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: Inhibitors or activators used in this study include PLX4720 (ab141362, Abcam; 1 μM or as indicated), Torin1 (Selleckchem S2827, 1 μM, 3 h), BafA1 (Sigma B1793, 100 nM, 6 h), CQ (C6628, Sigma, 20 μM, 24 h), ERK inhibitor FR180204 (Selleckchem S7524, 10 μM, 24 h), RSK inhibitor (SL0101; 50 μM, 24 h), CRM1 inhibitor LMB (L2913, Sigma-Aldrich, 20 nM, 2 h), PI3KC3 inhibitor 3-MA (M9281, Sigma, 100 nM, 24 h), TGF-βRI inhibitor SB431542 (Sigma-Aldrich S4317; 10 μM, 22 h), TGF-β1 (Cell Signaling #8915; 10 ng/ml, 48 h).

Techniques: Activation Assay, Inhibition, Staining, Translocation Assay, Western Blot, Control, Binding Assay, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Activity Assay, Derivative Assay

Journal: American Journal of Cancer Research

Article Title: SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer

doi:

Figure Lengend Snippet: SHMT2 promotes breast cancer cell proliferation by activating MAPK pathway. Western blotting analysis of proteins with phosphorylation or not related in MAPK signaling pathway in breast cancer cells with SHMT2 knockdown (A) and over-expression (B). Cell viability was analyzed by MTT assay in HCC1806 cells with SHMT2 knockdown treated with an ERK inhibitor FR180204 (C) or a p38 inhibitor SB202190 (D). ZR-75-1 cells with SHMT2 overexpression were treated with an ERK inhibitor FR180204 (E) or a p38 inhibitor SB202190 (F). # represents the comparison between inhibitor plus SHMT2 over-expression group and SHMT2 over-expression group. * represents the comparison between treatment group and control group. #, *P < 0.05. Student’s t-test. The data are presented as the mean ± SD. The error bars show the SD.

Article Snippet: ERK MAPK inhibitor FR180204 (HY-12275), p38 MAPK inhibitor SB202190 (HY-10295) were purchased from the Med Chem Express (MCE).

Techniques: Western Blot, Phospho-proteomics, Knockdown, Over Expression, MTT Assay, Comparison, Control