Additional file 3 of AUY922 induces retinal toxicity through attenuating TRPM1

Additional file 3: Figure S3. a Viability of A375 (blue line) and Mel1617 (red line) cells, after treatment with varying concentrations of AUY922 for 3 d. Data are presented as the mean ± s.e.m. (n = 3). b Representative images of clonogenic growth assays in Mel1617 (top) and A375 (bottom) cells treated with the indicated concentrations of AUY922 for 10 d. Three hundred cells/well were seeded in 6-well plates. n = 3. c Strategy for iTRAQ proteomic profiling of DMSO- and AUY922-treated 661 W cells. d Quantification of the AUY922 treatment experiments in 661 W, Mel1617 and A375 cells, related to Fig. 3d. Data are presented as the mean ± s.e.m. The P values were determined by two-tailed Student’s t-test, * P < 0.05; ** P < 0.01. n = 3. e ROS production assays were performed in 661 W cells treated with AUY922 for 48 h. Cells were labeled with DCFDA (20 μM) and analyzed by flow cytometry. Data are presented as the mean ± s.e.m. The P values were determined by two-tailed Student’s t-test, ** P < 0.01; n.s. = not significant. n = 3. f Quantification of the AUY922 treatment experiments in ARPE19 and Mel1617 cells expressing 3xF-TRPM1, related to Fig. 3e. Data are presented as the mean ± s.e.m. The P values were determined by two-tailed Student’s t-test, * P < 0.05; ** P < 0.01; *** P < 0.001. n = 3. g Representative western blot results of ARPE19 and Mel1617 cells stably expressing either an empty vector or 3xF-TRPM1 after treatment with 30 nM AUY922 for different time points. Quantification was performed for three independent experiments. Data are presented as the mean ± s.e.m. The P values were determined by two-tailed Student’s t-test, * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. = not significant. n = 3. h Representative western blot results of 661 W cells pretreated with 30 nM AUY922 for 48 h and treated with 100 nM MG132 for 4 h. Quantification was performed for three independent experiments. Data are presented as the mean ± s.e.m. The P values were determined by two-tailed Student’s t-test, * P < 0.05; ** P < 0.01. n = 3.