Fig. 6: Moderate cooling induces trypsin-like proteasome activity in human cells.
a, Trypsin-like activity in HEK293 cells at cold temperature (36 °C) for 24 h (mean ± s.e.m. relative slope to 37 °C, n = 12 independent experiments). b, Caspase-like activity in HEK293 cells (mean ± s.e.m. relative to 37 °C, n = 18 independent experiments). c, Chymotrypsin-like activity in HEK293 cells (mean ± s.e.m. relative to 37 °C, n = 9 independent experiments). d, Trypsin-like activity in HEK293 cells upon 25 µM HC-030031 for 24 h (mean ± s.e.m. relative to 37 °C + DMSO vehicle control, n = 7 independent experiments). e, mRNA levels of proteasome subunits in HEK293 cells (mean ± s.e.m. relative expression to 37 °C, n = 8 independent experiments). f, Western blot of PA28γ/PSME3, 19S PSMD11, and 20S α6/PSMA1 in HEK293 cells. Graph represents relative percentage values of proteasome subunits (corrected for β-actin loading control) to 37 °C (mean ± s.e.m., n = 5 independent experiments). g, Native gel electrophoresis of HEK293 cells followed by immunoblotting with anti-PSME3 antibody. Representative of 5 independent experiments. h, Knockdown levels in HEK293 cells expressing PSME3 shRNA (mean ± s.e.m. relative to non-targeting (NT) shRNA, n = 4 independent experiments). i, PSME3 protein levels in PSME3 shRNA-HEK293 cells. β-actin loading control. Representative of three independent experiments. j, Trypsin-like activity in PSME3-shRNA-HEK293 cells (mean ± s.e.m. relative to 37 °C NT shRNA, n = 5 independent experiments). k, PSME3 mRNA levels in HEK293 cells overexpressing (OE) PSME3 at 37 °C (mean ± s.e.m. relative to empty vector, n = 3 biological replicates). l, PSME3 protein levels in PSME3(OE)-HEK293 cells at 37 °C. α-tubulin loading control. Representative of four independent experiments. m, PSME3 overexpression increases trypsin-like activity at 37 °C and 36 °C (mean ± s.e.m. relative to 37 °C + empty vector, n = 4 independent experiments). In all the experiments, cells were cultured at 37 °C and then shifted to cold temperature (36 °C) or maintained at 37 °C for 24 h before the analysis. Statistical comparisons were made by two-tailed Student’s t-test for paired samples, except Fig. 6k (unpaired t-test). P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR (q value < 0.05). Source Data contains exact P and q values.
