Fig. 3.

Soluble α-Klotho (s-KL) inhibited osteogenic differentiation of human renal interstitial fibroblasts (hRIFs). HRIFs were transfected with lentivirus to overexpress or silence s-KL. A–D Alizarin Red staining (ARS) of transfected hRIF layers 14 days after osteogenic induction; n = 3. E Relative alkaline phosphatase (ALP) activity in hRIF lysates 7 days after osteogenic induction; n = 3. F–G Representative western blots and quantification of s-KL and osteogenic marker (OCN, MSX2, Runx2) expression in hRIFs 7 days after osteogenic induction; n = 3. H s-KL concentration determined by ELISA in the cell supernatants of hRIFs 7 days after osteogenic induction; n = 6. I Representative western blots of osteogenic markers in hRIFs cocultured with different concentrations of recombinant human KL (r-KL) 7 days after osteogenic induction; n = 3. J–K Representative western blots and quantification of osteogenic marker expression in Len-sh-KL-transfected hRIFs cocultured with 200 ng/ml r-KL in osteogenic medium (OM) 7 days after osteogenic induction; n = 3