Extended Data Fig. 8: VVD-118313 inhibits JAK1-dependent signaling ex vivo.
From: Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine
a, Left, Representative western blots showing recovery of JAK1-mediated STAT1-phosphorylation in human PBMCs that were treated with 5a (0.1 μM) or tofacitinib (1 μM) for 2 h, then compounds were removed by washing and PBMCs were stimulated with IFNα (100 ng/mL, 30 min) at the indicated timepoint post washout. Right, Quantification of pSTAT1 signal intensity normalized to DMSO-treated control. Data are mean values ± S.E.M. from n = 4 independent experiments. Significance determined by two-way-ANOVA with Tukey’s post-hoc test. b, Left, Representative western blots, and right, quantification of pSTAT1 signal intensity from equivalent experiment to that described in (a), except that media was not exchanged after the first 2 h. Data are mean values ± S.E.M from n = 3 independent experiments. Significance relative to stimulated DMSO-treated control at each time point determined by two-way-ANOVA with Tukey’s post-hoc test. For a and b, T = 0 refers to the time of the washout step performed in a. c, Western blots containing the results quantified in Fig. 4h, which represents ex vivo cytokine-stimulated STAT phosphorylation assays performed in splenocytes from mice treated with vehicle or compound 5 (25 mg/kg, 2 Ă—4 h). Splenocytes were stimulated with IFNα (1000 U/mL, 30 min), IL-2 (20 U/mL, 15 min), IL-6 (10 ng/mL, 30 min) or GM-CSF (10 ng/mL) prior to analysis of indicated STAT phosphorylation signals. #1-3 correspond to n = 3 individual mice per treatment groups. Blots are representative of n = 3 (IFNα, IL-2) or n = 1 (GM-CSF, IL-6) independent experiments. d, Quantification of ex vivo stimulation of splenocytes from mice treated with vehicle or 5 (25 mg/kg, s.c., 2 Ă—4 h) with IL-6 (10 ng/mL, 30 min) or GM-CSF (10 ng/mL, 15 min). Data are mean values ± S.E.M., from n = 3 mice analyzed in one experiment.
