Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 1. APOL1 is a lipid droplet-associated protein that can alter lipid droplet number and size. (A and B) Fluorescence micrographs of human primary podocytes transiently transfected with untagged APOL1 (G0, G1, or G2) (A) or with RFP-tagged APOL1 (B). Lipid droplets were labeled with BODIPY 493/503 (green) and nuclei with DAPI. (Scale bar, 10 μm.) (C and D) Percentage of human primary podocytes containing APOL1-positive LDs (C) (as in A) or APOL1-RFP–positive LDs (D) (as in B). Data from each group represent >50 podocytes from three independent experiments. Data are presented as means ± SD. ***P < 0.001; ns, non- significant. (E) Number of LDs per cell in APOL1-RFP–expressing human primary podocytes. Data from three independent experiments, each with at least 30 cells per group, are presented as means ± SD. ***P < 0.001; ns, nonsignificant. (F) Cross-sectional area (number of pixels) of human primary podocytes transfected with APOL1-RFP, quantified from pooled cells from three separate experiments for each group with untransfected (UT) n = 545, G0 n = 131, G1 n = 48, and G2 n = 75 presented as means ± SD. *P < 0.05, ***P < 0.001. (G) Distribution of lipid droplet diameters in untransfected human primary podocytes or podocytes transfected with APOL1-RFP (G0, G1, G2). (H) Indirect immunofluorescence of human primary podocytes transfected with APOL1-FLAG (G0-FLAG and G2-FLAG) and labeled with BODIPY 493/503 and DAPI. (Scale bar, 10 μm.) (I) Immunoblot analysis of purified APOL1-FLAG; 1% Nonidet P-40 lysates from human primary podocytes transfected 16 to 18 h previously with Apol1-FLAG (G0, G2) or empty vector were incubated with anti-FLAG agarose and eluted with 3× FLAG peptide. (J) APOL1-FLAG solid-phase lipid binding. Affinity-purified APOL1-FLAG (G0, G2) or 3× FLAG peptide (negative control) were used in a lipid overlay assay. APOL1- FLAG (G0) bound strongly to PI(4)P, PI(4,5)P2, and PI(3,4,5)P3 and less strongly to PPA and CL. APOL1-FLAG (G2) bound more strongly to PS and CL than to PI(4,5)P2 or PI(3,4,5)P3. One of two experiments with identical results performed with one of two immunopurified preparations of recombinant APOL1. 3-SGC, 3-sulfogalactosylceramide; Blank, solvent blank; Chol, cholesterol; CL, cardiolipin; DAG, diacylglycerol; PPA, phosphatidic acid; PC, phosphati- dylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI(4)P, phosphatidylinositol (4)-phosphate; PI(4,5)P2, phosphatidylinositol (4,5)-bisphos- phate; PI, phosphatidylinositol; PI(3,4,5)P3, phosphatidylinositol (3–5)-trisphosphate; PS, phosphatidylserine; SPH, sphingomyelin; TG, triglyceride.

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Fluorescence, Transfection, Labeling, Expressing, Immunofluorescence, Western Blot, Purification, Plasmid Preparation, Incubation, Binding Assay, Affinity Purification, Negative Control, Overlay Assay, Recombinant, Solvent

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 2. Differential localization of wild-type (G0) APOL1 to LDs and its risk variants (G1, G2) primarily to the ER. (A) SR-SIM micrographs of representative primary human podocytes transiently transfected with APOL1-RFP, labeled for perilipin-2 (light blue), and with BODIPY 493/503 and DAPI (blue). [Scale bars, 10 μm and 2 μm (Insets).] (B) SR-SIM micrographs of primary human podocytes transiently transfected with APOL1-GFP, labeled for mitochondria (MitoTracker in red), ER (calnexin in magenta), and DAPI (blue). [Scale bars, 10 μm and 2 μm (Insets).] (C) Colocalization analysis of APOL1-GFP with MitoTracker Red (mitochondria) or calnexin (endoplasmic reticulum). Data are presented as means ± SD. *P < 0.05. (D and E) Yellow arrow indicating colocalization of APOL1- RFP with GFP-Rab7. Airyscan micrographs of live primary human podocytes coexpressing APOL1-RFP (G0) with GFP-Rab7 and mito-BFP (D) or with GFP- PLIN2 and BFP-KDEL (E). [Scale bars, 10 μm and 2 μm (Insets).] See SI Appendix, Fig. S3 for time frame still images. See also Movies S1 and S2.

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Transfection, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 3. Treatment with oleic acid can enrich APOL1(G0)-RFP on LDs and redistribute risk variants G1 and G2 from a reticular pattern to LDs. SR-SIM mi- crographs of representative human primary podocytes transiently transfected with APOL1-GFP and treated with vehicle (fatty acid-free BSA) (A) or oleic acid (0.8 mM) (B), and then labeled with BODIPY 665/676 and DAPI. (Scale bars, 10 μm.)

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Transfection, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 4. Treatment of T-Rex-293 cells with oleic acid reduces cellular toxicity and autophagic flux. (A) Bright-field phase-contrast micrographs of represen- tative APOL1 T-Rex-293 cells stably expressing empty vector (EV), G0, G1, or G2 pretreated with vehicle (fatty acid-free BSA), 1 mM oleic acid, or 1 mM palmitic acid for 3 h and then induced for 18.5 h with tetracycline (10 ng/mL) to express APOL1. (Scale bar, 50 μm.) (B) Quantitation of T-Rex-293 cells stably expressing an empty vector or APOL1 variants G0, G1, or G2 treated with BSA, 1 mM OA, or 1 mM PA and induced to express APOL1 with tetracycline (10 ng/mL) for 16 h. ***P < 0.001. (C) Cell cytotoxicity/viability after 22-h treatment with tetracycline (10 ng/mL). Data from two independent experiments, each with eight experimental replicates, are presented as means ± SD. ***P < 0.001; ns, nonsignificant. (D) Immunoblot of APOL1-FLAG, LC3, and vinculin from whole-cell lysates prepared after 22-h induction with tetracycline (10 ng/mL) and treatment with 1 mM OA or 1 mM PA. (E) Quantitation of band intensities by den- sitometry from two independent experiments for LC3I and LC3II mean densities (from C) displayed as a ratio (means ± SD).

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 5. Coexpression of APOL1 (G0) with its risk variants (G1 or G2) promotes G1/G2 accumulation on LDs and reduces cell death. (A) Diagram illustrating predicted localization of APOL1-RFP (red) or APOL1-GFP (green) variants to the LD or ER. (B) Immunoblot of whole-cell lysates from human primary podocytes transiently cotransfected with the indicated APOL1-RFP and APOL1-GFP variant DNAs in equal amounts. (C) Airyscan micrographs of human primary podocytes cotransfected with the indicated variants of APOL1-RFP (red), APOL1-GFP (green), and BODIPY 665/676 (blue). Merged images in magenta when overlapped with APOL1-RFP, APOL1-GFP, and BODIPY 665/676. (Scale bar, 10 μm.) (D) Quantitation of human primary podocytes containing detectable LDs positive for APOL1-RFP (G0, G1, G2) when cotransfected with the indicated APOL1-GFP variants. Data from three independent experiments per group, each with ≥50 cells analyzed, are presented as means ± SD. ***P < 0.001; ns, nonsignificant. (E) Number of LDs per cell in human primary podocytes cotransfected with APOL1-RFP and APOL1-GFP. Data presented from at least 25 cells per group are presented as means ± SD. ***P < 0.001; ns, nonsignificant. (F) Cross-sectional area of human primary podocytes cotransfected with APOL1-RFP and APOL1-GFP; quantified for each group, G0-RFP/G0-GFP n = 56, G0-RFP/G2-GFP n = 46, and G2-RFP/G2-GFP n = 65, presented as means ± SD. *P < 0.05, ***P < 0.001. (G) Quantitation from bright-field images using five fields from two representative experiments of tetracycline-inducible transgenic APOL1 T-Rex-293 cells stably expressing empty vector (C-terminal MYC and FLAG tags), G0 (APOL1-G0-T-Rex-293), G1 (APOL1-G1-T-Rex-293), or G2 (APOL1-G2-T-Rex-293) cells transfected with plasmids encoding RFP (control plasmid), G0-RFP (G0), or G2-RFP (G2) 4 h before induction of tetracycline (10 ng/mL)- inducible APOL1 for 16 h. **P < 0.01, ***P < 0.001. (H) Immunoblot of APOL1 T-Rex-293 whole-cell lysates after transient transfection with APOL1-FLAG (G0, G2) or empty vector 4 h before tetracycline (10 ng/mL) induction for 18 h and cell collection at 22 h. (I) Quantitation of cytotoxicity/viability of APOL1 T-Rex-293 cells transiently transfected with APOL1-FLAG (G0, G2) or empty vector for 4 h before tetracycline induction for 18 h. Data from three independent experiments, each with eight experimental replicates, are presented as means ± SD. ***P < 0.001, *P < 0.05; ns, nonsignificant.

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Western Blot, Variant Assay, Quantitation Assay, Transgenic Assay, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

doi: 10.1073/pnas.1820414116

Figure Lengend Snippet: Fig. 6. Model for APOL1-mediated cytotoxicity and its reduction by ma- neuvers promoting LD formation and enlargement. Under normal physio- logic conditions, podocytes express low levels of APOL1 and do not cause cytotoxicity. Innate immune activation by environmental stress or viral in- fection are thought to up-regulate APOL1. Accumulating levels of G1 and G2 may reach a threshold level causing burden on organelles such as the ER and mitochondria, possibly via communication via mitochondrial-associated membranes. Excess APOL1 will activate cell stress pathways including stress- activated protein kinases, ER stress pathways, and mitochondrial oxidation pathways. LD formation might (partially or totally) relieve stress pathways caused by excessive APOL1 risk variant accumulation in the ER. 1: For G0, APOL1 can be efficiently diverted away from the ER to the LD (a relatively inert reservoir for lipophilic proteins) and the podocyte can process APOL1 to levels below a threshold causing toxicity. APOL1 directed to lipid droplets can interact with other organelles, including endosomes and mitochondria, which may aid the processing, transport, and metabolism of APOL1. 2: Coover- expression of wild-type APOL1 can recruit APOL1 risk variants to lipid drop- lets. 3: Treatment with OA can increase LD size and number, acting as shuttles to sequester toxic APOL1 risk variant polypeptides away from the ER.

Article Snippet: APOL1 cDNA was purchased from OriGene.

Techniques: Activation Assay, Variant Assay, Expressing

Journal: Scientific Reports

Article Title: A translational protocol optimizes the isolation of plasma-derived extracellular vesicle proteomics

doi: 10.1038/s41598-025-08366-8

Figure Lengend Snippet: Size exclusion chromatography using Sepharose CL-4B shows high EV enrichment. ( a ) A set of three peripheral blood samples was each collected in n =4 biological replicates in Streck Cell-Free DNA BCT. In all cases, platelet-poor plasma (PPP) was first processed by density gradient, followed by SEC. After SEC, the EV-enriched fractions were concentrated by ultrafiltration. EV-enriched fractions were analyzed by nanoparticle tracking analysis (NTA), bicinchoninic acid assay (BCA) and western blot (WB). ( b ) Total particle recovery, ( c ) median particle size and ( d ) total amount of protein in EV-enriched fractions after size exclusion chromatography using different SEC resins, measured by ( b ), ( c ) NTA or ( d ) BCA. In ( b )-( d ) symbols signify independent biological replicates. In ( b ) and ( d ) mean of values normalized to Izon columns, is displayed, in ( c ) mean values are displayed for each SEC column. In ( b ) statistical significance is assessed using one sample t test (Bonferroni-Holm-adjusted p-values) with * p =0.0302 (Izon vs. Sepharose) and p =0.1745 (Izon vs. Sephacryl). In ( d ) statistical significance is assessed using one sample t test with * p =0.0182 (Izon vs. Sepharose) and * p =0.0152 (Izon vs. Sephacryl). ( e ) Representative images of standards run through each column to pre-assess their elution profiles. Dextran Blue (2 g/L), high molecular weight polymer, was used to predict the void volume and locate the EV-enriched peak. Thyroglobulin (2 g/L) and bovine serum albumin (BSA, 4 g/L), were used as representatives of a high and low molecular weight proteins, respectively. The signal corresponding to dextran blue was measured by absorbance at 595 nm. For protein quantification Bradford reagent was used and absorbance was measured at 595 nm. ( f ) Representative immunoblotting of 1mL fractions collected from each size exclusion chromatography to asses elution profiles using plasma as starting material. CD9 was selected as EV marker, and ApoB and ApoA1, as lipoprotein markers. Original blots are presented in Supplementary Fig. S12 online. ( g ) CD9/ApoA1 ratio calculated based on immunobloting signal quantification ( n =2 independent biological replicates). Mean values are displayed.

Article Snippet: The following primary antibodies were used for immunoblotting: CD9 (Cell Signalling Technology, #13403, 1:1,000), GAPDH (Proteintech, #60004-1, 1:1,000), ALIX (abcam, ab275377, 1:1,000), ApoA1 (R&D Systems, MAB36641, 1:2,500), ApoB (R&D Systems, MAB41242, 1:1,000), CD235a (Invitrogen, JC159, 1:500), CD41 (Integrin α−IIb, abcam, ab83961, 1:1,000), Hsc70 (abcam, ab51052, 1:2,500).

Techniques: Size-exclusion Chromatography, Clinical Proteomics, Acid Assay, Western Blot, High Molecular Weight, Polymer, Molecular Weight, Marker

Journal: Journal of the American Society of Nephrology

Article Title: Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

doi: 10.1681/asn.2019080829

Figure Lengend Snippet: Figure 1. APOLs 1, 2, and 6 are expressed in kidney podocytes. (A) RT-PCR of five normal human kidneys with probes specific for each APOL family member. Data are normalized to RPL19 and the mean and SD of the five kidneys are plotted over the individual data points (each kidney is assigned a unique symbol and the mean of the relative expression [22dCT] of up to three in- dependent RT-PCRs is plotted for each sample). See Supplemental Figure 1A for APOL probe specificities. (B) RT-PCR of immortalized differentiated (Diff, blue, grown at 38°C) or undifferentiated (Undiff, red, grown at 33°C) human podocytes (mean6SD of three separate passages, each analyzed in duplicate) using the same probes as in (A). The variability is likely attributable to our observation that APOL1 levels decline during passaging. Open triangles, untreated; closed triangles, IFNg treated (100 ng/ml for 24 hours). APOL3 became detectable after IFNg treatment in undifferentiated, but

Article Snippet: Unfortunately, all three commercial rabbit antibodies used in previous reports8,21,22,25,26 do crossreact with APOL2 (Figure 2, A and B), the Proteintech polyclonal additionally recognizing APOL3 (and weakly APOL4, not shown) by IF.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Passaging