Tamaoki et al., 2020 - Splicing- and demethylase-independent functions of LSD1 in zebrafish primitive hematopoiesis. Scientific Reports   10:8521 Full text @ Sci. Rep.

Fig. 3

Phenotypic rescue experiments using human and zebrafish LSD1 variants. (A) The gata1a expression at 15 hpf in the lateral plate mesoderm (arrowheads) of wild-type or kdm1ait627 embryos injected with or without mRNAs encoding indicated human LSD1 variants was detected by WISH analysis. +2a, LSD1+2a; +8a, LSD1+8a; +2a8a, LSD1+2a8a. The numbers in each picture indicate the numbers of embryos with similar staining profiles and tested embryos. WISH was performed in at least two separated trials. (B) Immunoblot analysis using anti-FLAG antibody of FLAG-tagged LSD1 variants in 15-hpf wild-type embryos injected with or without mRNAs encoding indicated human LSD1 variants. Total protein loading was shown by CBB staining. (C) The gata1a expression at 15 hpf in the lateral plate mesoderm (arrowheads) of wild-type or kdm1ait627 embryos injected with or without mRNAs encoding indicated zebrafish LSD1 variants. +2al, LSD1+2al; +8al, LSD1+8al; +2a8al, LSD1+2a8a. (D) Immunoblot analysis using anti-FLAG antibody of FLAG-tagged LSD1 variants in 15-hpf wild-type embryos.

Fig. 4

Phenotypic rescue experiments using demethylase-deficient human LSD1 mutant. (A) The gata1a expression at 15 hpf in the lateral plate mesoderm (arrowheads) of wild-type or kdm1ait627 embryos injected with or without mRNAs encoding indicated wild-type or K661A mutant-type human LSD1 was detected by WISH analysis. (B) Immunoblot analysis using anti-FLAG antibody of FLAG-tagged LSD1 proteins in 15-hpf wild-type embryos. Total protein loading was shown by CBB staining. (C) Expression of alas2 and hbbe1.1at 20 hpf in the intermediate cell mass (arrowheads). (D) Expression of etv2 at 15 hpf in the lateral plate mesoderm (arrowheads).

Fig. 5

Phenotypic rescue experiments using demethylase-deficient zebrafish LSD1. (A) Multiple alignments of vertebrate LSD1 proteins around Lys661 in human LSD1. Red “K” indicates highly conserved lysine residues in the catalytic center of LSD1. (B) Bacterially expressed zebrafish LSD1+8al proteins used in a demethylation assay. Arrowheads indicate full-length proteins. (C) A demethylation assay of indicated zebrafish LSD1+8al proteins using bulk histones from calf thymus as substrates. Methylated proteins were detected by immunoblotting using specific antibodies. Bar graphs indicate the relative intensity of immunoblot signals standardized to the level of a negative control LSD1+8alΔ609. Error bars represent standard deviations. The experiments was repeated 3 times. Asterisks denote p values < 0.05. (D) The gata1a expression at 15 hpf in the lateral plate mesoderm (arrowheads) of wild-type or kdm1ait627 embryos injected with or without mRNAs encoding wild-type or K638A mutant-type zebrafish LSD1+8al. (E) Immunoblot analysis using anti-FLAG antibody of FLAG-tagged LSD1 proteins in 15-hpf wild-type embryos. Total protein loading was shown by CBB staining.

Acknowledgments:
ZFIN wishes to thank the journal Scientific Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Sci. Rep.