Extended Data Fig. 3: Characterization of VVD-118313 inhibitory activity against JAK1 in 22Rv1 cells.
From: Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine
a, Quantification of western blotting data measuring cytokine-stimulated STAT phosphorylation in 22Rv1 cells expressing WT-, C810A-, or C817A-JAK1 variants compared to mock-transfected 22Rv1 cells (see Fig. 3b for representative western blots). Cells were treated with IFNα (100 ng/mL, 30 min), IL-6 (50 ng/mL, 30 min) or prolactin (PRL, 500 ng/mL, 15 min) after which the indicated phosphorylated STATs (pSTATs) were measured. Signal intensities were normalized to unstimulated 22Rv1 cells expressing WT-JAK1. Data are mean values ± S.E.M. from n = 3 independent experiments. Significance was determined by two-way ANOVA with Tukey’s post hoc test and reported for select comparisons. IFNα and IL-6-stimulated STAT1/3 phosphorylation was significantly enhanced by expression of any of the three JAK1 variants (P < 0.0001), while prolactin-stimulated STAT5 phosphorylation was unaffected by JAK1 expression. b, c, Western blots showing concentration-dependent effects of VVD-118313 (5a) on IFNα-stimulated STAT1 phosphorylation (b) and IL-6-stimulated STAT3 phosphorylation (c) in 22Rv1 cells expressing WT-, C810A-, or C817A-JAK1 variants. Blots are representative of n = 2 independent experiments. d, Quantification of concentration-dependent effects of VVD-118313 (5a) on IFNα-stimulated pSTAT1 (left), IL-6-stimulated pSTAT3 (middle), and pJAK1 (integrated from both IFNα- and IL-6-stimulations) in 22Rv1 cells expressing WT-JAK1. Data are mean values ± S.E.M. from n = 2 (pSTAT1, pSTAT3) or n = 3 (pJAK1) independent experiments.
