Fig 2: 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.

Fig 3: Akt1-mediated mtROS activates PGC-1a via phosphorylation of p38 MAPK. A, Nuclear immunoblot analysis for p-PGC-1a (S570) and PGC-1a in transfected macrophages. B, PPARGC1A promoter activity in transfected macrophages (n = 8). Inset, p-Akt1 and p-Akt2 immunoblot. C, Nuclear immunoblot analysis in transfected macrophages. D, Schematic of Akt2 phosphorylation and mutation sites on PGC-1a. E, NFE2L2 mRNA analysis in macrophages expressing empty (emp), Akt1C(A) or Akt2CA together with PGC-1aWT (WT) or PGC-1aS570A (S570A) (n = 3). Macrophages were transfected with empty or Akt1CA together with MKK6(Glu) or p38DN. F, PPARGC1A mRNA expression (n = 3), G, nuclear immunoblot analysis, H, quantification of PGC-1a expression (n = 3); inset, Akt1 immunoblot analysis, I, NRF1 mRNA expression (n = 3). J, Schematic of p38MAPK phosphorylation and mutation sites on PGC-1a. K, NFE2L2 mRNA analysis of macrophages expressing empty or Akt1CA together with PGC-1aWT (WT), PGC-1aT262A (T262A), PGC-1aS265A (S265A), PGC-1aT298A (T298A) (n = 3). L, Immunoblot analysis of BAL cells from saline or bleomycin-exposed Akt1fl/fl and Akt1-/-Lyz2-cre mice. Macrophages were transfected with scramble or PGC-1a siRNA and empty or Akt1CA. M, Mitochondrial DNA content (n = 3) and N, Tfam mRNA expression (n = 3). Inset, PGC-1a mRNA expression. O, Nuclear immunoblot analysis in macrophages expressing empty or Akt1CA and treated with vehicle or mitoTEMPO (10 µM, overnight). **P < .001; ***, P < .0001. Values shown as mean ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was utilized. CPD = Cdc4 phosphodegron motifs

Fig 4: Pulmonary fibrosis does not require Akt2. Akt2fl/fl or Akt2−/−Lyz2‐cre mice were exposed to saline or bleomycin (Bleo). BAL cells were isolated 21 days later. A, TUNEL staining (n = 5), B, TUNEL quantification (n = 4‐5), and C, caspase‐3 activity (n = 5). Scale bar represent 50 μm. mRNA analysis of D, Ppargc1a (n = 5‐6), E, Tfam (n = 5‐6), F, Cs (n = 5‐6), and G, Cox4i1 expression (n = 5‐6) in BAL cells. H, Histology of lung sections with Masson’s trichrome staining (n = 5) and I, hydroxyproline analysis of homogenized lung (n = 5‐6). ***P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized

Fig 5: Deletion of PGC-1a in monocyte-derived macrophages protects against pulmonary fibrosis. Ppargc1afl/fl or Ppargc1a-/-Csf1rMeriCreMer mice were administered tamoxifen (TAM) or regular (Reg) chow and exposed to saline or bleomycin (bleo). BAL cells were isolated 21 days later. A, mRNA analysis of PGC-1a in BAL cells (n = 5-6). B, TUNEL staining (n = 5-6), and C, TUNEL quantification (n = 5-6). Scale bar represents 50 µm. D, Tfam (n = 5), E, Cs (n = 5-6), and F, Cox4i1 mRNA expression (n = 5-6). G, mtDNA/nDNA in BAL cells (n = 4-5). H, Masson’s trichrome staining of lung sections (n = 5-6) and I, hydroxyproline analysis (n = 5). BAL cells were isolated from IPF patients by BAL. **P < .001; ***P < .0001. Values shown as mean ± S.E.M. One-way ANOVA followed by Tukey’s multiple comparison test was utilized