Figure 8. TEM4 depletion results in the... | Rockefeller University Press

Figure 8.

$TEM4 depletion results in the reduction of YAP protein levels. (A) HeLa cells were depleted of YAP by increasing the concentration of siRNA (0–100 nM of each duplex), and lysates were probed with the indicated antibodies. Band intensity in the linear range relative to α-tubulin is indicated in Fig. S7 B. (B) Control and TEM4-depleted cells were fixed probed with the indicated antibodies. Scale bar = 5 μm. (C) The relative total intensity of YAP from B. Data show mean ± SEM, n = 3 independent experiments, N ≥ 45 cells per condition. A one-way ANOVA and Brown-Forsythe test for significance was performed followed by Dunnett’s T3 multiple comparisons test. (D) Control and TEM4-depleted lysates were probed for the indicated proteins by western blotting. YAP signal intensity in the linear range relative to GAPDH is indicated. * denotes the YAP paralog TAZ. (E) Normalized YAP intensity in the indicated cell lines grown at low versus high density. Pairwise comparisons are shown between control and TEM-depleted cells. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 33 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test to determine P values. (F) Normalized nuclear (N) and cytoplasmic (C) YAP intensity in the indicated cell lines. Pairwise comparisons are shown between control and TEM-depleted cells, and between nuclear and cytoplasmic fractions for each cell line. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 35 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test. (G) Control and TEM4 depleted cells were treated with DMSO, MG132, or cycloheximide (CHX) for 5 h before lysis and western blotting with the indicated antibodies. YAP signal intensity in the linear range relative to GAPDH is indicated. (H) Mitotic index in the indicated cell lines treated with either DMSO (D) or the YAP inhibitor verteporfin (0.5 μM for 24 h) with nocodazole treatment in the final 12 h. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 600 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test. (I) Model of TEM4 regulation of YAP. See discussion for details. Source data are available for this figure: SourceData F8. Refer to the image caption for details.$

TEM4 depletion results in the reduction of YAP protein levels. (A) HeLa cells were depleted of YAP by increasing the concentration of siRNA (0–100 nM of each duplex), and lysates were probed with the indicated antibodies. Band intensity in the linear range relative to α-tubulin is indicated in Fig. S7 B. (B) Control and TEM4-depleted cells were fixed probed with the indicated antibodies. Scale bar = 5 μm. (C) The relative total intensity of YAP from B. Data show mean ± SEM, n = 3 independent experiments, N ≥ 45 cells per condition. A one-way ANOVA and Brown-Forsythe test for significance was performed followed by Dunnett’s T3 multiple comparisons test. (D) Control and TEM4-depleted lysates were probed for the indicated proteins by western blotting. YAP signal intensity in the linear range relative to GAPDH is indicated. * denotes the YAP paralog TAZ. (E) Normalized YAP intensity in the indicated cell lines grown at low versus high density. Pairwise comparisons are shown between control and TEM-depleted cells. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 33 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test to determine P values. (F) Normalized nuclear (N) and cytoplasmic (C) YAP intensity in the indicated cell lines. Pairwise comparisons are shown between control and TEM-depleted cells, and between nuclear and cytoplasmic fractions for each cell line. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 35 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test. (G) Control and TEM4 depleted cells were treated with DMSO, MG132, or cycloheximide (CHX) for 5 h before lysis and western blotting with the indicated antibodies. YAP signal intensity in the linear range relative to GAPDH is indicated. (H) Mitotic index in the indicated cell lines treated with either DMSO (D) or the YAP inhibitor verteporfin (0.5 μM for 24 h) with nocodazole treatment in the final 12 h. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 600 cells per condition. A one-way ANOVA test for significance was performed followed by Tukey’s multiple comparisons test. (I) Model of TEM4 regulation of YAP. See discussion for details. Source data are available for this figure: SourceData F8.