Figure 7. Chemical inhibitor analysis of NTR/Mtz-mediated rod cell death in zebrafish.

Box plots of rod cell survival effects of eight PARP inhibitors (green), a necroptosis inhibitor (blue), four apoptosis inhibitors (magenta), and three Tdp1 inhibitors (orange) in Mtz-treated rho:YFP-NTR zebrafish larvae (targets are listed above for each category). Assays performed as per confirmation tests (see Figure 2A) with all inhibitors tested across 10 concentrations using a twofold dilution series (see Methods for highest concentration tested per compound). Conditions resulting in a statistically significant increase in survival effects relative to +Mtz controls are marked with an asterisk. Survival effects (normalized YFP %), 95% confidence intervals, p-values, and sample sizes (N) for each condition are shown in the table below. Student’s t-test was used to calculate p-values for each condition relative non-ablated controls (0 mM Mtz). Bonferroni correction for multiple comparisons resulted in an adjusted significance level of 0.003 (α=0.003). A minimum of two experimental repeats was performed for each condition (Figure 7—source data 1). No statistical differences in larval survival were observed for tested compounds relative to their respective +Mtz controls, except for PM (67%; Fisher’s exact test, p<0.05). Target abbreviations: Parp, Poly (ADP)-ribose polymerase; Ripk1, Receptor-interacting serine/threonine-protein kinase (1); Tdp1, Tyrosyl-DNA phosphodiesterase 1; Mtz, metronidazole. Inhibitor abbreviations: AG, AG-14361; NMS, NMS-P118; BMN, talazoparib; ABT, veliparib; OLA, olaparib; MK, niraparib; RUC, rucaparib; NEC, necrostatin-1; AC, Ac-DEVD-CHO; CASI, caspase3/7 inhibitor I; CASVII, caspase three inhibitor VII; PM, paromomycin; ThS, thiostrepton; MD, methyl-3,4-dephostatin. Other abbreviations: CI, confidence interval; Mtz, Metronidazole.

Figure 7—figure supplement 1. Additive survival effects of paired PARP and necroptosis inhibitors.

Box plots of rod cell survival effects of BMN (PARP inhibitor) and NEC (necroptosis inhibitor) tested alone and in pairs at three different concentrations in rho:YFP-NTR zebrafish larvae (assays performed as per confirmation tests, see Figure 2A). Seven of nine pairs promoted a statistically significant increase in survival relative to both individual compound controls (++), while one pair performed better than a single compound control (+). Survival effects (normalized YFP %), 95% confidence intervals, p-values, and sample sizes (N) for each condition are shown in the table below. For comparison, the survival effect of each pair (Effect %) and the summed effect of the respective individual compound controls (Sum %) are provided. Additive effects were defined as any pair that promoted survival at equal to or greater than the sum of the corresponding individual compound effects ±10%. By this criterion, eight pairs produced additive effects (bolded). A minimum of two experimental repeats were performed for each condition and data polled across replicates (Figure 7—figure supplement 1—source data 1). See also Supplementary file 1b for Student’s t-test calculated p-values for paired conditions relative to ablated controls (+Mtz) and to respective individual compound controls. Inhibitor abbreviations: BMN, talazoparib; NEC, necrostatin-1. Other abbreviations: CI, confidence interval; Mtz, Metronidazole; NA, not applicable.