Fig. 4: RagD mutants impair TFEB response to mitochondrial injury. | Nature Communications

Fig. 4: RagD mutants impair TFEB response to mitochondrial injury.

From: RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome

Fig. 4

a Representative immunofluorescence images of HK-2 cells transfected with HA-RagD WT or mutants (S77L or P88L) and treated with 10 μg/ml Oligomycin and 5 μg/ml Antimycin A (O/A) for 3 h. Cells were stained with anti-TFEB and anti-HA. Staining for Tomm20 was used as mitochondrial marker. Scale bar, 10 μm. b Graph shows the TFEB nuclear-cytosolic ratio intensity of the HA positive (RagD WT or mutants S77L, P88L) cells treated with O/A (mean ± s.d. of n > 1000 cells from n = 3 independent experiments). Quantification performed as in Fig. 2f. Ordinary one-way ANOVA Tukey multiple comparison test (**p < 0.01, ****p < 0.0001). c Representative immunofluorescence images of HK-2 cells transfected with HA-RagD WT or mutants (S77L or P88L) and treated with 10 μg/ml Oligomycin and 5 μg/ml Antimycin A (O/A) for 6 h. Cells were stained with anti-Parkin (Rabbit polyclonal) and anti-HA (Rat monoclonal). Staining for Tomm20 (Mouse monoclonal) was used as a mitochondrial marker. Scale bar, 10 μm. d Graph shows the mitochondria-Parkin co-localization of the HA positive (RagD WT or mutants S77L, P88L) cells treated with O/A (mean ± s.d. for n = 195 cells of 3 independent experiments). Ordinary one-way ANOVA Tukey multiple comparison test (****p < 0.0001). e Representative western blot images showing: PINK1, Parkin, and Actin in HK-2 cells transiently transfected with HA-RagD-WT or RagD mutants, S77L, P88L, treated ± O/A for 6 h. Graphs show the PINK1/Actin (f) and Parkin/Actin ratios (g). Data are representative of n = 3 biologically independent experiments. Ordinary one-way ANOVA Tukey multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001).

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