Tensin3 regulates β1 integrin activity in the presence of talin. (A) Images of TLNKO cells expressing GFP-vector control, GFP-TLN1, or GFP-TNS3. F-actin was visualized by phalloidin staining. (B) Representative integrin activation (β1) profiles of TLNKO cells expressing GFP-vector, GFP-TLN1, or GFP-TNS3 as measured by flow cytometry analysis. Red profiles are from cells expressing the indicated constructs, and gray profiles are from the non-transfected cells in the same samples. (C) Integrin activation index (normalized to cells expressing GFP-vector) calculated from triplicate experiments of B. (D) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3 constructs (shown in Fig. 5 E), respectively. Activated β1 integrin was visualized by staining with 9EG7 antibody. (E) Quantification of 9EG7-positive adhesions in D pooled from three independent experiments; n = 42 (vector), 38 (WT), 41 (L702E), 46 (ΔPTB), and 40 (L702E+ΔPTB) cells. (F) Representative integrin activation profiles of TLNKO cells co-expressing GFP-TLN1 with different mCh-TNS3 constructs. (G) Mean fluorescence intensity of F. Red values are from transfected cells, and gray values are from the nontransfected cells in the same samples. Note that the quantification of the integrin activation index pooled from three replicates is shown in Fig. 5 H. (H) Representative images of U2OS TNS3KO cells expressing GFP-TNS3 constructs (WT, L702E, ΔPTB, or L702E+ΔPTB). Cells were cultured on FN-coated glass overnight before being treated with DMSO or blebbistatin (50 μM, shown in Fig. 5 J) for 60 min. Actin and β1 integrin were visualized by staining with phalloidin and 9EG7 antibody. Note that all four GFP-TNS3 constructs were localized to adhesions when cells were treated with DMSO. (I and J) NIH3T3 cells transfected with GFP-TNS3 constructs (same as those used in H) were treated with DMSO (I) or blebbistatin (50 μM, J) for 60 min before fixation and stained for actin. (K) Quantification of GFP-TNS3–positive adhesions in J. Note that GFP-TNS3-WT– and GFP-TNS3-ΔPTB–positive adhesions largely remain after blebbistatin treatment, whereas GFP-TNS3-L702E–positive and GFP-TNS3-Cterm–positive adhesions mostly disappear. n = 64 (WT), 49 (L702E), 62 (ΔPTB), and 71 (Cterm) cells. All error bars are the SEM. ** indicates P < 0.01, and **** indicates P < 0.0001 (C: ordinary one-way ANOVA with Turkey’s multiple comparisons, E and K: Kruskal–Wallis test with Dunn’s multiple comparisons test). Data are collected from three independent experiments. Scale bars in A, D, and H–J, 10 μm.
Figure S3.

Tensin3 regulates β1 integrin activity in the presence of talin. (A) Images of TLNKO cells expressing GFP-vector control, GFP-TLN1, or GFP-TNS3. F-actin was visualized by phalloidin staining. (B) Representative integrin activation (β1) profiles of TLNKO cells expressing GFP-vector, GFP-TLN1, or GFP-TNS3 as measured by flow cytometry analysis. Red profiles are from cells expressing the indicated constructs, and gray profiles are from the non-transfected cells in the same samples. (C) Integrin activation index (normalized to cells expressing GFP-vector) calculated from triplicate experiments of B. (D) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3 constructs (shown in Fig. 5 E), respectively. Activated β1 integrin was visualized by staining with 9EG7 antibody. (E) Quantification of 9EG7-positive adhesions in D pooled from three independent experiments; n = 42 (vector), 38 (WT), 41 (L702E), 46 (ΔPTB), and 40 (L702E+ΔPTB) cells. (F) Representative integrin activation profiles of TLNKO cells co-expressing GFP-TLN1 with different mCh-TNS3 constructs. (G) Mean fluorescence intensity of F. Red values are from transfected cells, and gray values are from the nontransfected cells in the same samples. Note that the quantification of the integrin activation index pooled from three replicates is shown in Fig. 5 H. (H) Representative images of U2OS TNS3KO cells expressing GFP-TNS3 constructs (WT, L702E, ΔPTB, or L702E+ΔPTB). Cells were cultured on FN-coated glass overnight before being treated with DMSO or blebbistatin (50 μM, shown in Fig. 5 J) for 60 min. Actin and β1 integrin were visualized by staining with phalloidin and 9EG7 antibody. Note that all four GFP-TNS3 constructs were localized to adhesions when cells were treated with DMSO. (I and J) NIH3T3 cells transfected with GFP-TNS3 constructs (same as those used in H) were treated with DMSO (I) or blebbistatin (50 μM, J) for 60 min before fixation and stained for actin. (K) Quantification of GFP-TNS3–positive adhesions in J. Note that GFP-TNS3-WT– and GFP-TNS3-ΔPTB–positive adhesions largely remain after blebbistatin treatment, whereas GFP-TNS3-L702E–positive and GFP-TNS3-Cterm–positive adhesions mostly disappear. n = 64 (WT), 49 (L702E), 62 (ΔPTB), and 71 (Cterm) cells. All error bars are the SEM. ** indicates P < 0.01, and **** indicates P < 0.0001 (C: ordinary one-way ANOVA with Turkey’s multiple comparisons, E and K: Kruskal–Wallis test with Dunn’s multiple comparisons test). Data are collected from three independent experiments. Scale bars in A, D, and H–J, 10 μm.

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