Fig. 7 | Nature Communications

Fig. 7

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

Fig. 7

Effect of TFEB S142 phosphorylation on PLX4720-resistant BRAFV600E melanoma cells. a Colony formation assay of PLX4720-resistant A375R melanoma cells stably expressing vector, WT TFEB, S142A, or S142E TFEB mutants as indicated. Bars are mean ± SD percentage of colonies for each group after 21 days. n= 3 independent experiments. b Western blot analysis of TFEB expression in cells shown in (a). Actin served as a loading control. Data are from one experiment that is representative of three independent experiments. c Tumor volume of xenografts formed after subcutaneous injection of NOD/SCID mice with A375R melanoma cells stably expressing the indicated TFEB constructs. Results are the mean volume ± SD for 5–6 mice per group per time point. d Tumor weight from experiment shown in (c) upon autopsy at day 30. Representative images of tumor size of the indicated A375R xenograft tumor genotypes are shown below. e Western blot analysis of autophagy (p62 and LC3-I/II), lysosome (CTSD), TGF-β/Smad2/3 signaling, EMT-related [E-cadherin (E-Cad.), N-cadherin (N-Cad.), and Vimentin, Twist1, ZEB1, Snail, and Slug], γ-H2AX, ERK and mTORC1 activation in the indicated xenografts (three randomly chosen samples per group; similar results observed in all 10–12 samples per condition). Actin served as a loading control. See Supplementary Fig. 13 for uncropped data. For all quantification, data represent the mean ± SD derived from indicated number of independent experiments. Comparisons were made using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001