Journal: Nature Communications
Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
doi: 10.1038/s41467-019-09634-8
Figure Lengend Snippet: PLX4720 promotes TFEB activation through ERK inhibition. a Confocal analyses of the subcellular distribution of endogenous TFEB, TFE3, and MITF in A375 cells treated with DMSO or PLX4720 (1 μM, 12 h). Nuclei were stained with DAPI (blue). n = 3 independent experiments. b Quantification of nuclear translocation of TFEB, TFE3, and MITF in cells in ( a ). n = 150 cells, pooled from three independent experiments. c Immunoblots for TFEB, MITF, and TFEB3 in the cytoplasmic/nuclear fractions of A375 cells treated with PLX4720 (1 μM, 12 h). Lamin B1 is the control for the nuclear fractions, whereas LAMP1 and Tubulin are the controls for the cytoplasmic fractions. d Immunoblotting of endogenous TFEB and p-14-3-3-binding motif of TFEB from PLX4720 (1 μM, 12 h-treated) A375 cells. WCL whole-cell lysate. e PLX4720 disrupts TFEB interaction with 14-3-3. WCLs of A375 cells were immunoprecipitated (IP) with anti-TFEB, followed by immunoblotting (IB) with antibodies against 14-3-3 and TFEB. f Representative images (top) and quantification (middle) of nuclear translocation of TFEB in A375 cells stably expressing mTORC1 (E2419K), RagB GTPase (Q99L), or ERK (R67S/D321N), or DEPDC5-specific shRNA, with or without the treatment of PLX4720 (1 μM, 12 h), or ERK inhibitor FR180204 (ERKi, 10 μM, 24 h). IB showed protein expression as indicated with the corresponding mTORC1 activity (p-p70S6K and p-4E-BP1). n = 4 independent experiments. g Representative confocal images (top) and quantification (bottom) of nuclear localization of endogenous TFEB in A375 and MeWo cells treated with FR180204 or with ERK shRNA. n = 4 independent experiments. h Immunoblots for endogenous TFEB, TFE3, MITF, and ERK in cytoplasmic/nuclear fractions of A375 cells treated with DMSO or FR180204 (10 μM, 24 h) or with ERK shRNA. Scale bars, 10 μm. Data in c , d , e , f , and h 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 < 0.001; n.s. not significant. See Supplementary Fig. for uncropped data of c , e , f , h
Article Snippet: The full-length complementary DNA (cDNA) clones of human TFEB (plasmid #99955), BRAF V600E (plasmid #15269), mTOR (E2419K) (plasmid #19994), ERK (R67S/D321N) (plasmid #53203), and RagB (Q99L) (plasmid #19315), were purchased from Addgene (USA).
Techniques: Activation Assay, Inhibition, Staining, Translocation Assay, Western Blot, Control, Binding Assay, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Activity Assay, Derivative Assay
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