Collagen IV expression in VIC. (A). Representative Western blots depicting collagen IV protein expression in VIC exposed for 2, 7, 14, and 21 days to normal glucose (NM), high glucose (HG) concentrations, medium containing osteogenic factors (OM), and medium containing HG and osteogenic factors (HGOM). Below, is the quantification of the level of collagen IV protein expression as reported to β-actin protein level at different intervals and normalized to the day 2 values. Results represent the means ± S.D. of three independent experiments performed in duplicate (n = 6). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus values determined on day 2; #p < 0.05, ##p < 0.01 versus values determined on day 7; & p < 0.05 versus values determined on day 14. (B). Representative fluorescence images indicating the presence of collagen IV (red) on 2D support-cultured VIC and grown in NM or HGOM for 2, 7, and 14 days. Nuclei are stained with DAPI (blue). Scale bar: 100 μm. The mean fluorescence of collagen IV was quantified on fluorescence microscopy images (ImageJ software version 1.8.0) and the graph is shown below the images. Results are presented as means ± S.D. of two independent experiments performed in triplicate, three fields per sample. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C, left). Representative fluorescence and phase contrast images disclosing the presence of collagen IV (red) in VIC seeded in 3D hydrogel and grown in NM or HGOM for 7 days. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. (C, right). Quantification of the mean fluorescence of collagen IV on fluorescence microscopy images (ImageJ software version 1.8.0). Results are the means ± S.D. of five fields per sample (n = 3). ∗∗∗p < 0.001 versus values determined on VIC incubated with NM medium.