Fig. 4: PER2 regulates OSCC cuproptosis through PER2/HSP70/AKT complex formation and AKT pathway. | Cell Death & Disease

Fig. 4: PER2 regulates OSCC cuproptosis through PER2/HSP70/AKT complex formation and AKT pathway.

From: PER2 interaction with HSP70 promotes cuproptosis in oral squamous carcinoma cells by decreasing AKT stability

Fig. 4

A Western blotting showed that DLAT, PDHB, and SLC31A1 protein expression was significantly reduced by the addition of SC79 to OE-PER2-SCC25 cells. B Copper Colorimetric Assay Kit assay showed a significant reduction in copper concentration with the addition of SC79 to OE-PER2-SCC25 cells. C Non-denaturing gel electrophoresis assay detected a significant reduction of DLAT oligomers after addition of SC79 to OE-PER2-SCC25 cells. D Immunofluorescence assay detected a significant reduction in DLAT oligomers after addition of SC79 to OE-PER2-SCC25 cells (yellow, DLAT oligomer; green, DLAT; red, Mitotracker; blue, DAPI; scale bars = 50 μm; three independent experiments). E CCK-8 assay showed, cell proliferation levels were significantly increased by the addition of SC79 to OE-PER2-SCC25 cells. F MTT assay showed, cell proliferation levels were significantly increased by the addition of SC79 to OE-PER2-SCC25 cells. G Thomas blue-stained gel showed, Flag antibody fishing for candidate proteins that may bind to PER2 in SCC25 cells transfected with Flag-PER2. Schematic shows that three subunits (HSPA1, HSPA4 and HSPA8) of HSP70 in immunoprecipitation mass spectrometry results are all candidate proteins for binding to PER2 (screening criterion is unique peptide ≥2), suggesting that PER2 may have strong binding to HSP70. H Co-IP assay for PER2, HSP70 and AKT binding in SCC25 and 293 T cells. I GST pull-down assay for detecting direct binding of PER2 to HSP70 and AKT in vitro and direct binding of HSP70 to AKT in vitro. J Schematic showed that the major structural domains in UniProt and InterPro databases where PER2 binds to protein interactions are PAS1, PAS2, and C-terminal structural domain. K Construction of three plasmids with Flag-tagged PER2 deletion mutations in the structural domains of PAS1 (region 181V-248L), PAS2 (region 321Y-387Q) and CT (region 500S-1255T), respectively. L Co-IP assays were performed to detect the binding of HSP70 and AKT after three deletion mutant plasmids transfection into SCC25 cells, respectively. All data represent three replicate independent experiments. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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