Fig. 5: PER2 promotes OSCC cuproptosis by downregulating HSP70 binding to AKT.
A Co-IP assay for PER2/HSP70 and HSP70/AKT complexes in Vector-SCC25 and OE-PER2-SCC25 cells. B CHX chase assay for half-life of HSP70 and AKT in Vector-SCC25 and OE-PER2-SCC25 cells. C Western blot and strip assay for levels of AKT ubiquitination in Vector-SCC25 and OE-PER2-SCC25 cells. D Western blotting assays for AKT, p-AKT, DLAT, PDHB and SLC31A1 protein expression in Vector-SCC25 and OE-PER2-SCC25 cells. E Copper Colorimetric Assay Kit for detecting copper concentration in Vector-SCC25, OE-PER2-SCC25 and Mut-PER2ΔCT-SCC25 cells. F Non-denaturing gel electrophoresis assay for detection of DLAT oligomers in Vector-SCC25, OE-PER2-SCC25 and Mut-PER2ΔCT-SCC25 cells. G Immunofluorescence assay for DLAT oligomers in Vector-SCC25, OE-PER2-SCC25 and Mut-PER2ΔCT-SCC25 cells (yellow, DLAT oligomer; green, DLAT; red, Mitotracker; blue, DAPI; scale bars = 50 μm; three independent experiments). H Micro-mitochondrial Complex I and II Activity Assay Kit to measure activity of mitochondrial complexes I and II in Vector-SCC25, OE-PER2-SCC25, and Mut-PER2ΔCT-SCC25 cells. I Fumarate Assay Kit and α-KG Assay Kit to examine fumarate and α-ketoglutarate concentrations in Vector-SCC25, OE-PER2-SCC25, and Mut-PER2ΔCT-SCC25 cells. J CCK-8 assay for proliferation levels of Vector-SCC25, OE-PER2-SCC25 and Mut-PER2ΔCT-SCC25 cells. K MTT assay for proliferation levels of Vector-SCC25, OE-PER2-SCC25 and Mut-PER2ΔCT-SCC25 cells. L Co-IP showed a significant increase in HSP70/AKT complex in Mut-PER2ΔCT-SCC25 cells compared with OE-PER2-SCC25 cells. M Western blotting demonstrated significant increases in AKT and p-AKT and significant decreases in DLAT, PDHB and SLC31A1 protein expression in Mut-PER2ΔCT-SCC25 cells compared with OE-PER2-SCC25 cells. 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.
