Journal: The Journal of Clinical Investigation
Article Title: APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease
doi: 10.1172/JCI172262
Figure Lengend Snippet: ( A ) Fura-2 Ca 2+ sensor shows that 8 hours of APOL1 G1 expression attenuates thapsigargin-induced surge of cytosolic Ca 2+ , which is rescued by VX-147 cotreatment ( n = 8). ( B ) Thapsigargin-induced Ca 2+ release plotted as bar graphs ( n = 8). ( C ) Direct measurement of ER Ca 2+ with fluorescent sensor (D1ER) shows that 8 hours of APOL1 G1 expression depletes basal ER Ca 2+ , which is restored by VX-147 cotreatment ( n = 8–12). Addition of CPA, which further depletes ER Ca 2+ , has marginal effect on ER Ca 2+ of APOL1 G1–expressing cells. ( D ) Basal ER Ca 2+ level before CPA addition shown as bar graph ( n = 12–30). ( E ) Real-time live-cell fluorescence shows increased DAG biosynthesis after 8 hours of G1 induction. G1-induced DAG synthesis is blocked by VX-147, U73122 (PLC inhibitor), and FR*359 (Gαq inhibitor) ( n = 8). ( F ) Fluorescent Ca 2+ sensor shows that combined inhibition of IP3R with XSpC and of RYR with JTV-519 rescues thapsigargin-induced Ca 2+ response in G1-expressing T-REx-293 cells (12 hours of treatment), similar to the effect of VX-147 ( n = 3–6). ( G ) Measurement of live-cell basal cytosolic Ca 2+ levels shows that VX-147, XSpC, and JTV-519 cotreatment prevents G1-induced increase in cytosolic Ca 2+ ( n = 6–12). ( H ) Multitox assay shows that JTV-519, XSpC, and VX-147, but not SKF96365, rescue APOL1 G1–induced cytotoxicity in T-REx-293 after 24 hours of treatment ( n = 4–12). ( I ) Immunoblot shows successful CRISPR/Cas9 knockout of IP3R1 and IP3R3 in T-REx-293 G1 cells (C1 and C10 are 2 independent clones). ( J ) Multitox assay shows loss of IP3R-mediated APOL1 G1–induced cytotoxicity in T-REx-293 after 24 hours of treatment ( n = 5–7). ( K ) Schematic summary of , showing how each step of this signaling cascade is reversible. All data are represented as mean ± SD. * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001, ordinary 1-way ANOVA with Tukey’s multiple-comparison test.
Article Snippet: D1ER plasmid, a genetic coded calcium indicator (GCCI) of SR/ER Ca 2+ , was electroporated using a neon electroporation system (Thermo Fisher) into T-REx-293 G1 cells and seeded on a 35 mM MatTeck plate.
Techniques: Expressing, Fluorescence, Inhibition, Western Blot, CRISPR, Knock-Out, Clone Assay, Comparison
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