Fig. 1 | Nature Communications

Fig. 1

From: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance

Fig. 1

BRAFV600E inhibitor triggers autophagy–lysosomal activation through TFEB. a, b Western blot analysis (a) and densitometric quantification (b) of the LC3-II/LC3-I and the p62/Actin ratios in A375 and MeWo cells treated with the indicated concentrations of PLX4720 for 12 h. Actin served as a loading control. n= 4 independent experiments. c Representative images of LysoTracker Red staining of A375 and MeWo cells treated for 12 h with DMSO or PLX4720 (1 μM). Quantification of relative fold induction of lysosomes by PLX4720 is shown in the right panel. n= 3 independent experiments. d Relative lysosome NAG activity in PLX4720-treated A375 cells. n= 3 independent experiments. e Expression analysis of the autophagy–lysosome relevant genes in PLX4720-treated A375 cells in the presence or absence of TFEB (shRNA). n= 3 independent experiments. f, g Western blot analysis (f) and densitometric quantification (g) of the LC3-II/LC3-I and p62/Actin ratios in PLX4720-treated A375 cells with shRNA-mediated depletion of the indicated genes. Expression of indicated proteins is also shown. Actin served as a loading control. n= 3 independent experiments. h, i Representative images (h) and quantification (i) of LysoTracker Red (red) and LAMP1 (green) immunostaining of PLX4720 (1 μM, 12 h-treated) A375 cells with depletion of the indicated genes. Note the reduced lysosome staining in PLX4720-treated cells upon TFEB depletion. n= 3 independent experiments. Scale bars, 10 μm. Data in a and f are from one experiment that is representative of three independent experiments. For all quantification, data represent the mean ± SD derived from the indicated number of independent experiments. Comparisons were made using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s. not significant. See Supplementary Fig. 13 for uncropped data of a, f