Huang et al., 2021 - Tanshinone I, a new EZH2 inhibitor restricts normal and malignant hematopoiesis through upregulation of MMP9 and ABCG2. Theranostics   11:6891-6904 Full text @ Theranostics

Fig. 1 (A) Sudan Black staining of Caudal hematopoietic tissue (CHT) regions of the DMSO or Tan I treated embryos at 72 hpf. (B) Fluorescent images of CHT regions (the framed region of whole embryos in the right bottom) of the Tg(lyz:EGFP) zebrafish embryos at 72 hpf treated with DMSO or Tan I. (C) Images of the embryos treated with DMSO or Tan I WISH analyzed with c-myb, lyz, hbαe1 probes or stained with O-Dianisidine. Scale bars: 200 µm.

Fig. 2 (A) Fluorescent images of Tg(c-mybhyper:GFP) zebrafish embryos with DMSO or Tan I treatments. (B) Fluorescent images of Tg(lyz:EGFP);pu.1G242D zebrafish embryos treated with DMSO or Tan I. (C) Images of Tg(c-mybhyper:GFP) embryos treated with DMSO or Tan I by -c-myb, lyz, and hbαe1 WISH and O-dianisidine staining. (D) Images of Tg(lyz:EGFP);pu.1G242D embryos treated with DMSO or Tan I evaluated by c-myb, lyz, and hbαe1 WISH or O-dianisidine staining. All treatments were continuously starting from 30 hpf. Embryos in all images were at 72 hpf of development. Scale bars: 200 µm.

Fig. 3 (A-D) Flow cytometric analyses of Annexin V and 7-AAD staining after treatments of HL-60 (A, B) or NB4 (C, D) cells with DMSO or the indicated concentrations of Tan I for 3 days. (E-H) Flow cytometric analyses of CD11b expression after treatments of HL-60 (E, F) or NB4 (G, H) cells with DMSO or the indicated concentrations of Tan I for 3 days. (I) Cartoon illustrating steps of the embryonic zebrafish xenograft assay, hpi (hours post-injection), hpt (hours post-treatment). (J) Fluorescent images of HL-60-cell grafted zebrafish embryos at indicated times post transplantation with DMSO or Tan I treatments. (K) Statistic analysis of HL-60 cell number in the CHT regions of images in panel J, Error bars represent standard error of means (SEM). (L) Fluorescent images of NB4-cell grafted zebrafish embryos at indicated times post transplantation with DMSO or Tan I treatments. (M) Statistic analysis of NB4 cell number in the CHT regions of embryos shown in panel L, Error bars represent standard error of means (SEM). Scale bars: 200 µm.

Fig. 4 (A) Venn diagram showing the numbers of differentially expressed genes between DMSO and Tan I treated NB4 cell (blue) or Tg(c-mybhyper:GFP) zebrafish (orange), the overlapped gene number also shown (three of the human genes have two homologs each in zebrafish). (B-E) GSEA (Gene Set Enrichment Analysis) of the expressing profile of NB4 cells treated with DMSO or Tan I using a leukemia stem cell-associated upregulated signature (B), a hematopoietic stem cell and progenitor-associated signature (C), an apoptosis-associated signature (D) and a myeloid differentiation-associated upregulated signature (E). (F) RT-qPCR assay on the mRNA levels of a number of genes after treatments of NB4 cells with DMSO or 10 µM Tan I for 3 days. (G-H) Heatmap of 36 differentially expressed genes between DMSO and Tan I treatment in both human NB4 cell (G) and Tg(c-mybhyper:GFP) zebrafish (H).

Fig. 5 (A-B) c-mybhyper zebrafish embryos were treated with DMSO or indicated concentrations of Tan I. The histone H3K27me1, me2, me3 levels were measured by western blotting, total histone H3 was used as the control. (B) is the quantification of the western blotting. (C-D) SPR analysis of the binding between EZH2 and Tan I at different concentrations. The open bar in (D) indicated the duration of Tan I perfusion in SPR. (E-F) In vitro inhibition of PRC2 complex activities by Tan I. In detail, the indicated proteins (0.28 μg of the PRC2 complex and 0.2 µg of histone H3) were mixed with Tan I and SAM. The reaction products were separated by 15% SDS-PAGE and analyzed by western blotting using antibodies against histone H3K27me1, me2, me3, H3K36me2, histone H3, and EZH2.

Fig. 6 GSEA of the expression profile of NB4 cells treated with DMSO or Tan I using a histone H3K27me3-associated signature “BENPORATH_H3K27ME3” (A) and an EZH2 target genes-associated signature “LU_EZH2_TARGETS_UP (M2139)” (B). (C) Genome browser track representing the binding sites of EZH2 at MMP9 gene locus in human LNCaP cells. (D-F) ChIP-qPCR assay of H3K27me3 (D), EZH2 (E) or IgG (F) occupancy at MMP9 gene locus in NB4 cells treated with DMSO or 10 µM Tan I for 3 days. (G) Genome browser track representing the binding sites of EZH2 at ABCG2 gene locus in human LNCaP cells. (H-J) ChIP-qPCR assay of H3K27me3 (H), EZH2 (I) or IgG (J) occupancy at the ABCG2 gene locus in NB4 cells treated with DMSO or 10 µM Tan I for 3 days. (K) Horizontal lines with Arabic numeral 1 to 2 indicate the regions at the mmp9 gene locus amplified by qPCR in zebrafish ChIP assay. (L-M) ChIP-qPCR assay for H3K27me3 (L) or IgG (M) occupancy at the mmp9 gene locus in zebrafish embryos treated with DMSO or 60 µM Tan I for 3 days. (N) Horizontal lines with Arabic numeral 1 to 2 indicate the regions at the abcg2d gene locus amplified by qPCR in zebrafish ChIP assay. (O-P) ChIP-qPCR assay for H3K27me3 (O) or IgG (P) occupancy at the abcg2d gene locus in zebrafish embryos treated with DMSO or 60 µM Tan I for 3 days. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01. All results are from three independent experiments.

Fig. 7 (A) Rescue Tan I induced phenotype by overexpression of ezh2 in the zebrafish embryo. Fluorescent images of c-myb GFP+ cells in Tg(c-mybhyper:GFP) and Tg(hsp70l:ezh2:CG) double transgenic zebrafish embryos, with DMSO or Tan I treatments and with or without a 37 ºC heat shock for 1h, as indicated. dpf (days post-fertilization) (B) Rescue Tan I induced phenotype by knockdown of mmp9 and abcg2 in the zebrafish embryo. Fluorescent images of c-myb GFP+ cells in Tg(c-mybhyper:GFP) transgenic zebrafish embryos. As indicated, mmp9 and abcg2d morpholino was injected separately or together in DMSO or Tan I treated embryos. (C-E) Overexpression MMP9 in NB4 cells. RT-qPCR analyses of MMP9 mRNA level on the established NB4 cells transduced with an empty vector or MMP9-expressing vector (C). Flow cytometric analyses of Annexin V/7-AAD staining (D) and CD11b expression (E) in control or MMP9-overexpressed NB4 cells; (F-H) Overexpression of ABCG2 in NB4 cells. RT-qPCR analyses of ABCG2 mRNA level on the established NB4 cells transduced with empty vector or ABCG2-expressing vector (F). Flow cytometric analyses of Annexin V/7-AAD staining (G) and CD11b expression (H) in control or ABCG2-overexpressed NB4 cells. Scale bars: 200 µm.

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