Fig 2: Protein levels of RDH10 and DHRS3 in hepatic membrane fractions from fed versus 6 h-fasted mice.A, western blot analysis of RDH10 and DHRS3 protein content in mitochondria (MT), microsomes (MS), and lipid droplets (LD) isolated from 3-month-old male mice (n = 3). The loading was as described under Figure 2. The blots were incubated with a mixture of RDH10 and DHRS3 antibodies diluted 1:3000 each followed by incubation with Cy5 secondary anti-rabbit antibodies (1:2500). After developing the blots, they were incubated with ADRP (perilipin 2) antibodies diluted 1:3000. The red boxes in fed liver samples denote perilipin 2 (ADRP) and DHRS3 proteins that increase in LDs upon fasting (compare to green boxes). The unchanged RDH10 bands are enclosed in black boxes. B, relative quantities of RDH10 and DHRS3 proteins in lipid droplets from fed and 6 h-fasted mice. The blots were scanned using ImageQuant and the volume is presented as the total pixel intensity. Error bars, S.D. Abbreviations are: LDhf, lipid droplet fraction from the livers of mice on high-fat diet; RP, recombinant untagged human RDH10 and DHRS3 proteins overexpressed in HEK293 cells. The middle lane contains PageRulerTM Prestained Protein Ladder (Fermentas, catalogue #SM0671); *denotes a nonspecific protein band recognized by RDH10 antibodies in mitochondrial membranes. C, assays of NAD+-dependent retinol dehydrogenase activity using membrane fractions from fed and 6 h-fasted male mice. D, QPCR analysis of gene expression. *p < 0.05; **p < 0.01.

Fig 3: Elevated uNK cell density is associated with blunted expression of the MR-dependent genes in decidualizing HESCs. A, The uNK cell densities in 21 biopsies correlated inversely to the induction of DHRS3 transcripts in primary HESCs decidualized for 4 days (left panel) or 8 days (middle panel). The right panel shows the mean (±SEM) induction of DHRS3 transcripts in biopsies deemed to have normal or elevated uNK cell counts. B, The uNK cell densities in 21 biopsies correlated inversely to the induction of RETSAT transcripts in primary HESCs decidualized for 4 days (left panel) or 8 days (middle panel). The right panel shows the mean (±SEM) induction of RETSAT transcripts in biopsies deemed to have normal or elevated uNK cell counts. *, P < .05; **, P < .01.

Fig 4: Protein levels of RDH10 and DHRS3 in hepatic membrane fractions from wild-type versus Ins2Akita mice.A, western blot analysis of RDH10 and DHRS3 protein content in mitochondria (MT), microsomes (MS), and lipid droplets (LD). Each membrane fraction was resuspended in 200 µl, the MS and MT membrane fractions (but not LD fraction) were diluted fivefold and 10 µl of each fraction was loaded onto two separate gels. The last sample set represents the average of fractions from individual mice mixed together (WT av and AKITA av). The blot was first incubated with a mixture of RDH10 and DHRS3 antibodies, exposed to film (upper panel), and then incubated with ADRP antibodies (lower panel); Asterisk (*) denotes a nonspecific band recognized by RDH10 antibodies in mitochondrial membranes. The results shown are from three individual wild-type C57BL/6J (WT) and three C57BL/6-Ins2Akita/J (heterozygous for Ins2Akita) male mice (Akita), all at 6 months of age. A representative Coomassie-stained gel and Ponceau-stained blot showing protein amount in each fraction is depicted in Fig. S5, B and C. B, quantification of RDH10 and DHRS3 proteins in hepatic membrane fractions from WT and Akita mice. The blots were scanned using UN-SCAN-IT and the data are presented as total pixel density. Abbreviations are: LDhf, lipid droplet fraction from the livers of mice on high-fat diet; RP, recombinant untagged human RDH10 and DHRS3 proteins overexpressed in HEK293 cells. *p < 0.05; ***p < 0.001. Error bars, S.D.

Fig 5: Protein levels of RDH10 and DHRS3 in hepatic membrane fractions from fed versus fasted mice.A–C, side-by-side western blot analysis of hepatic subcellular fractions from mice fasted for 16 h. Subcellular fractions were isolated from four individual 3-month-old male mice and loaded as follows: 40 µg of hepatic microsomes (A); 40 µg of mitochondria (B); and lipid droplets loaded by wet weight (10 µl out of 200 µl) (C). The blot was incubated with a mixture of RDH10 and DHRS3 antibodies diluted 1:3000 each. After developing the blots, they were incubated with the corresponding markers of each subcellular fraction: P450 reductase (P450red), antibodies diluted 1:4000; pyruvate dehydrogenase complex subunits E2 and E1a, antibodies diluted 1:5000; and ADRP (perilipin 2), antibodies diluted 1:3000. D, western blot analysis of RDH10 and DHRS3 protein content in mitochondria (MT), microsomes (MS), and lipid droplets (LD) isolated from 3-month-old male mice (n = 3) fed or fasted for 24 h. Each membrane fraction was resuspended in 200 µl, the MS and MT membrane fractions (but not LD fraction) were diluted fivefold and 10 µl of each fraction was loaded onto two separate gels. The last sample set represents the average of fractions from individual mice mixed together (FED av and FASTED av). A representative Ponceau-stained blots showing protein amount in each lane are depicted in Fig. S5A. E, quantification of RDH10 and DHRS3 proteins in hepatic membrane fractions from fed and 24 h-fasted mice. The blots were scanned using UN-SCAN-IT and the data are presented as total pixel density. *p < 0.05; **p < 0.01. Error bars, S.D. Abbreviations are: LDhf, lipid droplet fraction from the livers of mice on high-fat diet; RP, recombinant untagged human RDH10 and DHRS3 proteins overexpressed in HEK293 cells. Positions of molecular mass standards are indicated by numbers; asterisks (*) denote a nonspecific protein band recognized by RDH10 antibodies in mitochondrial membranes.