Figure 5.

Chmp1A knockdown abolished ATRA mediated growth inhibition and the increase of protein levels of Chmp1A, CRBP, P53 and phospho-P53. (A) Chmp1A shRNA was cloned into shRNA RNAintro™ pSM2 retroviral vector to generate stable PanC-1 clones expressing Chmp1A shRNA. Western blot analysis demonstrated the knockdown efficiency of two colonies (KD1 and KD2). Non-silencing shRNA was used as control. (B) Chmp1A expression was reduced in Chmp1A silenced PanC-1 cells more than 50% on Day 1 and 40% on Day 2 regardless of ATRA treatment, compared with non-silencing shRNA expressing cells that were treated with vehicle DMSO. ATRA treatment increased Chmp1A expression slightly. (C) Silencing of Chmp1A induced growth promotion in the presence or absence of ATRA, compared with control cells that were treated with either DMSO or ATRA. ATRA did not have an effect on growth upon stable expression of Chmp1A shRNA compared to control shRNA. (D) Control shRNA expressing cells showed an increase in P53 and phospho-P53 at serine 15 and 37 without exhibiting an increase in CRBP-1. However, Chmp1A shRNA expressing cells exhibited a decline in CRBP-1 and phospho-P53 expression at serine 15 and 37 when compared with control shRNA expressing cells that were treated with DMSO. ATRA increased P53 expression when Chmp1A was silenced, however, Chmp1A depletion did not decrease P53 expression. D: DMSO, R: ATRA