Skip to main content
. 2016 Aug 19;12(11):2183–2196. doi: 10.1080/15548627.2016.1219013

Figure 4.

Figure 4.

Knockdown of PEBP1 expression stimulates starvation-induced autophagy. (A) HeLa cells were infected with the adenoviral vectors encoding PEBP1 shRNA (Ad-shPEBP1) or control vector (Ad-GFP). After incubation for 72 h, cells were starved for the indicated times, and whole cell extracts were prepared for analysis by western blot. ACTB was used as a loading control. Protein levels were quantified using NIH ImageJ software. Numbers below each lane indicate the relative signal intensity ratio between LC3-II, SQSTM1, or PEBP1 and ACTB, as calculated from 3 independent experiments. (B) Detection of GFP-LC3 puncta in PEBP1-knockdown cells. HeLa cells were transiently transfected with shRNA against PEBP1 (shPEBP1) or control vector DNA (shVector). After 48 h, the cells were infected with Ad-GFP-LC3 (MOI 10) for another 24 h before starvation in HBSS medium for 2 h. The GFP-LC3 puncta were then examined by fluorescence microscopy and quantified as shown in (C). Scale bar: 10 μm. Numbers of GFP-LC3 puncta per cell were determined by counting 20 cells for each sample, which were selected from 6 different areas. Data represent the mean (± SD) of 3 independent experiments (**p < 0.01). (D) Determination of the autophagic flux in PEBP1-knockdown cells. PEBP1 expression in HeLa cells was knocked down by transfection with Ad-shPEBP1 or Ad-GFP vectors for 72 h. Then, cells were starved for 2 h in the absence or presence of 20 μM chloroquine (CQ). Production of LC3-II was analyzed by western blot analysis and quantified as shown in (E). ACTB was used as an internal control. Data represent the relative mean values (± SD) of 3 independent experiments to the control (CTL, the first column; Ad-GFP, no CQ, no starvation). *p < 0.05, **p < 0.01 (compare with control); #p < 0.05 (compared with the presence and absence of CQ under starvation conditions). (F) LC3-II net flux was determined by subtracting the densitometric value of LC3-II in nontreated samples (LC3-II -CQ) from the value corresponding to the chloroquine-treated samples (LC3-II +CQ). **p < 0.01.