Figure 2.

PU.1-dependent WIPI-1 regulation in NB4 APL cells. (a) Schematic representation of a 5.8-kb human WIPI-1 promoter fragment. Using MatInspector, 4 putative PU.1 binding sites (circles) are found in the human WIPI-1 promoter. (b) In vivo binding of PU.1 to the 4 binding sites was shown by ChIP in NB4 cells using antibodies against PU.1. Antibodies against acetyl-histone H3 and IgG served as positive and negative controls, respectively. GAPDH amplification was shown as a negative control for the different pull-downs. (c) Upper panel: WIPI-1 mRNA expression was measured in NB4 shPU.1 knockdown cells upon ATRA treatment at day 4. Lower panel: PU.1 western blot analysis of NB4 SHC002 control and PU.1 knockdown cells. Total protein expression was used as loading control. (d) NB4 cells, transduced with an inducible PU-1-ER expressing vector were treated with 4-OHT to induce PU.1 translocation to the nucleus. WIPI-1 mRNA levels were assessed by qPCR and normalized to the HMBS housekeeping gene. Results are given as n-fold regulation compared with untreated, control transduced NB4 pBabe cells. M.W.U, ***P<0.001. M.W.U, Mann–Whitney U-test