Bulk exposure of mouse primary BMDMs and larval zebrafish to HNE/DMF triggers Bax-dependent macrophage/neutrophil apoptosis.a Left: western blots analyzing changes in the endogenous levels of cleaved PARP (hallmark of apoptosis) in two different pairs of lentiviral shWdr1-knockdown BMDMs and knockdown-control BMDMs, subsequent to HNE or staurosporine treatment (3 h). Right: ImageJ quantification of relative extent of cleaved PARP in HNE-treated, compared to the respective DMSO-control set, in Wdr1-knockdown BMDMs (red squares) vs. shControl (blue dots) lines. In each dataset, normalization was performed against α-tubulin loading control. Slopes from linear regression of shControl vs. shWdr1, Upper plot: 0.00043 ± 0.00012 and 0.00006 ± 0.00003, respectively. Lower plot: 0.0059 ± 0.0022 and 0.0023 ± 0.0008, respectively. Fits derived from fitting to y = mx. bTg(lyz:TagRFP) embryos expressing Halo-TEV-Keap1 were treated at 30 hpf with HNE or DMF or HDE for 6 h at indicated concentrations (up to maximum tolerable amount prior to cytotoxicity), and then neutrophils were counted. P values were calculated with ANOVA and Dunnett’s multiple comparisons test. Note that for the left two plots, the non-treated data are the same, but are presented in each plot for clarity. c Similar experiment as in (a) except with or without simultaneous administration of Bcb. Inset at right: Fold changes (treated/non-treated) in neutrophil counts. P values were calculated with two-tailed Student’s unpaired t-test. Note that the non-treated data are the same, but are presented in each plot for clarity. All data present mean ± SEM. All sample sizes are listed in Supplementary Methods. Source data are provided as a Source data file.
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