Fig. 5

High 20E titer induces PGK1 acetylation to initiate autophagy and apoptosis. A High 20E titer induces PGK1 acetylation via ARD1. dsARD1 knockdown (500 ng/larva administered in the sixth-instar at 6 h, three times over 24 h intervals) and then injected with 500 ng/larva 20E for 3 h. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. *p < 0.05, n = 3. B Insulin stimulates PGK1 deacetylation through HDAC3. dsRNA knockdown (500 ng/larva administered in the sixth-instar at 6 h, three times over 24 h intervals) and then injected with 5 μg/larva insulin for 3 h. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. p < 0.05, n = 3. C High 20E titer facilitates but insulin suppresses PGK1 acetylation, and high concentration of insulin represses 20E-induced PGK1 acetylation. PGK1-His were overexpressed in HaEpi cells, followed by treatment with insulin or 20E for 3 h. PGK1 proteins were purified by Ni–NTA His bound beads. p < 0.05, n = 3. D PGK1 WT-His and its mutants (K214R, K218R, and K386R) were overexpressed in HaEpi cells for 48 h and then treated with 5 μM 20E for 3 h. PGK1 proteins were purified by Ni–NTA His bound beads. p < 0.05, n = 3. E and E’ Levels of ATG8-II and cleaved-caspase-3 in the PGK1 WT and PGK1 K386R mutant. The PGK1 WT and PGK1 K386R mutant were transfected into HaEpi cells for 48 h and then treated with 5 μM 20E for 6 h. Western blot analysis with anti-ATG8 and anti-caspase-3 antibodies. *p < 0.05, n = 3. F and F’ GFP-ATG8 was transiently expressed in HaEpi cells. Representative images of GFP-ATG8 puncta are shown. *p < 0.05, n = 3. G and G’ Apoptosis signals were examined via caspase-3 activity using a caspase-3 activity detection kit. *p < 0.05, n = 3. Blue: nucleus stained with DAPI. The data represent means ± SD for three independent experiments with one six-well cell culture plates or five larvae per replicate. Statistically significant differences were calculated using two-tailed Student’s t tests (*p < 0.05). Different letters indicate statistically significant differences (p < 0.05) based on one-way ANOVA tests. Ac, antibodies against pan anti-acetyl lysine