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The MCM5/stat1 complex is required for Stat1 phosphorylation and downstream gene bcl2a transcription. A The proteins Stat1 and p-Stat1 were evaluated in siblings and mcm5−/− embryos at 3.5 dpf. p-Stat1 but not Stat1 was downregulated in mcm5−/− embryos. B The expression of bcl2a in embryos with different treatments. The data showed that stat1a MO injection further downregulated bcl2a expression in mcm5 mutants, and stat1a MO injection also decreased bcl2a expression in embryos treated with camptothecin. C In situ staining for the rag1 probe in embryos. stat1a MO injection further decreased the expression of rag1 in mcm5 mutants (c2, n = 9). Compared to controls (c1, n = 33), embryos injected with stat1a MO displayed decreased expression of rag1 (c3, c4, n = 27). Compared to controls (c5, n = 29), stat1a MO injection led to a more severe decrease in rag1 expression when camptothecin treatment was applied (c6, c7, n = 38). D Quantification of the area of rag1 expression in different kinds of embryos. E The expression of rag2 in the thymus. Compared to controls (e1, n = 35), the expression of rag2 was decreased in embryos injected with stat1a MO (e2, e3, n = 27). Injection of bcl2a mRNA rescued the expression of rag2 in WT embryos injected with Stat1a MO (e4, n = 31). The DsRed-labeled T cells were examined in different kind of embryos (e5-e8). Compared to controls (e5, n = 30), the expression of DsRed in thymus was decreased in embryos injected with stat1a MO (e6, e7, n = 30). The expression of DsRed in stat1a morphants was also rescued by injecting bcl2a mRNA (e8, n = 29). F Quantification of the expression of rag2 in e1 to e4. G Examination of the interaction of zebrafish MCM5 and zebrafish Stat1a using a Co-IP experiment. In first lane, IgG was used as negative control to carry out IP experiment, anti-Flag was used to examine the interation between Stat1 and MCM5, anti-HA was used to perform west blotting staining. The staining showed that MCM5 binds to Stat1a. H The expression of rag1 in the thymus was greatly downregulated in mcm3−/− embryos but not as strongly as that in mcm5−/− embryos. I The expression of bcl2a in siblings and mcm3−/− embryos was examined using WISH, WB and RT‒qPCR. For (A, D, F, H, I), the data were presented as means ± SD; The P-values (t-test; two-tailed); NS not significant. “*”P < 0.05, “**”P < 0.01, “***”P < 0.001.
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