Fig. 7: ATF3 promotes OSCC cuproptosis dependent on PER2.

A RT-qPCR to detect PER2 mRNA expression in Vector-SCC25 and OE-ATF3-SCC25 cells. B Western blotting to detect PER2 protein expression in Vector-SCC25 and OE-ATF3-SCC25 cells. C Copper Colorimetric Assay Kit for measuring copper concentration in Vector-SCC25 and OE-ATF3-SCC25 cells. D Non-denaturing gel electrophoresis assay for analyzing DLAT oligomers in Vector-SCC25 and OE-ATF3-SCC25 cells. E Immunofluorescence assay for DLAT oligomers in Vector-SCC25 and OE-ATF3-SCC25 cells (yellow, DLAT oligomer; green, DLAT; red, Mitotracker; blue, DAPI; scale bars = 50 μm; three independent experiments). F TEM to observe the number of vacuolated mitochondria in Vector-SCC25 and OE-ATF3-SCC25 cells (red arrows indicate mitochondria; three independent experiments). G Fumarate Assay Kit and α-KG Assay Kit to detect fumarate and α-ketoglutarate concentrations in Vector-SCC25 and OE-ATF3-SCC25 cells. H Micro-mitochondrial Complex I and II Activity Assay Kit to detect activity of mitochondrial complexes I and II in Vector-SCC25 and OE-ATF3-SCC25 cells. I CCK-8 assay for proliferation levels of Vector-SCC25 and OE-ATF3-SCC25 cells. J MTT assay for proliferation levels of Vector-SCC25 and OE-ATF3-SCC25 cells. K RT-qPCR assay showed significantly lower mRNA expression of PER2 in OE-ATF3-SCC25+sh-PER2#3 cells compared with OE-ATF3-SCC25 cells. L Western blotting showed a marked reduction in PER2 protein expression in OE-ATF3-SCC25+sh-PER2#3 cells compared with OE-ATF3-SCC25 cells. M Non-denaturing gel electrophoresis assay to examine significant decreases in DLAT oligomers in OE-ATF3-SCC25+sh-PER2#3 cells compared with OE-ATF3-SCC25 cells. N Immunofluorescence assay showed significant decreases of DLAT oligomers in OE-ATF3-SCC25+sh-PER2#3 cells compared with OE-ATF3-SCC25 cells. O TEM observed significant decreases in the number of vacuolated mitochondria, as well as decreases in mitochondrial deformation and swelling in OE-ATF3-SCC25+sh-PER2#3 cells compared with OE-ATF3-SCC25 cells (red arrows denote mitochondria, three independent experiments). 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.