Fig. 4
ZNF322A modulates glucose uptake via the IRS1/PI3K/AKT pathway in A549 lung cancer cells. a Schematic representation of the inhibitory transcription regulation of ZNF322A on PIM3. b Relative mRNA levels of ZNF322A and PIM3 were examined by RT-qPCR analysis, and normalized by GAPDH. c-d Proteins were extracted from ZNF322A-silenced (siZNF322A) A549 cells after 48 h transfection. Protein expressions of ZNF322A and PIM3 were examined by immunoblot analysis. Representative western blot (c) and associated densitometric analysis (d) for ZNF322A and PIM3 expressions in A549 lung cancer cells. e-f Protein expressions of IRS1S1101 and AKT S473 phosphorylation were examined by immunoblot analysis. Phosphorylation levels were determined and normalized to the level of total proteins. The normalized values were compared between siRNA control (siControl) and siZNF322A groups. g-h Effect of 2-NBDG uptake upon ZNF322A perturbation in A549 lung cancer cells. Representative fluorescence microscopy images of 2-NBDG uptake ability in siRNA control (upper) and siZNF322A-silenced (bottom) A549 cells. 2-NBDG: green; DAPI: blue; scale bar: 10 μm (g). Mean fluorescence intensity (left) and number (right) of 2-NBDG in siControl and siZNF322A cells (h). (i) Schematic representation of the depletion of ZNF322A by siRNAs in A549 lung cancer cells transcriptionally regulates PIM3 kinase to induces IRS1Ser1101 phosphorylation, which attenuates PI3K/AKT signaling pathway and inhibits AKTS473, leading to glucose uptake blockade. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001