Fig. 3

The regulation of AKR1C1 on hASCs differentiation in vitro depended on its enzyme activity. a ALP staining indicated that overexpression of AKR1C1 by wild type plasmid (WT) in the AKR1C1 knockdown cells (shAKR1C1-1) downregulated the ALP activity whereas mutant plasmids (E127D, H222I and R304L) had no significant influence. The result of ALP quantification was consistent with the result of ALP staining. b The results of ARS staining and quantification were consistent with the results of ALP staining and quantification. c Overexpression of AKR1C1 by wild type plasmid (WT) in the AKR1C1 knockdown cells (shAKR1C1-1) downregulated the mRNA expression of RUNX2 and BGLAP whereas mutant plasmids (E127D, H222I and R304L) made no significant difference. d Western blot showed that transfection of wild type plasmid and mutant plasmids upregulated the protein expression of AKR1C1. The protein expression of RUNX2 was inhibited by transfection of wildtype plasmid but not mutant plasmids. e Oil red O staining revealed that in the AKR1C1 knockdown cells (shAKR1C1-1), more lipid droplet formed in WT group, whereas mutant plasmids (E127D, H222I and R304L) made no significant difference. f Overexpression of AKR1C1 by wild type plasmid (WT) in the AKR1C1 knockdown cells (shAKR1C1-1) upregulated the mRNA expression of PPARγ and CEBPα whereas mutant plasmids (E127D, H222I, and R304L) had no significant influence. g Western blot showed that transfection of wild type plasmid and mutant plasmids upregulated the protein expression of AKR1C1. The protein expression of PPARγ was promoted by transfection of wild type plasmid but not mutant plasmids. **p < 0.01, ***p < 0.001