Fig. 7: Inhibition of ROCK1 and ROCK2 enhances IFNα-induced anti-neoplastic responses in MPN cells.

a Immunoblotting analysis of ROCK1 and ROCK2 in lysates from control (Ctrl) siRNA or ROCK1 and ROCK2 (ROCK1/2) siRNA-transfected JAK2^V617F-positive SET-2 cells. b Proliferation and cell viability of control (Ctrl) siRNA or ROCK1 and ROCK2 (ROCK1/2) siRNA-transfected JAK2^V617F-positive SET-2 cells either left untreated, or treated with IFNα was measured using a WST assay. Data are expressed as percent cell viability relative to Ctrl siRNA-transfected untreated cells (Ctrl siRNA), and represent means ± SEM of five independent experiments. Each symbol on the graphs represents an independent biological replicate. c Proliferation of JAK2^V617F-positive (left panel) SET-2 or (middle and right panels) HEL cells treated with ROCK1/2 inhibitor (GSK429286A or Fasudil) and/or IFNα vs. DMSO-vehicle control (Ctrl) was measured using a WST assay. Each symbol on the graphs represents an independent biological replicate. Data are expressed as percent cell viability relative to Ctrl-treated cells and represent means ± SEM of four, five and three independent experiments, as indicated. d Clonogenic capability of JAK2^V617F-positive HEL cells treated with the ROCK1/2 inhibitor GSK429286A and/or IFNα vs. DMSO-vehicle control (Ctrl). Each symbol on the graphs represents an independent biological replicate. Data are expressed as percent colony formation relative to Ctrl-treated cells and represent means ± SEM of three independent experiments. e Clonogenic capability of Ctrl siRNA or ROCK1/2 siRNA-transfected peripheral blood mononuclear cells from patients with PV, either left untreated or treated with IFNα. Data are expressed as percent colony formation relative to control siRNA-transfected untreated cells (Ctrl siRNA) and represent means ± SEM of four independent experiments, using cells from four different patients with PV. f Clonogenic capability of peripheral blood mononuclear cells from patients with PV treated with the ROCK1/2 inhibitor Fasudil and/or IFNα vs. DMSO-vehicle control (Ctrl). Data are expressed as percent colony formation relative to Ctrl-treated cells and represent means ± SEM of four independent experiments, as indicated, using cells from four different patients with PV. e, f Each data point on the graphs represents an independent biological replicate and data for an individual patient is represented by the same symbol for each experimental condition. b–d Data were normalized to the control group (Ctrl siRNA or Ctrl) and statistical analyses comparing the other three groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Adjusted p-values are reported. e, f Data were analyzed using linear mixed effects models, with % colony formation relative to the control group as the outcome, treatment (the three remaining groups) as the fixed effect, and subject as a random effect to account for within-subject correlation between multiple conditions. Kenward-Roger degrees of freedom adjustment was used, which improves performance when sample size is small. Pairwise group comparison tests were adjusted for multiple comparisons using Tukey’s method. p-values are reported. Source data are provided as a Source Data file.