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Fig. 3 | BMC Biology

Fig. 3

From: 20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

Fig. 3

Phosphorylation of PGK1 at Y194 enhances PGK1 glycolytic activity and PTEN dephosphorylates PGK1. A Phosphorylation of PGK1 at Y194 promotes glycolysis based on the detection of Vmax of 3-PG production in vitro. Wild type PGK1 (WT) and mutant PGK1 (Y194F) were overexpressed in HaEpi cells for 48 h, respectively, and then incubated with insulin (5 μg/mL) for 3 h. PGK1 proteins were purified by Ni–NTA agarose beads to detect the Vmax of 3-PG production. *p < 0.05, n = 6. B and B’ PGK1 Y194 phosphorylation promotes cell proliferation. HaEpi cells were overexpressed with PGK1 WT or PGK1 Y194F, and incubated with 5 μg/mL insulin for 3 h, followed by proliferation signal detection using an EdU kit (B). The ratio of proliferation cells (red) to total cells (blue, nuclei stained with DAPI) in the field of view was assessed, and data were expressed as mean ± SD from 100 × 6 cells for statistical analysis. **p < 0.01, n = 6. Western blot validation of PGK1 WT and PGK1 Y194F mutant overexpression efficiency in HaEpi cells (B’). n = 3. C PGK1 is dephosphorylated by 20E and phosphorylated by insulin. HaEpi cells overexpressing PGK1-His were treated with 20E or insulin for 3 h. PBS or DMSO were used as controls. PGK1 proteins were purified with Ni–NTA agarose beads. *p < 0.05, n = 3. D 20E dephosphorylates PGK1 via PTEN. dsRNA knockdown (500 ng/larva administered in the sixth-instar at 6 h, three times over 24 h intervals), followed by incubation with 2 μM 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. E PTEN overexpression decreased the phosphorylation levels of PGK1. HaEpi cells overexpressing PGK1-GFP-His were transfected with PTEN-His or not, followed by incubation with 2 μM 20E for 3 h. Anti-GFP antibody immunoprecipitated PGK1-GFP-His, followed by detection using the antibody against phosphorylated tyrosine. *p < 0.05, n = 3. Data represent means ± SD for more than 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 and **: p < 0.01) or one-way analysis of variance (ANOVA, p < 0.05) tests. pY, the antibody against phosphorylated tyrosine

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