HeLa and FUNDC1 knockout HeLa cells were treated with 5 μM selenite in the absence or presence of bafilomycin A1 (BA1) for the indicated times, lysed and analyzed by immunoblotting with the indicated antibodies. Asterisk indicates a nonspecific band detected by the anti-Ser13-FUNDC1 antibody. f HeLa and PGAM5 knockout HeLa cells were treated with 5 μM selenite or vehicle for 12 h, lysed and analyzed by immunoblotting with the indicated antibodies