Figure 4.
Figure 4. RhoA, ROCK, and myosin II are required for lobopodia-based 3D migration inside CDM. (A) A representative Western blot demonstrating the specificity of siRNA-mediated knockdown of Rac1, Cdc42, or RhoA. HFFs were transfected with the indicated siRNAs and lysed 72 h after transfection, and the lysates were blotted with the indicated antibodies. (B) Quantification of Western blots represented in A. (C) RhoA siRNA treatment switches HFFs to lamellipodia-based 3D migration in the CDM. The percentage of lobopodia-bearing HFFs migrating inside the CDM after the indicated treatments. (A and B) *, P < 0.001 versus the siGLO control. 48 h after siRNA treatment, HFFs were transfected with GFP-actin and imaged migrating in the 3D CDM. (D) Quantification of the velocity of siRNA-treated HFFs in the CDM 72–84 h after transfection. *, P < 0.05 versus the siGLO control. (E) ROCK dependence distinguishes HFF migration in collagen from the CDM. Quantification of the velocity of HFFs migrating in the CDM or 1.7 mg/ml collagen treated with FBS or FBS + 10 µM Y-27632. *, P < 0.03 versus the FBS control. (F) Quantification of HFF velocity in the CDM when treated with FBS or FBS + 25 µM blebbistatin. Blebbistatin treatment resulted in two subsets of HFFs, rapidly moving spread cells on top of the CDM (2D), and slowly moving elongated cells inside the CDM (3D). *, P < 0.001 versus the FBS control. (G and H) Myosin II is required for 3D lobopodia-based migration. (G) Cortactin localization in HFFs in the 3D CDM, either untreated or treated with 25 µM blebbistatin. The arrow indicates the local accumulation of cortactin at the leading edge. Bars, 5 µm. (H) The mean cortactin intensity profile, measured from the leading edge (0 µm) toward the cell center, of cells treated with FBS or FBS + 25 µM blebbistatin. Each cortactin intensity profile was averaged from 13 cells, with three measurements per cell. All cells are oriented with the leading edge toward the right of the figure. Error bars show means ± SEM. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; a.u. arbitrary unit.

RhoA, ROCK, and myosin II are required for lobopodia-based 3D migration inside CDM. (A) A representative Western blot demonstrating the specificity of siRNA-mediated knockdown of Rac1, Cdc42, or RhoA. HFFs were transfected with the indicated siRNAs and lysed 72 h after transfection, and the lysates were blotted with the indicated antibodies. (B) Quantification of Western blots represented in A. (C) RhoA siRNA treatment switches HFFs to lamellipodia-based 3D migration in the CDM. The percentage of lobopodia-bearing HFFs migrating inside the CDM after the indicated treatments. (A and B) *, P < 0.001 versus the siGLO control. 48 h after siRNA treatment, HFFs were transfected with GFP-actin and imaged migrating in the 3D CDM. (D) Quantification of the velocity of siRNA-treated HFFs in the CDM 72–84 h after transfection. *, P < 0.05 versus the siGLO control. (E) ROCK dependence distinguishes HFF migration in collagen from the CDM. Quantification of the velocity of HFFs migrating in the CDM or 1.7 mg/ml collagen treated with FBS or FBS + 10 µM Y-27632. *, P < 0.03 versus the FBS control. (F) Quantification of HFF velocity in the CDM when treated with FBS or FBS + 25 µM blebbistatin. Blebbistatin treatment resulted in two subsets of HFFs, rapidly moving spread cells on top of the CDM (2D), and slowly moving elongated cells inside the CDM (3D). *, P < 0.001 versus the FBS control. (G and H) Myosin II is required for 3D lobopodia-based migration. (G) Cortactin localization in HFFs in the 3D CDM, either untreated or treated with 25 µM blebbistatin. The arrow indicates the local accumulation of cortactin at the leading edge. Bars, 5 µm. (H) The mean cortactin intensity profile, measured from the leading edge (0 µm) toward the cell center, of cells treated with FBS or FBS + 25 µM blebbistatin. Each cortactin intensity profile was averaged from 13 cells, with three measurements per cell. All cells are oriented with the leading edge toward the right of the figure. Error bars show means ± SEM. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; a.u. arbitrary unit.

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