Fig 2: APOL1 traffics to the PM prior to Ca2+ influx.(a) Confocal images of transfected and permeabilized CHO cells depict RUSH-APOL1 (red) localized to the ER (stained via calnexin, green) without biotin followed by partial PM localization after 90 min of biotin treatment. Representative cells from n = 3 independent experiments. (b) RUSH-APOL1 localizes to and forms punctae at the PM within 90 min of biotin treatment. CHO cells were treated and imaged as in (a) except without permeabilization. Here anti-calnexin (green) was used as a control for cell permeabilization (depicted in the merged images, no permeabilization was detected). Representative cells from n = 4 independent experiments. (c) High-throughput confocal microscopy reveals that RUSH-APOL1 begins localizing to the PM within 60–90 min. Cells were randomly imaged at 20x, capturing =10 fields of view per well from 3 replicate wells for each condition. Calnexin signal was used to filter out permeabilized cells. Each dot represents a single cell (n = 462,918 cells analyzed). (d–e) RUSH-APOL1 localization to the PM steadily increases until 90 min post release from the ER. (d) The mean intensity of all cells in (c) was normalized to the respective no biotin controls. (e) The percentage of cells expressing RUSH-APOL1 at the PM was determined using a threshold set by untransfected wells, and then normalized to the respective no biotin controls. For analysis of (d) and (e), a generalized linear model was used to make pairwise comparisons between all samples. Comparisons were performed between biotin treated and untreated cells within each respective genotype. All data are represented as mean ± s.d. (a–b) Scale bars = 10 µm.

Fig 3: A, Expression of TP63, RAB24, APOL1, and PTK6 in tissues by western blotting (n = 4 for each group). B, Relative values of TP63, RAB24, APOL1, and PTK6 in the tissues mentioned in (A). C-F, Prognostic values of dysregulated TP63, RAB24, APOL1, and PTK6 in cancer tissues, as analyzed by GEPIA. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: Comparison of helix boundaries among the determined ApoL1 and ApoL2 structures.ApoL1-NTD and ApoL2-NTD sequences are aligned with the Fab epitopes in boxes and the conserved epitope residues highlighted in pink color. The helix boundaries, determined by using CCP4 and ProCheck, are shown for the four crystal structures and the ApoL1 NMR structure. The kink in the long ApoL1-helix 5 in the two Fab3B6 structures, due to the helix-breaking P145 residue, is shown as gray box.

Fig 5: Expression of the RRVs leads to a Ca2+ influx that precedes cell swelling and death.(a) Fluorescence traces of representative GCaMP6f-positive cells demonstrating that G1 and G2 cause a Ca2+ influx prior to cell swelling. GCaMP6f-transfected FT293 cells were incubated with DRAQ7 followed by 50 ng/mL doxycycline to induce APOL1 expression and then imaged via widefield every 10 min for 4.5–30 hr post induction. Traces represent levels of cytoplasmic Ca2+ over time as measured by GCaMP6f fluorescence (no DRAQ7 was observed in depicted cells). Cells are from Video 1. Scale bars = 20 µm. (b) High-throughput analysis revealed a significant increase of cytoplasmic Ca2+ levels driven by G1 and G2 compared to G0. Each point is the ?F/F0 for an individually tracked cell and bars represent the cell population mean of GCaMP6f fluorescence. Cells were analyzed from 4 fields of view per condition, n = 1748. A one-way ANOVA multiple comparisons test was performed to compare the RRVs with G0 at the indicated timepoints.Figure 3—source data 1.FT283 cells GCaMP6f microscopy, 30 hours after induction one way ANOVA.