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. Author manuscript; available in PMC: 2016 Jun 28.
Published in final edited form as: Oncogene. 2014 Mar 31;34(10):1323–1332. doi: 10.1038/onc.2014.60

Figure 2.

Figure 2

STAT5B and p53 both are necessary for transcriptional activation of the LPP/miR-28 promoter. (a) Chromatin immunoprecipitations (ChIP) using antibodies against RNA pol II, STAT5B or p53 were performed on LPP/miR-28 non-expressing (UT7) or expressing (HEL) cell lines. Both STAT5B and p53 are bound on the LPP/miR-28 promoter in HEL cells whereas only STAT5B is bound in UT7 cells (*Po1e − 4; P = 3.7e − 3 and P = 1.4e − 3, respectively). The beta-actin amplification produced by primers in the beta-actin gene body was used as a negative control for STAT5B and p53 binding. (b) (Upper left) quantitative RT–PCR for p53 transcript in p53 knockdown HEL cell line compared with control HEL cell line (*P = 1.5e − 5). (Middle left) western blotting of p53 from HEL cell lines expressing an shRNA against p53 or a control shRNA. (Bottom left) quantitative RT–PCR for LPP transcript in HEL cell lines knockdown for p53 (*P = 4.4e − 3) or control. (Middle right) western blot analysis of p53 and STAT5 levels and tryrosine phosphorylated STAT5 in the presence and absence of JAK2 inhibitor in total lysates of HEL cells. (Bottom right) quantitative RT–PCR for LPP transcript in HEL cell lines treated with the JAK2 inhibitor INCB018424 (*P = 8.2e − 3). (c) Coimmunoprecipitation of endogenous STAT5 and p53 from enlarged spleens of JAK2 V617F knockin heterozygous mice presenting MPN phenotype (d) Co-immunoprecipitation of endogenous STAT5 and p53 in HEL cells, in the presence (18 h pretreatment, 10 μM INCB018424) or absence of JAK2 inhibitor. (e) ChIP of RNA pol II, STAT5B and p53 (*P = 2.7e − 2) in p53 knockdown HEL cells and control cells, and in HEL cells treated with the JAK2 inhibitor INCB018424 or solvent alone (DMSO) (STAT5B: *P = 1.1e − 2; p53: *P = 1.8e − 5). Error bars represent s.d. for the qPCR performed in triplicate on sh-Ctrl and sh-p53 cells, or s.e.m. from two independent experiments performed on HEL cell after treatment by the JAK2 inhibitor qPCR, quantitative PCR; RT–PCR, PCR in reverse transcription. DMSO, dimethyl sulfoxide.