Fig. 3: USF1 is a downstream target of DUSP18. | Nature Communications

Fig. 3: USF1 is a downstream target of DUSP18.

From: Inhibition of DUSP18 impairs cholesterol biosynthesis and promotes anti-tumor immunity in colorectal cancer

Fig. 3

a Candidate transcription factor prediction was performed by GSEA of regulatory target gene sets. The top 8 TFs ranked by GeneRation scores and p values are shown. b Inhibition of USF1 suppresses the indicated protein levels. c qPCR ChIP analysis of USF1 binding to SREBF2 promoter regions in HCT116 cells (n = 3). d A luciferase vector driven by a WT SREBF2 promoter, but not by a non-USF1-binding mutant promoter, is responsive to inhibition of USF1 (n = 3). e GSEA analysis for USF signature in shCtrl versus shDUSP18 cells. f DUSP18 inhibition suppresses USF1 protein levels. g USF1 threonine dephosphorylation mediated by DUSP18. HA-USF1 was co-transfected with Flag-DUSP18 (WT or phosphorylase Dead) into HEK293T cells, and the cell lysates were subjected to immunoprecipitation. h Dephosphorylation of USF1 T100 by DUSP18. Flag-USF1s (WT, T10A, T100A, T131A, T153A and T194/195A) were co-transfected with HA-DUSP18WT or DUSP18Dead into HEK293T cells, and the cell lysates were subjected to immunoprecipitation. i Stability of Flag-USF1 (WT, T100A and T100D) proteins in HEK293T cells (left) in the presence of CHX block. Flag-USF1 protein quantified by densitometry, with β-actin as a loading control (right) (n = 3). j Decreased Flag-USF1 (T100A) polyubiquitination. Myc-Ub was co-transfected with Flag-USF1 (WT, T100A and T100D) into HEK293T cells, and the cell lysates were subjected to immunoprecipitation. k Overexpression of Flag-DUSP18 has no effect on the SREBP2 protein level mediated by USF1 inhibition. l Subcutaneous xenograft experiments in C57BL/6 were performed in the indicated MC38 cells group. Tumor growth curves and weights are shown (n = 4). The percentage of infiltrating CD8+ T cells (n = 3) (m), effector molecules (n = 3) (n), and exhausted molecules (n = 3) (o) in the indicated group. Data are presented as mean ± SD (c, d, i, l–o). P values were calculated by modified Fisher’s exact tests (a), Kolmogorov–Smirnov tests (e), unpaired two-tailed t-tests (c, i), one-way ANOVA (d, l–o); ns, not significant. All IB data are representative of three independent experiments. Source data are provided as a Source Data file.

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