Extended Data Fig. 5: Monocultures of vascular cells in diabetic medium.

a, iPS cell-derived (NC8) endothelial cells cultured in nondiabetic conditions, in 75 mM d-glucose (hyperglycaemia), 1 ng ml⁻¹ TNF and 1 ng ml⁻¹ IL-6 or diabetic medium (hyperglycaemia, TNF and IL-6) were stained for CD31 and collagen type IV. Left, representative images. Right, quantification of collagen type IV intensity of individual endothelial cells. Data are mean ± s.d. of n = 3 independent experiments. P values were calculated using a one-way ANOVA. b, iPS cell-derived (NC8) vascular smooth muscle cells (vSMCs) were cultured in the presence of normal medium, hyperglycaemia, TNF and IL-6 or diabetic medium (hyperglycaemia, TNF and IL-6). Cultures were stained for SMA, collagen type IV and fibronectin (FN). Collagen type IV and fibronectin intensity of individual cells was quantified. Data are mean ± s.d. of n = 3 independent experiments. P values were calculated using a one-way ANOVA. c, G1S1 endothelial cells were cultured in nondiabetic normal medium and diabetic medium (hyperglycaemia, TNF and IL-6) and stained for fibronectin, laminin or collagen type IV. Phalloidin was used to visualize the actin cytoskeleton. Experiments were repeated independently n = 3 times with similar results. d, COL4A1 expression was measured by RT–qPCR from G1S1 cells cultured in nondiabetic and diabetic media for different time points, as indicated. Expression was normalized to HPRT expression. n = 3 independent experiments. P values were calculated using a two-tailed Student’s t-test. Scale bars, 100 μm (a–c).