Fig. 8: Cold temperature prevents neurodegeneration in ALS iPSCs-derived motor neurons. | Nature Aging

Fig. 8: Cold temperature prevents neurodegeneration in ALS iPSCs-derived motor neurons.

From: Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes

Fig. 8

a, Western blot with anti-FUS antibody in HEK293 cells expressing wild-type FUS (WT) or ALS-related mutant FUSP525L. Graphs represent the relative percentage of FUS levels (corrected for β-actin loading control) to the corresponding 37 °C + non-targeting (NT) shRNA (mean ± s.e.m, FUS(WT): n = 3; FUS(P525L): n = 4). b, Filter trap with anti-FUS of HEK293 cells expressing wild-type or mutant FUS. Representative of five independent experiments. c, Western blot with anti-FUS of PSME3(OE)-HEK293 cells expressing mutant FUS. Graph represents the relative percentage of FUS levels (corrected for β-actin) to 37 °C + Empty vector (mean ± s.e.m. of 4 independent experiments). d, Filter trap with anti-FUS of PSME3(OE)-HEK293 cells expressing mutant FUS(P525L). Representative of four independent experiments. e, Immunocytochemistry of ALS (FUSP525L/P525L) and isogenic control (FUSWT/WT) iPSC-derived motor neurons. Cleaved caspase-3 (red), MAP2 (green) or Hoechst (blue) staining was used as a marker of apoptosis, neurons and nuclei, respectively. Scale bar: 20 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of eight biological replicates, FUS(WT) 37 °C: 998 total nuclei; FUS(WT) 36 °C: 661; FUS(P525L) 37 °C: 331, FUS(P525L) 36 °C: 265). f, Western blot with anti-PSME3 of ALS-iPSCs expressing PSME3 shRNA. β-actin loading control. Representative of three independent experiments. g, Trypsin-like activity in ALS iPSC-derived motor neurons on PSME3 knockdown (mean ± s.e.m. relative slope to 37 °C NT shRNA, n = 3 independent experiments). h, Immunocytochemistry of ALS iPSCs-derived motor neurons with anti-cleaved caspase-3 (red), anti-MAP2 (green) and Hoechst (blue). Scale bar: 20 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of nine biological replicates, 37 °C + NT shRNA: 822 total nuclei; 37 °C + psme-3 shRNA: 669; 36 °C + NT shRNA: 803; 36 °C + psme-3 shRNA: 567). 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. Two-tailed Student’s t-test for paired (a,c,g) or unpaired samples (e,h). 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.

Source data

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