Fig. 5: YAP1 inhibition and cell-cycle arrest after cell fusion depend on active clathrin-mediated endocytosis. | Nature Communications

Fig. 5: YAP1 inhibition and cell-cycle arrest after cell fusion depend on active clathrin-mediated endocytosis.

From: YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion

Fig. 5: YAP1 inhibition and cell-cycle arrest after cell fusion depend on active clathrin-mediated endocytosis.

a SUM-159 cells, transfected with VSV-G, were incubated with inhibitors of endocytosis (PitStop2 or Dynasore) or transfected with a dominant negative form of the clathrin adapter AP-180 (AP180-C), a specific inhibitor of CME. Cells were then induced to fuse, fixed at indicated time points, immunostained with anti-YAP1 antibody and imaged by confocal microscopy to determine the YAP1 localization. b The ratio of nuclear to cytoplasmic YAP1 was measured at each timepoint after fusion in control (untreated) or endocytosis inhibited cells washed with fusion buffer. Error bars represent the SEM of cells examined over three independent experiments. For untreated cells (magenta line): n = 44, n = 19, n = 21, and n = 23 cells at 0, 5, 15, and 60 min, respectively. For Dynasore treated cells (red line): n = 38 (ns), n = 25 (**p = 0.0092), n = 32 (**p = 0.0095), and n = 31 cells (***p < 0.0001) at 0, 5, 15, and 60 min, respectively. For PitStop2 treated cells (blue line): n = 101 (ns), n = 36 (**p = 0.0021), n = 44 (**p = 0.0003), and n = 50 cells (***p < 0.0001) at 0, 5, 15, and 60 min, respectively. For cells transfected with AP180-C (black line): n = 24 (*p = 0.019), n = 24 (ns), n = 25 (***p < 0.0001), and n = 28 (***p < 0.0001) at 0, 5, 15, and 60 min, respectively. p values represent the differences between the control and each endocytic inhibitor at the indicated time point. c Confocal images of P21-positive nuclei in untreated and PitStop2 treated cells fixed 24 h after fusion. Arrow heads points at nuclei within fused cells negative for P21. d Quantification of the percentage of P21-positive nuclei in both fused (blue bar) and non-fused (black bar) cells in the untreated samples and the fused cells in the PitStop2 treated samples (white bar) are graphed. Error bars represent the SEM, n = 21, n = 25, and n = 28 cells examined over three independent experiments for non-fused, fused, and PitStop2 treated fused cells, respectively. ***p < 0.0001, and **p = 0.0012. e Violin plots depict the fold change in CDKN1A (P21) expression measured by qRT-PCR and compared in unwashed control cells (black), fused cells (blue), and fused cells treated with PitStop2 (white). n = 3 independent experiments. *p = 0.0412, *p = 0.0109, and **p = 0.0007. ns not significant (p > 0.05). Statistical significance calculations were performed using a two-tailed unpaired Student’s t test. Scale bar size = 10 μm.

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