Fig 2: FH modulates expression of mitophagy and mitochondria dynamics genes. hTERT-RPE1 cells were seeded, left to attach overnight and silenced for 24 hours with negative control (siNeg) or CFH specific (siCFH) siRNA. Cells were exposed for 90 minutes to 200 µM H2O2 or PBS and RNA was collected after 48 hours. (a) gene expression analysis by qRT-PCR of genes involved in mitophagy processes: PTEN Induced Kinase 1 (PINK1) and E3 Ubiquitin-Protein Ligase Parkin (PARKIN) SEM is shown, n = 3. (b) Gene expression analysis by qRT-PCR of genes involved in mitochondria dynamics: OPA1 Mitochondrial Dynamin Like GTPase (OPA1) and Dynamin-Related Protein 1 (DRP1). SEM is shown, n = 3. Data are normalized to housekeeping gene PRPL0 using ? ?Ct method. Significance was assessed by Student’s t-test *p < 0.05, **p < 0.01.

Fig 3: FH reduction leads to extracellular C3/C3b accumulation. hTERT-RPE1 cells were seeded, left to attach overnight and silenced for 24 hours with negative control (siNeg) or CFH specific (siCFH) siRNA. Cells were exposed for 90 minutes to 200 µM H2O2 or PBS and cell pellets and cell culture supernatants were collected for further processing after 48 hours. (a) Monitoring of CFH expression by qRT-PCR analyses in silencing negative control (siNeg) and specific CFH silenced (siCFH) hTERT-RPE1 cells. Data are normalized to the housekeeping gene PRPL0 using ? ?Ct methods. SEM is shown, n = 3. (b) Western blot analyses of FH protein levels in cell culture supernatants of hTERT-RPE1 in the same conditions as (a). Quantification of signal density of 4 independent experiments is shown. (c) Monitoring of C3 expression by qRT-PCR analyses in silencing negative control (siNeg) and specific CFH silenced (siCFH) hTERT-RPE1 cells. Data are normalized to housekeeping gene PRPL0 using ? ?Ct method. SEM is shown, n = 4. (d) Western blot analyses of C3 a-chain and ß-chain protein levels in cell culture supernatants of hTERT-RPE1 cells. Quantification of signal density of 3 independent experiments is shown. (e) C3/C3b ELISA analyses of cell culture supernatants of hTERT-RPE1 cells. SEM is shown, n = 4. Western Blot images were cropped, and full-length blots are presented in Supplementary Fig. S5. Significance was assessed by Student’s t-test. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001.

Fig 4: FH loss impairs mitochondrial respiration in RPE cells. (a) Schematic representation of oxidative phosphorylation chain and targeted steps during Seahorse analyses (1,2,3) (b) hTERT-RPE1 cells were seeded, let attach overnight and silenced for 24 hours with negative control (siNeg) or CFH specific (siCFH) siRNA. 30.000 cells were transferred to seahorse plates overnight and pre-treated for 90 minutes with 200 µM H2O2 or PBS. Curves show oxygen consumption rate (OCR) measured after 48 hours in hTERT-RPE1. SEM is shown, n = 4–8. Arrows indicate injection of oligomycin (1), FCCP (2) and antimycin and rotenone (3). (c–e) Parameters of mitochondrial function are calculated from data shown in (b) and are expressed as total values for the 3 measurements (15 minutes). Basal respiration (c), maximal respiration (d) and reserve respiratory capacity (e). Significance was assessed by Student’s t-test (single effect) and two-way ANOVA (combined effects) as described in the methods section *p < 0.05.

Fig 5: FH modulates glucose uptake and expression of glucose metabolism genes. hTERT-RPE1 cells were seeded, let attach overnight and silenced for 24 hours with negative control (siNeg) or CFH specific (siCFH) siRNA. Cells were exposed for 90 minutes to 200 µM H2O2 or PBS. (a) Glucose uptake was measured 48 hours after H2O2 pre-treatment in siNeg control cells and in siCFH cells. SEM is shown, n = 3. (b) gene expression analysis by qRT-PCR of glucose transporter 1 (GLUT1/SLC2A1) and glycolysis enzyme gene lactate dehydrogenase A (LDHA). SEM is shown, n = 3. Data are normalized to housekeeping gene PRPL0 using ? ?Ct method. Significance was assessed by Student’s t-test (single effect) and two-way ANOVA (combined effects) as described in the methods section *p < 0.05.