Figure 7.

Defective contractile function of Tg26 podocytes. (A) Representative images of cells in three-dimensional collagen matrices. Images were obtained after 1 hour of gel polymerization before matrix was released from the substrate and allowed to float. Arrowheads point to WT podocyte extensions that are not present in Tg26 cells in three-dimensional culture. Scale bar, 75 μm. (B) Representative images of collagen gels at the end point of the floating matrix assay. (C) Quantitation of WT (white bars) and Tg26 podocyte (black bars) collagen matrix contraction. Data are from three independent experiments (n=3) performed in duplicate. Relative gel area after 4 hours of control or FBS treatment. *P<0.05 by ANOVA (GraphPad Prism). (D) Confirmation of equivalent numbers of cells in three-dimensional matrices (2×10⁴ cells) by a low-magnification immunofluorescence image of merged actin (phalloidin) and nuclei staining. Images were taken after 2 hours of 20% FBS treatment. Insets show confocal images of representative cells from the same respective matrices for enhanced observation of F-actin extensions that are visible in the WT cells. Arrowheads point to extensions in WT cells in control condition, which were retracted toward the cell body in FBS condition. Tg26 podocytes have negligible extensions in control and FBS conditions. Scale bar, 75 μm; 20 μm in insets. (E) Reduced three-dimensional gel contraction with graded replacement of WT podocytes by Tg26 podocytes. The same total number of cells (total 4×10⁴) was mixed with collagen gels in WT-to-Tg26 ratios ranging from 100:0 to 0:100. Gel area after contraction as shown in the representative image below the bar graph was calculated relative to the WT alone. *P<0.05 by two-way ANOVA followed by the Sidak multiple comparisons test; ***P<0.001 by two-way ANOVA followed by the Sidak multiple comparisons test; ****P<0.0001 by two-way ANOVA followed by the Sidak multiple comparisons test.