Fig. 5 | Nature Communications

Fig. 5

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

Fig. 5

Effect of TFEB S142 phosphorylation on BRAFV600E melanoma progression. a NOD/SCID mice bearing A375 xenografts tumors stably expressing vector, WT, TFEBS142A or TFEBS142E isolated on day 34 post-injection. b, c Tumor volume (b) and Tumor weight (g) upon autopsy at day 34 of A375 xenografts stably expressing the indicated TFEB. n = 5–6 mice per group per time point. d H&E and immunohistochemical (IHC) staining of Ki67, active Caspase 3, 4-HNE, and 8-oxo-dG in indicated A375 tumor genotypes. The levels of mitotic figures, necrosis, and multinucleated cells were quantified (right panels; n= 5–6 mice per group). N necrotic area, T tumor. Scale bars, 100 μm. e EM images of the indicated xenograft tumor genotypes. M mitochondria. Asterisks denote damaged mitochondria. Arrows denote autophagic vacuoles. Damaged mitochondria is quantified (bottom; n= 50). Scale bar, 200 nm. f IB of EMT-related factors and TGF-β/TGF-βR2/Smad2/3 signaling in indicated A375 xenograft tumor genotypes (two randomly chosen samples per group). Also shown (bottom) is the relative expression of indicated proteins in xenograft tumors. n= 3 independent experiments. g GSEA plot of A375 tumors showing the significantly changed autophagy–lysosome genes. The relevant complete GSEA data are in Supplementary Data 1a. FDR false discovery rate, NES normalized enrichment score. h Heat map depicting the expression of TGF-β target genes in indicated xenograft tumor genotypes. Each column represents a single replicate (n = 3). Also refer to Supplementary Data 1d. i IB of autophagy and EMT-related factors in A375 cells expressing TFEBS142A or TFEBS142E treated with TGF-β (10 ng/ml, 48 h), BafA1 (100 nM, 6 h), Beclin1 (BECN1)-shRNA, or the TGF-βRI inhibitor SB431542 (TGF-βRIi, 10 µM, 22 h). n= 3 independent experiments. Data in a, d, e are from one animal that is representative of 5–6 animals in each group. 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. See Supplementary Fig. 13 for uncropped data of f, i