Extended Data Fig. 5: YAP modulates the dynamics of cytoplasmic TDP-43 condensates.
From: YAP maintains the dynamics of TDP-43 condensates and antagonizes TDP-43 pathological aggregates
a,b, WT HeLa cells or YAP−/− HeLa cells reconstituted with YAP WT or S94A were transfected with TDP-43-GFP ΔNLS and treated with 250 µM NaAsO2 for 1 h and immunostained with YAP antibody. The co-localization between TDP-43-GFP ΔNLS and YAP was quantified (b). 6 images were pooled from 2 independent experiments for each condition. Data were shown as mean ± s.d. c, WT HeLa cells or YAP−/− HeLa cells reconstituted with either EV, YAP WT or S94A were transfected with TDP-43-GFP ΔNLS and treated with 250 µM NaAsO2 for 1 h. Cytoplasmic and nuclear fractions were prepared, respectively, and analysed by immunoblotting using the indicated antibodies. The experiment was repeated two independently times with similar results. d,e, WT HeLa cells or YAP−/− HeLa cells reconstituted with EV, YAP WT or S94A were treated with 250 µM NaAsO2 for 1 h and allowed to recover in fresh medium for the indicated time. Arrow heads highlighted the dead cells. The percentage of dead cells under each condition was quantitatively assessed (e). For WT HeLa, 229, 250, 236, 248, 319 cells for 0 h, 1 h, 2 h, 3 h, 4 h, respectively; for EV group, 230, 231, 251, 260, 266 cells for 0 h, 1 h, 2 h, 3 h, 4 h, respectively; for YAP WT group, 253, 302, 256, 263, 335 cells for 0 h, 1 h, 2 h, 3 h, 4 h, respectively; for YAP S94A group, 233, 272, 233, 260, 265 cells for 0 h, 1 h, 2 h, 3 h, 4 h, respectively. All the data were pooled from n = 3 independent experiments. mean ± s.e.m. f, HeLa cells transfected with the indicated siRNA were analysed by immunoblotting with the indicated antibodies. The experiment was repeated two independently times with similar results. g,h, WT HeLa transfected with siCtrl, or siTDP-43 were treated with 500 µM NaAsO2 for 1 h, then were processed for immunostaining using antibodies against endogenous YAP and G3BP1. Pearson’s correlation between YAP and G3BP1 was calculated (h). 6 images were pooled from 2 independent experiments for each condition. mean ± s.d. i,j, WT HeLa transfected with siCtrl, or siTDP-43 were treated with 0.2 M sorbitol for 1 h, and then were immunostained using the indicated antibodies. Cells with YAP condensates were quantified (i). 167, 173 cells for siCtrl and siTDP-43, respectively, from n = 3 independent experiments. mean ± s.e.m. k, WT 293T cells transfected with siCtrl, or siTDP-43 were treated with 0.2 M sorbitol for 3 h. The mRNA levels of CYR6 and CTGF in these cells were determined by RT-PCR. mean ± s.d. n = 3 technical replicates. Statistical significance was determined by two-way ANOVA (e), and two-tailed Student’s t-test (i).
