Figure 6. Effect of knockdown of 25-hydroxylases on 25OHD₃ generation.

hGF and hPDLC from donors 2, 4 and 5 were treated with vitamin D₃ at various concentrations indicated in the figure for 12 h after transfection with a siRNA oligonucleotide for CYP27A1, a siRNA oligonucleotide for CYP2R1, or a non-silencing control. 25OHD₃ production was measured in supernatants of hGF (A), supernatants of hPDLC (B), cell lysates of hGF (C), and cell lysates of hPDLC (D). When CYP27A1 or CYP2R1 was not knocked down, the production of 25OHD₃ increased with an increasing concentration of 25OHD₃. When CYP27A1 was knocked down in hGF and hPDLC, the generation of 25OHD₃ decreased significantly compared to when CYP27A1 was not knocked down. When CYP2R1 was knocked down in hGF (A, C), the generation of 25OHD₃ was not significantly different from that when CYP2R1 was not knocked down. When CYP2R1 was knocked down in hPDLC (B, D), the generation of 25OHD₃ was only slightly different at some time points from that when CYP2R1 was not knocked down. The data are presented as the mean ± SE. * hGF or hPDLC generated significantly less 25OHD₃ with the same amount of added vitamin D₃ when CYP27A1 or CYP2R1 was knocked down (p<0.05). # hGF or hPDLC generated significantly more 25OHD₃ with the same amount of added vitamin D₃ when CYP27A1 or CYP2R1 was knocked down (p<0.05).