Figure S1.
TEM4 decreases mitotic index in different cell lines independently of the microtubule status. (A) HeLa-T-REx were transfected with indicated TEM4 constructs before being fixed. Cells were stained with anti-GFP (green), Phalloidin-Atto 565 for F-actin (red), and Hoechst (blue). Scale bar = 15 μm for the main image and 2 μm for the zoomed image. (B) Control or HeLa-TREx cells depleted of TEM4 were treated with 15 nm taxol for 12 h and fixed. (C) Control or HeLa-TREx cells depleted for TEM4 were synchronized in mitosis after 10 h of release from double thymidine block. (D) Control or HeLa-TREx cells depleted for TEM4 were released from double thymidine and treated with MG132 at mitotic entry. (E) Expression of shTEM4-21 and shTEM-22 resistant TEM4 versus TEMWT proteins in control, TEM4-21 and TEM-22 cell lines. Lysates were blotted for the indicated antibodies. (F) Expression and localization of shRNA resistant TEM4WT versus TEMWT proteins in TEM4-21 and TEM4-22 cell lines. Cells we stained with anti-GFP and Phalloidin-Atto 565. Scale bar = 5 μm for the main image and 1 μm for the zoomed image. (G) Expression of ARHGEF17 mRNA in HCT-116 cells transduced with shTEM4-21 or shTEM4-22. ShRNA expression was induced for 72 h using 0.5 μg/ml doxycycline. (H) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for 72 h and released for 10 h from double a thymidine block. (I) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for a total of 72 h and cells were treated with 15 nm taxol for 12 h and fixed. (J) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for a total of 72 h and cells were released from double thymidine block and treated with the proteosome inhibitor MG132 to prevent cells from exiting mitosis. For B–D and G–J, the data shown are means ± SEM of three independent experiments (large data points). Small points represent the mitotic index of a single field of imaging and a total of 1,000–1,500 cells were quantified for each experiment. A one-way ANOVA-Kruskal–Wallis for significance was performed and the P values are shown from Dunn’s multiple comparisons. Source data are available for this figure: SourceData FS1. Refer to the image caption for details.

TEM4 decreases mitotic index in different cell lines independently of the microtubule status. (A) HeLa-T-REx were transfected with indicated TEM4 constructs before being fixed. Cells were stained with anti-GFP (green), Phalloidin-Atto 565 for F-actin (red), and Hoechst (blue). Scale bar = 15 μm for the main image and 2 μm for the zoomed image. (B) Control or HeLa-TREx cells depleted of TEM4 were treated with 15 nm taxol for 12 h and fixed. (C) Control or HeLa-TREx cells depleted for TEM4 were synchronized in mitosis after 10 h of release from double thymidine block. (D) Control or HeLa-TREx cells depleted for TEM4 were released from double thymidine and treated with MG132 at mitotic entry. (E) Expression of shTEM4-21 and shTEM-22 resistant TEM4 versus TEMWT proteins in control, TEM4-21 and TEM-22 cell lines. Lysates were blotted for the indicated antibodies. (F) Expression and localization of shRNA resistant TEM4WT versus TEMWT proteins in TEM4-21 and TEM4-22 cell lines. Cells we stained with anti-GFP and Phalloidin-Atto 565. Scale bar = 5 μm for the main image and 1 μm for the zoomed image. (G) Expression of ARHGEF17 mRNA in HCT-116 cells transduced with shTEM4-21 or shTEM4-22. ShRNA expression was induced for 72 h using 0.5 μg/ml doxycycline. (H) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for 72 h and released for 10 h from double a thymidine block. (I) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for a total of 72 h and cells were treated with 15 nm taxol for 12 h and fixed. (J) Mitotic index in HCT116 cells expressing shTEM4-21 or shTEM4-22. ShRNAs were induced for a total of 72 h and cells were released from double thymidine block and treated with the proteosome inhibitor MG132 to prevent cells from exiting mitosis. For B–D and G–J, the data shown are means ± SEM of three independent experiments (large data points). Small points represent the mitotic index of a single field of imaging and a total of 1,000–1,500 cells were quantified for each experiment. A one-way ANOVA-Kruskal–Wallis for significance was performed and the P values are shown from Dunn’s multiple comparisons. Source data are available for this figure: SourceData FS1.

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