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Fig. 4 Clock1a modulates genes associated with reactive oxygen generation Transcriptome sequencing was performed to determine the downstream genes of clock1a that regulate neutrophil recruitment. Total RNA was extracted from clock1a−/− and WT/AB zebrafish raised under normal LD conditions at 4 dpf. (A) The Venn diagram shows the number of unique RNAs in clock1a mutant zebrafish. (B and C) The heatmap and volcano plot show downregulated and upregulated genes in clock1a mutant zebrafish. (D and E) KEGG and GO enrichment analyses of DEGs classified the genes into different processes. (F) The expression levels of nfe212a, duox, mfn2, crp11a1, cyp2x8, and alas2 were confirmed with independent qPCR experiments. Data were analyzed with unpaired t tests. (G) The hydrogen peroxide content of the clock1a mutant increased significantly at both ZT1 and ZT15 (n = 20). Data were analyzed with one-way ANOVA. (H and I) Fluorescence staining revealed that clock1a mutation upregulated reactive oxygen production (n = 12). Scale bar: 50 μm. Data were analyzed with one-way ANOVA. All experiments were repeated three times, and the results showed the data from an independent experiment. Bar graphs represent the mean ± SEM (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).