Fig. 3: Reduced antagonism of CTRP5 on ADIPOR1 results in altered AMPK signaling in L-ORD-iRPE.
From: AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration
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 PGC1α expression in healthy and L-ORD-iRPE. f Quantification of Western blots for PGC1α. Healthy-iRPE (n = 4). L-ORD-iRPE (n = 4). g Representative Western blot for phospho (S570)-PGC1α in L-ORD-iRPE compared to healthy-iRPE. h Quantification of Western blots for phosphor (S570)-PGC1 α 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.
