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Files in this Data Supplement:
Fig. S1. ZRP-1-depleted cells lack actin stress fibers and mature focal adhesions. (A) siRNA-mediated suppression of ZRP-1 expression. Lysates of HeLa cells treated with or without ZRP-1-siRNA2 or ZRP-1-siRNA3 were immunoblotted with anti-ZRP-1 antibody (upper panel) or anti-α-tubulin antibody (lower panel, loading control). (B) HeLa cells transfected with or without ZRP-1-siRNA2, ZRP-1-siRNA3 or 10 nM ZRP-1-siRNA were fixed and stained with anti-ZRP-1 antibody and Rhodamine-phalloidin. Bars, 15 μm. (C,D) HeLa cells transfected with or without ZRP-1-siRNA2 or ZRP-1-siRNA3 were grown to confluence and wounded. Ten hours later, cells at wound sites were fixed and stained with anti-ZRP-1 and anti-paxillin (C) or anti-FAK (D) antibodies.
Fig. S2. ZRP-1-depleted cells exhibit bursts of actin polymerization. (B) HeLa cells transfected with or without ZRP-1-siRNAs were fixed and stained with anti-ZRP-1 antibody and Rhodamine-phalloidin. Bars, 15 μm.
Fig. S3. The expression of N-cadherin. Lysates of HeLa cells treated with or without ZRP-1-siRNA were immunoblotted with anti-N-cadherin antibody (upper panel) or anti-α-tubulin antibody (lower panel, loading control).
Fig. S4. Effects of FAK overexpression on actin stress fiber and focal adhesion. Both control (upper panels) and ZRP-1-depleted (lower panels) HeLa cells were transfected with expression vectors for EGFP and HA-FAK(Δ1−100) (constitutively active form of FAK) at a ratio of 1:9. These cells then were fixed and stained with phalloidin (A) or antibody against FAK (B). Bars, 15 μm.
Movie 1. A6 cells constitutively expressing EGFP-ZRP-1. A6 cells constitutively expressing EGFP-ZRP-1 were grown to confluence and wounded. After 6 hours, cells were imaged at 5-minute intervals.
Movie 2. (A) EGFP-actin-expressing control HeLa cells. HeLa cells were treated with control-siRNA for 24 hours. Cells were then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, cells were imaged at 1-minute intervals. (B) A EGFP-actin expressing ZRP-1-RNAi HeLa cell. HeLa cells were treated with ZRP-1-siRNA for 24 hours. Cells were then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, cells were imaged at 1-minute intervals.
Movie 3. (A) Actin dynamics at cell-cell contact (control). HeLa cells were treated with control-siRNA for 24 hours. Cells were then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, actin dynamics at cell-cell contacts was imaged at 1-minute intervals. (B) Actin dynamics at cell-cell contact (ZRP-1-RNAi). HeLa cells were treated with ZRP-1-siRNA for 24 hours and then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, actin dynamics at cell-cell contacts was imaged at 1-minute intervals.
Movie 4. ZRP-1-RNAi HeLa cells invading under neighboring cells. HeLa cells were treated with ZRP-1-siRNA for 24 hours. Cells were then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, relative migration between neighboring cells was imaged at 1-minute intervals.
Movie 5. (A) Wound healing assay (control). HeLa cells treated with control-siRNA were subjected to wound-healing assay as described in the Materials and methods. Four hours after wounding, cells were imaged at 1-minute intervals for 12 hours. (B) Wound healing assay (ZRP-1-RNAi). HeLa cells treated with ZRP-1-siRNA were subjected to wound-healing assay as described in the Materials and methods. Four hours after wounding, cells were imaged at 1-minute intervals for 12 hours.
Movie 6. (A) Actin dynamics in Rac1T17N-expressing control cells. HeLa cells were transfected with expression vectors for Myc-Rac1T17N and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. (B) Actin dynamics in Rac1T17N-expressing ZRP-1-RNAi cells. HeLa cells were transfected with Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-Rac1T17N and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals.
Movie 7. (A) Actin dynamics in RhoA-expressing control cell-. HeLa cells were transfected with expression vectors for Myc-WT-RhoA and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. (B) Actin dynamics in RhoA-expressing ZRP-1-RNAi cell-. HeLa cells were transfected with Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-WT-RhoA and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals.
Movie 8. EGFP-expressing ZRP-1-RNAi cell-. HeLa cells were transfected with ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for EGFP. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals.
Movie 9. Actin dynamics in a Rac1 G12V expressing HeLa cells. HeLa cells were transfected with expression vectors for Myc-Rac1G12V and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals.
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