Figure 4.

Attenuated differentiation of WIPI-1 and WIPI-2 knockdown APL cells is in part due to impaired autophagy. (a) Autophagic flux was measured in SHC002 and shWIPI-1 or shWIPI-2 expressing NB4 cells upon 1 μM ATRA treatment at day 4 in the presence or absence of bafilomycin A1 by measuring endogenous LC3-II protein levels on western blot and four independently performed experiments were quantified. GAPDH was used as loading control. (b) Stable EGFP-Cherry-LC3 expressing NB4 cells were transduced either with SHC002, shWIPI-1 or shWIPI-2 and cells were treated with 1 μM ATRA for 4 days. Bafilomycin A1 was added after 3 days. Representative pictures of EGFP-Cherry-LC3 puncta from three independently performed experiments are shown. (c) The rate of long-lived protein degradation was measured in control and ATRA-treated SHC002 control cells or WIPI-1, WIPI-2 knockdown cells, in the presence or absence of the lysosomal inhibitor bafilomycin A1.²¹ The degradation rate for long-lived proteins was calculated as the percentage of radioactivity in the TCA-soluble fraction relative to the total radioactivity in nonsoluble fractions. Further, values were subtracted from the corresponding sample treated with bafilomycin A1. (d) Long-lived protein degradation in NB4 SHC002 control and PU.1 knockdown cells. Analysis as in c. M.W.U, *P<0.05. **P<0.01. M.W.U, Mann–Whitney U-test