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. Author manuscript; available in PMC: 2014 Dec 23.
Published in final edited form as: Dev Cell. 2013 Dec 23;27(6):607–620. doi: 10.1016/j.devcel.2013.11.013

Figure 2. Calpain Contribution to Megakaryocytic P-TEFb Activation and Differentiation.

Figure 2

(A) Analysis of Calpain 2 association with P-TEFb. Extracts from K562 cells treated with either DMSO (−) or 25nM TPA (+), underwent immunoprecipitation (IP) with antibody to cyclin T1 (CT1) or control IgG followed by immunoblotting for calpain 2 (Capn2), Cdk9 (arrow), GATA-1 (G1) (arrow), and cyclin T1 (CT1).

(B) Effects of calpain inhibition on megakaryocytic MePCE downregulation and HEXIM1 upregulation. Primary human progenitors either undifferentiated (Un) or cultured in megakaryocytic medium (Mk) with DMSO, 40μM calpeptin or 20μM calpain inhibitor III (Capn inh III), underwent immunoblot and densitometry as in Figure 1A. Results from three independent experiments are shown as mean ± SEM for signals relative to those in undifferentiated cells. In addition, all signals are normalized to tubulin. * P < 0.05; ** P < 0.01; *** P < 0.005; NS, not significant.

(C) Effects of calpain inhibition on P-TEFb dissociation from HEXIM1. Extracts from HPC7 cells grown 48 hours in expansion (Un), erythroid (Ery), or megakaryocytic (Mk) medium underwent immunoprecipitation for cyclin T1 (CT1) followed by immunoblotting for HEXIM1 (H1) and CT1. The cells undergoing megakaryocytic culture were treated in the final 16 hours with either DMSO, 50μM calpeptin (Calp), or 25μM Capn inh III (CI III).

(D) Effects of calpain inhibition on megakaryocytic differentiation. Primary human progenitors grown for 6 days in megakaryocytic medium with DMSO or 40μM calpeptin were analyzed by flow cytometry for CD41 expression, and DNA content by propidium iodide staining (PI). Cell morphology was assessed by light microscopy of Wright-stained cytospins (200X). Graph represents mean ± SEM for CD41 expression in three independent experiments; * P < 0.05.

(E) Role of calpain S1 in megakaryocytic differentiation. Primary human progenitors transduced with lentiviral shRNA constructs targeting Calpain S1 (CAPNS1) underwent megakaryocytic culture for 5 days followed by analysis as in (C). For documentation of knockdown see Figure S2E. See also Figure S2.