Both knock-down and over-expression of AGS3 lead to an increased LC3II level in HEK293 and HeLa cells.

<p>Cells transfected with DNA (0.4 µg/ml) or siRNA (20 nM) were lysed 24 or 48 hrs after transfection, repectively, unless otherwise noted. β-actin was used as a loading control. (A) The effect of rapamycin (100 nM, 4 hrs) on the level of LC3II in HEK293 cells. (B) The impact of AGS3 over-expression on LC3II in HEK293 cells. Cells transfected with an empty vector (pcDNA3) were used as a negative control. (C) The influence of AGS3 knock-down on LC3II in HEK293 cells. Cells transfected with a non-targeting siRNA were used as a negative control. (D) The influence of AGS3 knock-down on LC3II in HeLa cells. Western blot LC3II band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (E) HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and either starved (2 hrs) or not. (F) GFP-LC3 stable HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and endogenous LC3 was assayed by western blot. Western blot band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (G) HEK293 cells stably expressing GFP-LC3 were treated with AGS3 siRNA1 (50 nM, 72 hrs) and either starved (2 hrs) or not; a higher AGS3 knock-down efficiency was obtained for the HEK293 GFP-LC3 stable cells when using a higher siRNA concentration and a longer incubation time. This could be due to differences in cell origin. The number of GFP positive puncta structures were quantified as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#s2" target="_blank">Materials and Methods</a>”. For the sake of clarity normalized GFP-LC3II counts/cell (n = 6, approximately 10,000 cells/condition) are shown. Two statistical tests (ANOVA and single-sample Student's t-test) performed on LOG transformed data revealed that the AGS3 effect was significant across all experimental conditions (p<0.05 for Starved, p<0.01 for all others). The images on the left side are representative full size acquired images, the inset to the right shows a close-up of one cell with indication of detected GPF puncta (red circles). The green dotted lines enclose the cell nuclei. Representative blots and images are shown (AGS3 was detected using a rabbit polyclonal antibody characterized below). Data presented in bar graphs is the average result from at least three independent experiments except in (G). Error bars: standard error of the mean. Asterisks - * = p<0.05, ** = p<0.01, paired Student's t-test.