Fig. 5: SLC38A4 upregulates HMGCS2 expression via repressing Wnt/β-catenin/MYC.

a qRT-PCR analyses of the expressions of ten candidate genes, whose expressions were positively or negatively correlated with SLC38A4 in HCC tissues, in Hep3B cells with SLC38A4 stable overexpression or control. b qRT-PCR analyses of the expressions of ten candidate genes, whose expressions were positively or negatively correlated with SLC38A4 in HCC tissues, in Huh7 cells with SLC38A4 stable knockdown or control. c Western blot analyses of HMGCS2 expressions in Hep3B cells with SLC38A4 stable overexpression or control. d Western blot analyses of HMGCS2 expressions in Huh7 cells with SLC38A4 stable knockdown or control. e β-catenin reporter TOPFlash was co-transfected with pRL-TK into Hep3B cells with SLC38A4 stable overexpression or control. Luciferase activities were measured 48 h after transfection. Results were presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. f β-catenin reporter TOPFlash was co-transfected with pRL-TK into Huh7 cells with SLC38A4 stable knockdown or control. Luciferase activities were measured 48 h after transfection. Results were presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. g Western blot analyses of nuclear β-catenin expression in Hep3B cells with SLC38A4 stable overexpression or control. h Western blot analyses of nuclear β-catenin expression in Huh7 cells with SLC38A4 stable knockdown or control. i Immunofluorescence staining of β-catenin in Hep3B cells with SLC38A4 stable overexpression or control showed reduced nuclear β-catenin after SLC38A4 overexpression. Scale bar, 15 μm. j Immunofluorescence staining of β-catenin in Huh7 cells with SLC38A4 stable knockdown or control showed increased nuclear β-catenin after SLC38A4 silencing. Scale bar, 15 μm. k qRT-PCR and western blot analyses of MYC expressions in Hep3B cells with SLC38A4 stable overexpression or control, and Huh7 cells with SLC38A4 stable knockdown or control. l Huh7 cells with SLC38A4 stable knockdown or control were treated with 20 mM LiCl for 24 h. Then, nuclear β-catenin expression and total HMGCS2 and MYC expression were detected by western blot. Histone H3 and GAPDH were used as a nuclear protein and total protein loading controls, respectively. m Western blot analyses of AXIN1 expressions in Huh7 cells with SLC38A4 stable overexpression or control. n Western blot analyses of AXIN1 expressions in Huh7 cells with SLC38A4 stable knockdown or control. o Huh7 cells with SLC38A4 stable knockdown or control were treated with 50 µg/ml CHX or 10 µM MG132 for 24 h, followed by detection of AXIN1 protein levels. Data are shown as mean ± s.d. of n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, by Student’s t test.