Fig 2: Mfrprd6 mice lack ADIPOR1 in their RPE layer. (a) IHC on P32 Mfrpwt and Mfrprd6 mouse eyes. Anti-ADIPOR1 staining was performed on Mfrpwt controls and Mfrprd6 mouse eyes. Anti-MFRP staining below confirms genotypes. Inset shows enlarged image near RPE layer. ADIPOR1 signal is lost in the RPE of Mfrprd6 animals. ADIPOR1/MFRP is observed via the red reaction product. Scale bar = 20 µm. (b) Western blot for ADIPOR1 using Mfrpwt and Mfrprd6 mouse eyes of age P21. α-TUBULIN was used as a loading control. Each lane represents an eye from a mouse of that genotype and eyes were loaded in pairs from individual animals (5 Mfrpwt and 5 Mfrprd6 individual animals), n = 10 eyes for both genotypes. The membrane was cut and probed separately for the analyzed protein and the loading control. Below, a zoomed in image of lanes 8–13 highlights the absence of the top ADIPOR1 band from the Mfrprd6 animals. (c) Western blot for ADIPOR1 comparing samples from a mouse eye, human stem cell derived RPE, and HEK293T cells. RPE and HEK293T ADIPOR1 runs at the same molecular weight as the top band from the mouse eye ADIPOR1 doublet. RPE cells were derived from H9 hESCs and matured for three months before analysis.

Fig 3: Reduced antagonism of CTRP5 on ADIPOR1 results in altered AMPK signaling in L-ORD-iRPE.a pAMPK levels determined by ELISA in L-ORD-iRPE (n = 15; 120.6% ± 0.08) compared to healthy-iRPE (n = 21; 100% ± 0.04), cultured in 5% serum-containing media. b Effect of recombinant globular CTRP5 (0.2 µg/mL) on pAMPK levels in the presence (+) or absence (-) of serum in healthy-iRPE (81% ± 4, n = 9) and L-ORD-iRPE (99% ± 1, n = 6), measured by ELISA. Data were normalized to the untreated condition (0 µg/mL gCTRP5), considered as 100%. c Effects of recombinant full-length CTRP5 (0.2, 2, and 25 µg/mL) on pAMPK levels in healthy-iRPE (n = 6) and L-ORD-iRPE (n = 6) incubated in serum-free medium for 5 h, measured by ELISA. d Effect of increasing cytosolic AMP with AICAR (an AMP analog, 2 mM) treatment or decreasing ATP with BAM15 (a mitochondrial uncoupler, 500 nM) on AMPK activity in healthy-iRPE and L-ORD-iRPE. All data were normalized to the 0% serum-containing untreated condition (AICAR: n = 20 for healthy-iRPE, n = 16 for L-ORD-iRPE; BAM15: n = 8 for healthy-iRPE, and n = 6 for L-ORD-iRPE). e Representative Western blot of PGC1a expression in healthy and L-ORD-iRPE. f Quantification of Western blots for PGC1a. Healthy-iRPE (n = 4). L-ORD-iRPE (n = 4). g Representative Western blot for phospho (S570)-PGC1a in L-ORD-iRPE compared to healthy-iRPE. h Quantification of Western blots for phosphor (S570)-PGC1 a in healthy-iRPE (n = 4) and L-ORD-iRPE (n = 4). i APOE expression (red) in untreated and adenine 9-beta-d-arabinofuranoside (1 µM, ara-A) treated L-ORD-iRPE. Collagen IV (green) marks RPE basal surface and nuclei (blue) are labeled with Hoechst 33342. (n = 3 images each) Scale bar: 10 µm. j ELISA detection of apical and basal VEGF secretion in untreated and adenine 9-beta-d-arabinofuranoside (1 µM, ara-A) treated L-ORD-iRPE (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant, AU arbitrary units.