Fig. 6

VRK2 is important for the induction of autophagy. a HeLa cells (ATCC) transduced with VRK2 shRNA or control (scrambled) shRNA were washed twice with HBSS, then incubated with HBSS for additional 8 h to induce autophagy. Downregulation of VRK2 by shRNAs increased the amounts of lipidated forms of LC3 (LC3II) according to the ratio of LC3II/LC3I conversion (top panel, compare lanes 1 and 3). Notably, however, further induction of autophagy was inhibited as revealed by the compromised LC3II/LC3I conversion after HBSS-induced autophagy (top panel, lane 4). b Quantification of LC3II/LC3I conversion (a) from five independent VRK2 shRNA experiments is shown. c Inhibition of VRK2 by shRNA abrogated the induction of autophagy as assessed by the levels of p62 (third panel from the top). d Quantification of the relative expression of p62 (c) in three independent experiments is shown with statistical analysis by Student’s t-test. e Inhibition of VRK2 expression by VRK2 shRNAs, which caused a modest increase in LC3 puncta numbers in untreated cells (left-hand panels, the fourth panel from the top, arrowheads) or attenuated the induction of autophagy by HBSS (middle panels) or rapamycin-treated cells (right-hand panels). f Quantification of the numbers of LC3 puncta per cell from 30 samples is shown with statistical analysis by Student’s t-test