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. 2022 Jun 6;119(24):e2120083119. doi: 10.1073/pnas.2120083119

Fig. 3.

Fig. 3.

(A) GSIS was carried out in INS-1 CD59-KO cells transfected (transient transfection) with WT CD59, IRIS-1, or IRIS-2; n = 5. Each data point represents the average of three independent repeats for five different CD59-KO clones (untransfected [UT] or transfected with human CD59-WT, or IRIS-1, or IRIS-2). (B) GSIS (16.7 mM glucose in the presence of 200 μM diazoxide) carried out in mock control and INS-1 CD59-KO cells transiently expressing IRIS-1 and IRIS-2; n = 3 biological repeats. (C) Exocytosis by TIRF-M: Total K+ depolarization induced exocytosis during 40 s in control INS-1 cells (black, n = 18 cells, three preparations), CD59-KO cells (red, n = 33 cells, three preparations), and CD59-KO cells stably overexpressing IRIS-1 (blue, n = 27 cells, three preparations) or IRIS-2 (orange, n = 27 cells, three preparations). (D) Exocytosis by patch clamp: Average cell capacitance time course (ΔCm, ±SEM) during a train of 14 × 200-ms depolarizations from −70 mV to 0 mV (n = 15, 30 cells assessed per clone). (E) Average changes in membrane capacitance from H, during the first depolarization, and total increase during the train of 14 depolarizations. (F) GSIS was carried out in EndoC-βH1 cells UT or transfected with siRNA against total human CD59 (siCD59) or negative control (siCtrl). n = 3 biological repeats. (G) GSIS inducing first (12 min) and second (1 h) phase of insulin exocytosis carried out in INS-1 CD59-KO cells stably overexpressing IRIS-1 and IRIS-2; n = 3, biological repeats. (H and I) Current–voltage relationship of inward currents in control INS-1 cells (black, n = 30 cells, four preparations), CD59-KO cells (red, n = 29 cells, four preparations), and CD59-KO cells stably overexpressing IRIS-1 (blue, n = 19 cells, three preparations) or IRIS-2 (orange, n = 15 cells, three preparations). (H) Average Ca2+ current during 5 to 45 ms of the depolarization and (I) peak Na+ current during the first 5 ms of the depolarization (p = 0.0001 for IRIS-1 and 0.001 for IRIS-2 vs. CD59-KO). Statistics (in A, B, and F): two-way ANOVA with Bonferroni’s posttest, (in C): one-way ANOVA with Fisher’s posttest, (in D, E, H, and I): two-tailed, unpaired t test. (in G): two-way ANOVA with Dunnett’s posttest. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.