Figure 5. Fis1, Rab7, and... | Rockefeller University Press

Figure 5.

$Figure 5. Fis1, Rab7, and autophagosome–lysosome fusion are dispensable for TFEB nuclear accumulation during mitophagy. (A) WT and mCherry-Parkin HeLa cells were transfected with control (Mock), Fis1, or Rab7 siRNA for 48 h before treatment with DMSO or O/A for 6 h, lysis, and immunoblotting. (B) WT (top) or mCherry-Parkin (bottom) HeLa cells were transfected and treated with DMSO or O/A as in A. Cell lysates were fractionated and localization of endogenous TFEB was analyzed by immunoblotting. Images in A and B are representative of n = 2 experiments. (C) WT and mCherry-Parkin HeLa cells were treated with O/A, CQ, and bafilomycin A1 (Baf) for 6 h as indicated, lysed, fractionated, and immunoblotted. (D) Quantification of data in C. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are the mean of n = 2 experiments for −Parkin; means ± SD (n = 3) for +Parkin. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (E) WT and mCherry-Parkin HeLa cells were treated as in C, lysed, and immunoblotted. C, cytosol; N, nuclear.$

Fis1, Rab7, and autophagosome–lysosome fusion are dispensable for TFEB nuclear accumulation during mitophagy. (A) WT and mCherry-Parkin HeLa cells were transfected with control (Mock), Fis1, or Rab7 siRNA for 48 h before treatment with DMSO or O/A for 6 h, lysis, and immunoblotting. (B) WT (top) or mCherry-Parkin (bottom) HeLa cells were transfected and treated with DMSO or O/A as in A. Cell lysates were fractionated and localization of endogenous TFEB was analyzed by immunoblotting. Images in A and B are representative of n = 2 experiments. (C) WT and mCherry-Parkin HeLa cells were treated with O/A, CQ, and bafilomycin A1 (Baf) for 6 h as indicated, lysed, fractionated, and immunoblotted. (D) Quantification of data in C. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are the mean of n = 2 experiments for −Parkin; means ± SD (n = 3) for +Parkin. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (E) WT and mCherry-Parkin HeLa cells were treated as in C, lysed, and immunoblotted. C, cytosol; N, nuclear.