Fig. 3: RagD mutants impair TFEB response to lysosomal damaging agents. | Nature Communications

Fig. 3: RagD mutants impair TFEB response to lysosomal damaging agents.

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

Fig. 3

a Representative immunofluorescence images of HeLa cells transfected with HA-RagD WT or mutants (S77L and P88L) and treated with 500 μM LLOMe (for 1 h). Cells were stained with anti-TFEB and anti-HA. Staining for Galectin-3 (Gal3) was shown to visualize lysosomal membrane permeabilization. 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 LLOMe (mean ± s.d. of n > 1000 cells from n = 3 independent experiments). Ordinary one-way ANOVA Tukey multiple comparison test (****p < 0.0001). Quantification performed as in Fig. 2f. c Western blot representing TFEB molecular weight shift, phospho-S6K and total S6K in HeLa cells transfected with HA-RagD WT or mutants (S77L and P88L) followed by LLOMe treatment (n = 3 independent experiments). d Representative immunofluorescence images of HeLa cells transfected with Ha-RagD WT or mutants (S77L and P88L) and treated with 30 μM MK6-83, a specific TRPML1 agonist, for 1 h. Cells were stained with anti-TFEB and anti-HA. Scale bar, 10 μm. e Graph shows the TFEB nuclear-cytosolic ratio intensity of the HA positive (RagD WT or mutants S77L, P88L) cells treated with MK6-83 (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 < 0.0001). f Western blot representing TFEB molecular weight shift, phospho-S6K and total S6K in HeLa cells transfected with HA-RagD WT or mutants (S77L and P88L) followed by MK6-83 treatment (n = 3 independent experiments). g Representative immunofluorescence staining for TFEB and sarcomeric marker ACTN2 in hiPSC-CMs transfected with mRNA-RagD-WT or -RagD mutants S76L, P88L, and T97P. Nuclei are stained with DAPI. Scale bar, 50 µm. h Graph showing the percentage of cells with TFEB nuclear signal/total nuclei in hiPSC-CMs transfected with mRNA-RagD-WT or -RagD mutants (S76L, P88L, T97P). n = 10 areas from 2 independent wells; one-way ANOVA followed by Tukey’s multiple comparisons test. Data are shown as mean ± SEM (****p < 0.0001).

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