Figure 8

Prepro-collagen α1 (I) and α-SMA expression are increased following mesangial cell attachment to collagen I through a Hic-5-dependant mechanism. (a) Spot density of prepro-collagen I expression following mesangial cell attachment to collagen I or collagen IV. Mesangial cell lysates were generated following 16 h of subculture on collagen I or collagen IV. Following 2D PAGE, spot density was determined by MELANIE software, and protein identity confirmed by MALDI-TOF. (b) RNA was isolated from mesangial cells and from Hic-5 siRNA knockdown cells and Hic-5 overexpressing cells grown in the same conditions as shown in (a) and first-strand cDNA prepared. Pro-collagen 1 levels were measured by real-time QPCR. Samples were run in quadruplicate (*P<0.03). (c) Wild-type mesangial cells, Hic-5 siRNA knockdown cells, and Hic-5 overexpressing cells were tested for susceptibility to apoptosis. Apoptosis was induced by serum starvation. Cells were visualized with acridine orange and morphologically apoptotic cells were counted. The proportion of cells that were apoptotic is shown, relative to the levels seen in control cells on collagen I. Significant differences from the control cells are marked as *P<0.05. (d) Lysates were generated from wild-type and Hic-5 knockdown mesangial cells subcultured onto collagen I or collagen IV for 16 h. Lysates were separated by SDS-PAGE, western blotted, and immunoprobed for α-SMA, Hic-5, and β-Actin (loading control). (e) Lysates from wild-type and Hic-5 overexpressing cells were immunoprobed in the same conditions as shown in (d).