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. 2018 May 16;6(2):181–198. doi: 10.1016/j.jcmgh.2018.05.002

Figure 5.

Figure 5

RB is required for E47-mediated cell-cycle arrest. (A) Transcripts encoding E2F target genes required for DNA replication are decreased. (B) Western blot analysis showed that phosphorylated RB (pSER807/811 and pSER608) decreased in response to E47. (C) Western blot analysis showing RB knockdown in BxPC-3/E47 cells. (D) BxPC-3 cells immunostained with Ki67 antibody after RB knockdown and induction of E47. Statistically significant increase in Ki67(+) cell numbers after knockdown of RB and induction of E47. Data represent the mean of 3 independent biological replicates analyzed by unpaired t test. Error bars are ± SEM. (E) Western blot analysis of BxPC-3/E47 whole-cell extracts from cells harboring shRB-1 that targets the human RB gene (panel B) after transduction with a second virus expressing hemagglutinin-tagged mouse Rb under control of the MSCV promoter. (F) Immunostaining of the BxPC-3 cells expressing shRB-1 that targets human RB and ± expression of MSCV-driven hemagglutinin-tagged mouse Rb after 72 hours of induction of E47. Cells were immunostained for Ki67 and counted. Data represent the mean of 6 separate fields, each with a minimum of 200 cells analyzed by unpaired t test. Error bars are ± SEM. (G) Western blot analysis showing that expression of CDKN1B/p27KIP1 and CDKN1A/p21CIP1 is unchanged after rescue of Rb expression. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001.