Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus.

doi: 10.1016/j.isci.2023.106830

Figure Lengend Snippet: Figure 1. Expression of APOL1 in the kidney and association of APOL1 risk genotypes with EC activation (A) Mean expression and fraction of cells expressing APOL1 in the single-cell transcriptomics dataset from 43 donors (healthy or with AKI or CKD) within the Kidney Precision Medicine Project (KPMP). (B) Volcano plot representing APOL1 differential expression between healthy individuals and those with AKI in peritubular capillary endothelial cells (adjusted p value = 0.05 represented by dotted line, APOL1 in red). (C) Heatmaps showing differential gene expression (log2 fold change) of EC markers from glomerulus (left heatmap) and tubulointerstium (right heatmap) kidney biopsies. Each heatmap shows expression profiles from individuals with nephrotic syndrome and either low-risk or high-risk APOL1 genotypes (see bottom panel).

Article Snippet: Caspase-Glo 8 Promega G8200 Caspase-Glo 3/7 assay system Promega G8090 XTT cell viability assay Sigma-Aldrich 11465015001 Taqman gene expression assay for ICAM1 ThermoFisher Hs00164932_m1 Taqman gene expression assay for PECAM1 ThermoFisher Hs01065279_m1 Taqman gene expression assay for HPRT ThermoFisher Hs02800695_m1 Taqman gene expression assay for CCL2 ThermoFisher Hs00234140_m1 Taqman gene expression assay for CCL5 ThermoFisher Hs00982282_m1 Taqman gene expression assay for NOS3 ThermoFisher Hs01574665_m1 Taqman gene expression assay for vWF ThermoFisher Hs01109446_m1 Taqman gene expression assay for APOL1 ThermoFisher Hs01066280_m1 Pierce BCA Protein Assay Kit ThermoFisher 23225 LysoTracker Deep Red ThermoFisher L12492 CD31 Micro-Bead Kit Miltenyi Biotec 130-091-935 CellTracker Red CMTPX Dye Invitrogen C34552 Incucyte Annexin V Dye for Apoptosis Sartorius 4641 STEMdiff Endothelial Differentiation Kit Stem cell Technologies 08005 STEMdiff Mesoderm Induction Medium Stem cell Technologies 05220 STEMdiff endothelial expansion medium Stem cell Technologies 08007 Complete classic medium with 10% serum and CultureBoost Cell-systems 4Z0-500

Techniques: Expressing, Activation Assay, Single-cell Transcriptomics, Clinical Proteomics, Quantitative Proteomics, Gene Expression

Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus.

doi: 10.1016/j.isci.2023.106830

Figure Lengend Snippet: Figure 2. In vitro expression of APOL1 iPSC-derived ECs promotes EC activation (A) Schematic representation of the EC differentiation from iPSCs, and representative histograms of the membrane expression of PECAM1 measured by flow cytometry at selected time points. (B) Gene expression analysis of key EC markers after APOL1 induction with 1uM doxycycline for 24h. (C) Membrane protein expression quantification of the adhesion molecule ICAM1 and representative histograms after 24h doxycycline exposure. (D) Membrane protein expression quantification of the endothelial structural protein PECAM1 after 24h exposure to doxycycline (1uM).

Article Snippet: Caspase-Glo 8 Promega G8200 Caspase-Glo 3/7 assay system Promega G8090 XTT cell viability assay Sigma-Aldrich 11465015001 Taqman gene expression assay for ICAM1 ThermoFisher Hs00164932_m1 Taqman gene expression assay for PECAM1 ThermoFisher Hs01065279_m1 Taqman gene expression assay for HPRT ThermoFisher Hs02800695_m1 Taqman gene expression assay for CCL2 ThermoFisher Hs00234140_m1 Taqman gene expression assay for CCL5 ThermoFisher Hs00982282_m1 Taqman gene expression assay for NOS3 ThermoFisher Hs01574665_m1 Taqman gene expression assay for vWF ThermoFisher Hs01109446_m1 Taqman gene expression assay for APOL1 ThermoFisher Hs01066280_m1 Pierce BCA Protein Assay Kit ThermoFisher 23225 LysoTracker Deep Red ThermoFisher L12492 CD31 Micro-Bead Kit Miltenyi Biotec 130-091-935 CellTracker Red CMTPX Dye Invitrogen C34552 Incucyte Annexin V Dye for Apoptosis Sartorius 4641 STEMdiff Endothelial Differentiation Kit Stem cell Technologies 08005 STEMdiff Mesoderm Induction Medium Stem cell Technologies 05220 STEMdiff endothelial expansion medium Stem cell Technologies 08007 Complete classic medium with 10% serum and CultureBoost Cell-systems 4Z0-500

Techniques: In Vitro, Expressing, Derivative Assay, Activation Assay, Membrane, Cytometry, Gene Expression

Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus.

doi: 10.1016/j.isci.2023.106830

Figure Lengend Snippet: Figure 3. Changes in cellular complexity in APOL1-expressing ECs (A) Cell morphology quantification by immunofluorescence of ECs. (B) Effect of APOL1 induction with doxycycline (1uM) for 24h in the forward scatter (FSC) and the side scatter (SSC) normalized against untreated control cells, with representative histograms. (C) Nuclear size quantification by immunofluorescence of ECs. (D) Quantification in live cells of acidic organelles by flow cytometry. (E) Mitophagy assessment in cells pre-loaded with a mitophagy dye for 30min followed by APOL1 induction for 24h, and the fluorescence intensity of punctate quantified. *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Data presented as fold change to non-doxycycline-treated cells. Each dot represents an independent experiment.

Article Snippet: Caspase-Glo 8 Promega G8200 Caspase-Glo 3/7 assay system Promega G8090 XTT cell viability assay Sigma-Aldrich 11465015001 Taqman gene expression assay for ICAM1 ThermoFisher Hs00164932_m1 Taqman gene expression assay for PECAM1 ThermoFisher Hs01065279_m1 Taqman gene expression assay for HPRT ThermoFisher Hs02800695_m1 Taqman gene expression assay for CCL2 ThermoFisher Hs00234140_m1 Taqman gene expression assay for CCL5 ThermoFisher Hs00982282_m1 Taqman gene expression assay for NOS3 ThermoFisher Hs01574665_m1 Taqman gene expression assay for vWF ThermoFisher Hs01109446_m1 Taqman gene expression assay for APOL1 ThermoFisher Hs01066280_m1 Pierce BCA Protein Assay Kit ThermoFisher 23225 LysoTracker Deep Red ThermoFisher L12492 CD31 Micro-Bead Kit Miltenyi Biotec 130-091-935 CellTracker Red CMTPX Dye Invitrogen C34552 Incucyte Annexin V Dye for Apoptosis Sartorius 4641 STEMdiff Endothelial Differentiation Kit Stem cell Technologies 08005 STEMdiff Mesoderm Induction Medium Stem cell Technologies 05220 STEMdiff endothelial expansion medium Stem cell Technologies 08007 Complete classic medium with 10% serum and CultureBoost Cell-systems 4Z0-500

Techniques: Expressing, Control, Cytometry

Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus.

doi: 10.1016/j.isci.2023.106830

Figure Lengend Snippet: Figure 4. APOL1 RV expression promotes primary glomerular EC activation (A) Gene expression analysis of key EC markers 24h after electroporation with green fluorescent protein (GFP) plasmid (C), G0 expressing plasmid with a GFP reporter (G0), or G1 expressing plasmid with a GFP reporter (G1). (B) PECAM1 protein expression quantification 24h after electroporation and representative images. (C) Macrophage adhesion quantification and representative images showing attached macrophages labeled with a cell mask (blue) helping in segmenting the macrophages and allowing quantification (data representative of three independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Each dot in the graph represents an independent experiment.

Article Snippet: Caspase-Glo 8 Promega G8200 Caspase-Glo 3/7 assay system Promega G8090 XTT cell viability assay Sigma-Aldrich 11465015001 Taqman gene expression assay for ICAM1 ThermoFisher Hs00164932_m1 Taqman gene expression assay for PECAM1 ThermoFisher Hs01065279_m1 Taqman gene expression assay for HPRT ThermoFisher Hs02800695_m1 Taqman gene expression assay for CCL2 ThermoFisher Hs00234140_m1 Taqman gene expression assay for CCL5 ThermoFisher Hs00982282_m1 Taqman gene expression assay for NOS3 ThermoFisher Hs01574665_m1 Taqman gene expression assay for vWF ThermoFisher Hs01109446_m1 Taqman gene expression assay for APOL1 ThermoFisher Hs01066280_m1 Pierce BCA Protein Assay Kit ThermoFisher 23225 LysoTracker Deep Red ThermoFisher L12492 CD31 Micro-Bead Kit Miltenyi Biotec 130-091-935 CellTracker Red CMTPX Dye Invitrogen C34552 Incucyte Annexin V Dye for Apoptosis Sartorius 4641 STEMdiff Endothelial Differentiation Kit Stem cell Technologies 08005 STEMdiff Mesoderm Induction Medium Stem cell Technologies 05220 STEMdiff endothelial expansion medium Stem cell Technologies 08007 Complete classic medium with 10% serum and CultureBoost Cell-systems 4Z0-500

Techniques: Expressing, Activation Assay, Gene Expression, Electroporation, Plasmid Preparation, Labeling

Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus.

doi: 10.1016/j.isci.2023.106830

Figure Lengend Snippet: Figure 5. APOL1 RV expression promotes apoptotic pathways in primary glomerular ECs (A) Cell death in HGECs 24 or 48 h after electroporation, quantified as the percentage GFP cells positive for annexin V staining. (B) Caspase 3/7 and caspase 8 quantification by luminescence 24h after electroporation. (C) TNFR1 protein expression quantification 24h after electroporation. (D) Quantification in live cells of acidic organelles by measuring the spot area of punctate 24h after electroporation. *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Each dot in the graph represents an independent experiment.

Article Snippet: Caspase-Glo 8 Promega G8200 Caspase-Glo 3/7 assay system Promega G8090 XTT cell viability assay Sigma-Aldrich 11465015001 Taqman gene expression assay for ICAM1 ThermoFisher Hs00164932_m1 Taqman gene expression assay for PECAM1 ThermoFisher Hs01065279_m1 Taqman gene expression assay for HPRT ThermoFisher Hs02800695_m1 Taqman gene expression assay for CCL2 ThermoFisher Hs00234140_m1 Taqman gene expression assay for CCL5 ThermoFisher Hs00982282_m1 Taqman gene expression assay for NOS3 ThermoFisher Hs01574665_m1 Taqman gene expression assay for vWF ThermoFisher Hs01109446_m1 Taqman gene expression assay for APOL1 ThermoFisher Hs01066280_m1 Pierce BCA Protein Assay Kit ThermoFisher 23225 LysoTracker Deep Red ThermoFisher L12492 CD31 Micro-Bead Kit Miltenyi Biotec 130-091-935 CellTracker Red CMTPX Dye Invitrogen C34552 Incucyte Annexin V Dye for Apoptosis Sartorius 4641 STEMdiff Endothelial Differentiation Kit Stem cell Technologies 08005 STEMdiff Mesoderm Induction Medium Stem cell Technologies 05220 STEMdiff endothelial expansion medium Stem cell Technologies 08007 Complete classic medium with 10% serum and CultureBoost Cell-systems 4Z0-500

Techniques: Expressing, Electroporation, Staining