Fig 1: G1 and G2 mice show evidence of podocyte loss. G0I384M/G0I384M, G1/G1 and G2/G2 mice were injected with pCpG-Muy for sustained levels of IFN-?. (A,B) Representative immunofluorescence images showing kidney tissue at week 1 and 3 post injection, immunostained for nephrin and APOL1 (Abcam) (A), and WT1 and APOL1 (Proteintech). (B). Asterisks indicate glomeruli without staining. Scale bars: 20 µM. (C) Quantification of WT1-stained cells in glomeruli of mice (as indicated) 1 and 3 weeks after injection. Each symbol represents one mouse (n=3 mice per genotype at each time point). Counts are the average number of WT1-positive cells in 35 glomeruli. **P<0.005, ****P<0.0001. At 3 weeks, G1/G1 and G2/G2 are significantly different from G0/G0, G1/G1 and G2/G2 mice at 1 week, and from G0/G0 at 3 weeks.
Fig 2: Knockdown of STING, TBK1, or IRF3 inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. AB8/13 podocytes were transfected with a non-targeting control siRNA (Co) or siRNA pool targeting STING (a–c), TBK1 (d–f), or IRF3 (g-i) for 48 h, and subsequently transfected with 1 µg ml-1 nsDNA for 18 h. Expression of indicated proteins (a,d,g) was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images in (a) were obtained from different gels. One blot was probed for IRF3 and re-probed for GAPDH. Blot images in (d) were obtained from different gels. One blot was probed with TBK1 and re-probed with GAPDH, while two other blots were probed for APOL1 and TBK1. The blot images in (g) were obtained from different gels. The blot probed for TBK1 was re-probed for GAPDH. Two other blots were probed individually for IRF3 and APOL1. Full images of the blots are shown in Supplementary Fig. S3. Expression of STING (b), TBK1 (e), IRF3 (h), and APOL1 (c,f,i) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).
Fig 3: APOLs Are Membrane-Associated Proteins Involved in Poly(I:C)-Induced Podocyte Death(A) APOL1 domains and mutations relevant for the study. SP, signal peptides (amino acids [aa] 1–27 and 28–53), PFD, pore-forming domain (aa 60–235), MAD, membrane-addressing domain (aa 238–304), hinge (aa 305–339), SRID, SRA-interacting domain (aa 340–398), HC/LZ 1/2, pairs of associated hydrophobic cluster and leucine zipper (aa 79–88/100–122 and 343–354/368–392), SID1/2, smallest interacting domains defined by Y2H interactions with the indicated bait sequences (aa 78–121 and 346–390).(B) APOLs immunodetection in total extracts from cells treated or not with poly(I:C). The arrows point to APOL3, located under a non-specific band. The bottom panel shows the immunodetection of endogenous V5- or TriFLAG-tagged APOL3 in the relevant cell lines. Actin immunodetection serves as a loading control.(C) Poly(I:C)-induced cell death in the different podocyte lines (error bars, SDs). See Figure S1 for additional data. WT, n = 11; 1KO, n = 7; 1?het, n = 7; 1?hom, n = 10; 3KO, n = 5; 1+3KO, n = 5; 2KO, n = 3; WT3V5, n = 6; 1?hom3V5, n = 6.(D) Immunodetection of recombinant APOLs (0.5 µg/mL) association with various lipids spotted on membrane strips. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1p, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine; TG, triglyceride; DAG, diacylglycerol; PG, phosphatidylglycerol; CL, cardiolipin; Chol, cholesterol; SM, sphingomyelin. Blank, no lipid.(E) Distribution of APOLs in cellular extracts from poly(I:C)-treated APOL3FLAG or APOL1?het podocytes, fractionated as indicated. Anti-FLAG antibodies were used to detect APOL3. Calnexin and Golgin97 are representative ER transmembrane and peripheral trans-Golgi membrane markers, which become soluble with, respectively, 0.1% NP-40 (N) and carbonate pH 10 (C). The loading of “s” lanes corresponds to half that of “p” lanes.(F) Distribution of APOLs in phosphatidylcholine liposomes containing or not containing 10% PI(4)P, following centrifugation at 10,000 × g. S, supernatant; p, pellet; equal amounts of initial material.
Fig 4: Deletion of MSALFL in yeast attenuates APOL1 toxicity and affects its localizationPlasmids containing the human APOL1 full-length and ?MSALFL fused to mCherry at the C-terminal, or empty vector (EV) were transformed into WT and vps38? yeast strains(A) Drop titration assay demonstrates that ?MSALFL APOL1 mutant expression is less toxic in WT and vps38? strains than full-length APOL1.(B) Immunofluorescence of APOL1-mCherry demonstrates that full-length APOL1 is expressed in the ER and vacuole; however, the ?MSALFL is expressed only in the vacuole.(C) Immunofluorescence of APOL1-mCherry in vps38? demonstrates that full-length APOL1 is diverted from the vacuole to the ER and plasma membrane while the ?MSALFL APOL1 has a homogeneous distribution in the cytoplasm and as expected was not translocated to the ER, plasma membrane, or vacuole.
Fig 5: Different ER insertion orientations accompany APOL1–APOL2 diversification. (A) The endogenous SP of the reporter enzyme SEAP (secreted alkaline phosphatase, gray box) was replaced by the N-termini of the APOL1 splice variants (vA, vB1, vB3, vC). The endogenous SP of SEAP (SPSEAP) and the well-known SP of human serum albumin (SPHSA) served as positive, and SEAP lacking its SP (?SP) as negative control. (B) SEAP secretion assay in HEK293T cells. Fluorescent SEAP secretion was detectable for positive controls (SPSEAP and SPHSA) and splice variants vA, vB1, and vC. SEAP with the N-termini of APOL1 vB3 and without the SEAP SP (?SP) were not secreted (***P < 0.0001). (C) Scheme: APOL1 splice variants vA, vB1, vB3, vC, a mutant lacking the complete APOL1 N-terminus (aa1-59; ?N59), and APOL2 were fused with an artificial C-terminal N-glycosylation tag (GT). (D) Western blot analyses of Glyco-tag (GT) APOL proteins (shown in C) before and after PNGaseF glycosidase digestion showed N-glycosylation and therefore ER luminal localization only for APOL1 vA, vB1, and vC (marked by black asterisks). (E) Scheme: C-terminally GFP-tagged APOL1 (G0 and renal risk variants G1 and G2), APOL1 lacking C-terminal aa305–398 (?C305), and APOL2. (F) Western blot analysis of these GFP-GT-tagged proteins (shown in E) before and after PNGaseF glycosidase digestion demonstrates luminal ER localization for these proteins (asterisks), except for APOL2-GFP-GT.
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