Fig 2: APOL1 expressions in proliferating APOL1 podocytesStable podocytes were expanded at 33°C. Cells were harvested by trypsinization. Cells were stained with APOL1 antibodies. Stained cells were subjected to screening at flow cytometer (BD LSRFortessa). FACS data were analysed by FlowJo. Data are expressed as a bar graph (mean ± S.D.). *P<0.05 compared with G0.

Fig 3: Protein GRP78 expression in APOL1 podocytesStable podocytes were cultured and expanded at 33°C. Cells were differentiated at 37°C for 4 days. Differentiated podocytes were harvested and cell lysates were prepared in RIPA buffer. Electrophoresis of cell lysates was done by loading equal amounts of protein and transferred on to a membrane. Blots were probed with GRP78 antibody and blots were stripped and re-probed for GAPDH (A). Densitometric scans of Western blots (n=3) were quantified and cumulative data (mean ± S.D.) are displayed as a bar graph (B). *P<0.05 compared with G0.

Fig 4: Schematic model of the effect of mutations on APOL1 variantsThe mutation may alter either RNA or protein pathway. Schematically, it appears that at RNA axis, APOL1 variants compromise APOL1 mRNA transcription and APOL1 transcript stability. At protein axis, we speculate that APOL1 variants may produce defects in protein stability and protein (APOL1) misfolding.

Fig 5: Overexpression of APOL1 into 293T cells by plasmidAn equal amount of APOL1 plasmids (vector, APOL1 G0, APOL1 G1 and APOL1 G2) were transfected into 293T cells using Effectene Transfection Regent Kit. After 24 h, cells were harvested and cell lysates were prepared in RIPA buffer. An equal amount of protein was subjected to electrophoresis and transferred on to the PVDF membrane. Blots were probed with APOL1 antibody for detection of APOL1 protein expression and blots were stripped and re-probed for actin protein as a loading control (A). Densitometric scanning of blots is displayed as a bar graph (B). Results represent (mean ± S.D.); *P<0.05 compared with G0.