Figure S5.
Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F(2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm2) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm2) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: SourceData FS5.

Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F(2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm2) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm2) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: SourceData FS5.

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