Fig 2: Procollagen production after ascorbate induction.Plotting the ng of procollagen in 35 µl of conditioned medium (10 ml total, 24 h incubation period) in cultures induced with ascorbate in different growth conditions over the course of 4 days (days 17–20 from the time of initial seeding of the cells). All the flasks contained 10 ml F12/DMEM + 0.2% FBS + 50 µg/ml ascorbate and then different amounts of SNZR PL, 0x (?), 3x (?), 5x (?), and 10x (?). The gels, an example on day 20 is shown in Fig. 5, were quantitatively analyzed using software that came with the imager (Azure 600; Azure Biosystems, Dublin, CA, USA; Table 1). The intensity of the fluorescence was converted to ng using a standard curve using RNase A as the standard (Fig. S2). All the points cluster around 25 ng on day 17. This is due in part because ascorbate had been given 24 h earlier and full induction requires 48 h. For 10x, only days 17 and 20 had sufficient levels to quantitate. The shape of the curves is less important for this analysis than the fact that the average level for three conditions (0x, 3x, and 5x) over 3 days (18, 19, 20) is 42 ± 12, a high level. The minus ascorbate levels would be six fold lower, a moderate levels, and be just detectable visually. A maintenance levels at 1% would be undetectable using this assay.