Figure 1.
TEM4 is required for mitosis. (A) Schematic illustration of the annotated domains of TEM4 protein. (B) Representative immunofluorescence image of mitotic HeLa-T-REx cells overexpressing the indicated GFP-TEM4 constructs. Cells were stained with anti-GFP (green), Phalloidin-Atto 565 for F-actin (red), and Hoechst (blue). Scale bar = 10 μm for the main image and 2 μm for the magnified image. (C) Fold change in ARHGEF17 mRNA in the indicated cell lines after 72 h shRNA induction. Gene expression was normalized to HPRT. Shown is mean ± SEM, n = 6 independent experiments. P values were determined by a one-way ANOVA test followed by the Holm-Šídák’s multiple comparisons test. (D) TEM4 western blot of lysates from the indicated cell lines after synchronization in mitosis with 100 ng/ml nocodazole at the end of a 72 h shRNA induction; n = 3 independent experiments. Note that the TEM4 antibody detects two different isoforms of TEM4 at ∼245 and 180 kDa. (E) The mitotic index of HeLa-T-REx control, TEM4-21, and TEM4-22 cells 72 h after shRNA induction. Cells were treated with 100 ng/ml nocodazole for 12 h prior to fixation. Data shown is mean ± SEM; n = 3 independent experiments, N ≥ 1,000 cells in total with individual points indicating the mitotic index in a single field of imaging. P values were determined by a one-way ANOVA test followed by the Holm-Šídák’s multiple comparisons test. (F) Control TEM4 shRNA and rescue cell lines were treated as indicated in E. Mean mitotic index ± SEM is shown, n = 3 independent experiments, individual points represent the mitotic index in a single field of imaging. P values are from a two-way ANOVA test followed by Tukey’s multiple comparison test. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

TEM4 is required for mitosis. (A) Schematic illustration of the annotated domains of TEM4 protein. (B) Representative immunofluorescence image of mitotic HeLa-T-REx cells overexpressing the indicated GFP-TEM4 constructs. Cells were stained with anti-GFP (green), Phalloidin-Atto 565 for F-actin (red), and Hoechst (blue). Scale bar = 10 μm for the main image and 2 μm for the magnified image. (C) Fold change in ARHGEF17 mRNA in the indicated cell lines after 72 h shRNA induction. Gene expression was normalized to HPRT. Shown is mean ± SEM, n = 6 independent experiments. P values were determined by a one-way ANOVA test followed by the Holm-Šídák’s multiple comparisons test. (D) TEM4 western blot of lysates from the indicated cell lines after synchronization in mitosis with 100 ng/ml nocodazole at the end of a 72 h shRNA induction; n = 3 independent experiments. Note that the TEM4 antibody detects two different isoforms of TEM4 at ∼245 and 180 kDa. (E) The mitotic index of HeLa-T-REx control, TEM4-21, and TEM4-22 cells 72 h after shRNA induction. Cells were treated with 100 ng/ml nocodazole for 12 h prior to fixation. Data shown is mean ± SEM; n = 3 independent experiments, N ≥ 1,000 cells in total with individual points indicating the mitotic index in a single field of imaging. P values were determined by a one-way ANOVA test followed by the Holm-Šídák’s multiple comparisons test. (F) Control TEM4 shRNA and rescue cell lines were treated as indicated in E. Mean mitotic index ± SEM is shown, n = 3 independent experiments, individual points represent the mitotic index in a single field of imaging. P values are from a two-way ANOVA test followed by Tukey’s multiple comparison test. Source data are available for this figure: SourceData F1.

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