Figure 4.

BRISC depletion enhances hematopoietic cell growth and increases JAK2 protein levels as well as JAK2 activation. (A-B) TF-1/hMPL cells stably expressing shRNAs to KIAA0157 or BRCC36 were established. Cells were cultured in different concentrations of MPL agonist Eltrombopag (A) or TPO (B). Live cell numbers after 3 days’ culture were determined by 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) absorbance. *P < .05; **P < .01; ***P < .001, 2 -tailed Student t test, shKIAA or shBRCC36 vs shRNA-luciferase (shLuc). (B, right panels) WB analysis shows efficient knockdown of individual BRISC components. shKIAA efficiently reduced BRCC36 protein levels. (C-D) TF1/hMPL cells depleted of various BRISC components (BRCC36 or KIAA0157) were starved and stimulated with TPO for different time points (C) or a graded dose of TPO for 10 minutes (D). (C-D, left panels) WB analysis with indicated antibodies is shown. (C, right) Relative pJAK2 levels upon TPO simulation to the shLuc controls at the 10-minute time point were quantified and plotted in the right panels. (D, right) Relative pJAK2 and total JAK2 protein levels to shLuc controls are quantified and plotted. N = 7. P values are determined by using the 2-tailed Student t test.