Fig. 7: CK1δ-mediated phosphorylation of USP46 regulates tumor progression of oncogenic NRAS-driven melanoma.
From: Pharmacological targeting of casein kinase 1δ suppresses oncogenic NRAS-driven melanoma
a NRAS and USP46 levels in SK-MEL-103 cells expressing shScramble (shScr) or shUSP46 treated with DMSO or PF670462 (5 μM) was examined. b NRAS and USP46 levels in SK-MEL-103 cells expressing shScr, shUSP46 and shCK1δ were examined. c SK-MEL-103 cells expressing empty vector (pLV3) or FLAG-USP46 were treated with DMSO or PF670462 (5 μM) and NRAS levels were examined. d Cells were transfected as indicated and treated with DMSO or PF670462 (5 μM) followed by MG132 treatment. Polyubiquitylated NRAS Q61R was examined. e Cells were cotransfected with empty vector, pIRES-NRAS Q61R and other plasmids. His-tagged ubiquitin was pulled-down by Ni-NTA beads and polyubiquitylated NRAS Q61R was examined. SK-MEL-103 and SK-MEL-30 cells were cotransfected with empty vector, pIRES-NRAS Q61R (f), pIRES-NRAS Q61K (g) and other indicated plasmids. His-tagged ubiquitin was pulled-down by Ni-NTA beads and polyubiquitylated NRAS Q61R/K was examined. h SK-MEL-103 cells stably expressing shScr or shUSP46 were transfected with empty vector (pLV5), USP46 WT or the 3A mutant. Cells were treated with DMSO or PF670462 and western blotting was performed. i SK-MEL-103 cells expressing shUSP46 were transfected with empty vector, USP46 WT or the 3A mutant and cycloheximide pulse-chase assay was performed. j SK-MEL-103 cells with endogenous USP46-deficiency were transfected with indicated plasmids and western blotting was performed. Proliferation (k), migration and invasion abilities (l) of cells (j) were examined and quantified. m SK-MEL-103 cells (j) were treated with DMSO or PF670462 (5 μM) and sensitivity to dacarbazine (DTIC) was determined. n, o SK-MEL-103 cells (j) were subcutaneously implanted into nude mice (5–6 weeks, n = 6). When tumors reached around 150–200 mm3 in size, mice were treated with saline, PF670462 (4 mg/kg), DTIC (8 mg/kg) or PF670462 plus DTIC. Tumors were collected (n) and weights were measured (o). The results represent the mean ± s.d. of data from six mice. Immunoblots are representative of three independent experiments (i, j). Data were presented as mean ± SD of three independent experiments (k–m). Data were analyzed by two-sided one-way ANOVA in (i, k, l, o). Source data are provided as a Source Data file.
