Fig 2: Silencing of ZNF322A was attributed to its induction of autophagy via protein phosphorylation. Silencing of ZNF322A transcriptionally regulates PIM3 to phosphorylate IRS1S1101 and further inhibits the PI3K/AKT/mTOR pathways, limiting access of lung cancer cell to glucose. Silencing of ZNF322A induces phosphorylation of HSP27S82 and leads to activation of the unfolded protein response (UPR). Glucose starvation and heat stress-elicited activation trigger autophagosome formation in ZNF322A-silenced A549 lung cancer cells

Fig 3: 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. ß-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

Fig 4: Manipulation of miR-33a levels directly affects PIM3 expression. (a) PIM3 protein levels in Tsc2 -/- MEFs over a time-course of treatment with a miR-33a inhibitor. Cells were transfected for 24, 48, or 72 hours with the final 16 hours starved overnight with vehicle or rapamycin treatment (Rap, 20 nM). (b) PIM3 protein and (c) mRNA levels in Tsc2 -/- MEFs upon treatment with a miR-33a mimic. Cells were reverse transfected for 24 hours and starved overnight with vehicle or rapamycin treatment (Rap, 20 nM) for the final 16 hours. N = 2 for qPCR; data are shown as mean ± s.e.m. (d) Proposed model of the results. *Statistical significance determined by a two-tailed t-test for qPCR data. Gels shown are cropped; full-length gels are presented in Supplementary Figure 5.

Fig 5: PIM kinases were expressed in HuH6 cells, the COA67 PDX, the parent tumor for COA67, and human hepatoblastoma tumor specimens(A) Immunoblotting for PIM1, PIM2, and PIM3 was completed on HuH6 and COA67 cell lysates with ß-actin controls. All three PIM proteins were present in both cell lines. (B) Immunohistochemistry for PIM3 was performed on human hepatoblastoma (top left panel, magnification on bottom left panel) and normal liver (top right panel) with negative staining in the IgG control is in bottom right insert, not bottom right panel as currently stated. PIM3 was more highly expressed in hepatoblastoma (top left panel) than normal liver (top right panel). Black arrow in bottom left panel indicates lack of staining in the peri-portal connective tissue in comparison to the PIM3 stained hepatoblastoma cells in the left half of the photomicrograph. (C) Hematoxylin and eosin (top panel) and immunohistochemistry for PIM3 (bottom panel) with IgG control (inserts, bottom panels) in parent tumor (left bottom panel) and COA67 PDX (right bottom panel) revealed that the COA67 PDX closely resembled the parent tumor histomorphologically and PIM3 was present in both the PDX and its parent.