Figure 1.
Figure 1. Integrin β3 localizes at CDRs after PDGF-BB stimulation and integrin β3–GFP translocation 4D tracing in a live cell. (A) Primary mouse fibroblasts were stimulated with or without PDGF-BB, fixed, and IF stained. Arrows denote integrin β3 at focal adhesions, and arrowheads denote integrin β3 at CDRs. (B) Primary mouse fibroblasts were stimulated with PDGF-BB, fixed, and IF stained. Confocal image stacks were scanned along the z axis. Arrows 1 and 2 denote remnant integrin β3 at focal adhesions. Arrowheads 3 and 4 denote integrin β3 at a large (early) CDR and a condensed (late) CDR, respectively. Crossed lines denote the orthogonal position. The z distance in the orthogonal views was exaggerated five times. (C) Primary mouse fibroblasts were stimulated with PDGF-BB for various times, fixed, and IF stained. The number of cells forming integrin β3 CDRs per 100 cells was counted (n = 5). (D) NIH3T3 cells stably expressing integrin β3–GFP were stimulated with PDGF-BB. The temporal and spatial translocation of integrin β3–GFP was traced by 4D time-lapse confocal live-cell imaging. Sections of confocal images were scanned along the cell z axis every 1 min. The ventral cell surface position was defined as z = 0 µm. A positive z distance defined the position distance above the ventral cell surface. t defined the time after PDGF-BB addition. Arrows denote integrin β3–GFP at focal adhesions. Large arrowheads denote integrin β3–GFP at early CDRs, late condensed CDRs, macropinosomes, and the perinuclear region. (E) The number of focal adhesions, diameter of CDRs or macropinosomes, and number of macropinosomes at various times after PDGF-BB stimulation were counted to determine the temporal and spatial translocation of integrin β3–GFP in D and Video 2. (F) The lifetimes of ventral surface integrin β3–GFP focal adhesions were quantified in PDGF-BB–stimulated cells and unstimulated control cells as in Video 2 and Video 3, respectively. A focal adhesion lifetime <30 min was recorded as actual time, and a lifetime >30 min was recorded as 30 min. The lifetimes of 30 focal adhesions per cell were recorded (n = 3). (G) Primary mouse fibroblasts or NIH3T3 cells stably expressing integrin β3–GFP were stimulated with PDGF-BB for various times. Integrin β3 protein levels from whole-cell lysates were analyzed by SDS-PAGE and Western blotting. Error bars represent means ± SD. ***, P < 0.05. Bars: (A) 50 µm; (B and D) 20 µm.

Integrin β3 localizes at CDRs after PDGF-BB stimulation and integrin β3–GFP translocation 4D tracing in a live cell. (A) Primary mouse fibroblasts were stimulated with or without PDGF-BB, fixed, and IF stained. Arrows denote integrin β3 at focal adhesions, and arrowheads denote integrin β3 at CDRs. (B) Primary mouse fibroblasts were stimulated with PDGF-BB, fixed, and IF stained. Confocal image stacks were scanned along the z axis. Arrows 1 and 2 denote remnant integrin β3 at focal adhesions. Arrowheads 3 and 4 denote integrin β3 at a large (early) CDR and a condensed (late) CDR, respectively. Crossed lines denote the orthogonal position. The z distance in the orthogonal views was exaggerated five times. (C) Primary mouse fibroblasts were stimulated with PDGF-BB for various times, fixed, and IF stained. The number of cells forming integrin β3 CDRs per 100 cells was counted (n = 5). (D) NIH3T3 cells stably expressing integrin β3–GFP were stimulated with PDGF-BB. The temporal and spatial translocation of integrin β3–GFP was traced by 4D time-lapse confocal live-cell imaging. Sections of confocal images were scanned along the cell z axis every 1 min. The ventral cell surface position was defined as z = 0 µm. A positive z distance defined the position distance above the ventral cell surface. t defined the time after PDGF-BB addition. Arrows denote integrin β3–GFP at focal adhesions. Large arrowheads denote integrin β3–GFP at early CDRs, late condensed CDRs, macropinosomes, and the perinuclear region. (E) The number of focal adhesions, diameter of CDRs or macropinosomes, and number of macropinosomes at various times after PDGF-BB stimulation were counted to determine the temporal and spatial translocation of integrin β3–GFP in D and Video 2. (F) The lifetimes of ventral surface integrin β3–GFP focal adhesions were quantified in PDGF-BB–stimulated cells and unstimulated control cells as in Video 2 and Video 3, respectively. A focal adhesion lifetime <30 min was recorded as actual time, and a lifetime >30 min was recorded as 30 min. The lifetimes of 30 focal adhesions per cell were recorded (n = 3). (G) Primary mouse fibroblasts or NIH3T3 cells stably expressing integrin β3–GFP were stimulated with PDGF-BB for various times. Integrin β3 protein levels from whole-cell lysates were analyzed by SDS-PAGE and Western blotting. Error bars represent means ± SD. ***, P < 0.05. Bars: (A) 50 µm; (B and D) 20 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal