Histone Methyltransferase

Treatment of c-Kit+ bone marrow (BM) cells from Setd2^f/f/Vav1-Cre mice with JQ1 500nM, EPZ-5676 1uM, BAY 1143572 400 nM for 24 h. Then cells were collected to determine the proteins levels of RNA pol II (Ser2P), pol II (Ser5P), Gata1, Gata3, Myc, and β-actin by immunoblotting.

Western blot analysis of day 7 lysates from LX92 lysates treated with various EZH2 inhibitors.

Representative confocal microscopy images of oocytes with DZNep or GSK343 treatment. The oocytes presented with a typical barrel-shaped spindle and well-aligned chromosomes on the metaphase plate not only in DZNep or GSK343 treated group but also in the control group. Spindle was recognized by α-tubulin (green) and DNA was recognized by PI (Propidium iodide, red). Scale bar = 4 μm.

The H3K9me2 inhibitor BIX01294 mimics the effects of BPA and DEHP on the expression of meiotic genes and Plzf in cultured testes. (A) representative double IF staining for Mvh (green) and Stra8 (red) and Scp3 (red) and (B) Plzf (red) in histological sections of control and BIX01294-treated testes. (C) RT-qPCR analysis of Stra8, Scp3, Rec8, Spo11 and plzf in testes from the control and BIX01294-treated groups. Data are represented as the mean ± SEM. ns, P > 0.05, *P < 0.05; **P < 0.01.

EZH2i-induced H3K27ac level change in cancer cells. Cells were treated with 1 mM EPZ-6438 and 1 mM GSK126 for 6 days, respectively. H3K27ac was detected by immunoblotting. Lysates from each cell line were blotted individually. EPZ: EPZ-6438.

H3K27me3 inhibitors inducing cell differentiation in the MDS-derived erythroid/myeloid cell line and primary MDS bone marrow cells via reducing H3K27me3 and increasing the expressions of PU.1 and its downstream genes. (a) ChIP experiments show a marked decrease in H3K27me3 at the PU.1 enhancer in the OCI-M2 cells treated with DZNep, compared with that in the cells treated with dimethyl sulfoxide (DMSO). (b) RT-PCRs show a significant increase in the expression of PU.1 mRNA in the OCI-M2 cells treated with DZNep in a dose-dependent manner. (c) Flow cytometric studies show that an increase in the expression of glycophorin A/B, a marker associated with erythroid differentiation, in the OCI-M2 cells treated with 0.25 μM of DZNep for 6 days. Red, isotype control; blue, no drug treatment (DMSO only); green, plus drug. (d) Increased PU.1 mRNA expression in primary MDS bone marrow cells treated with DZNep, compared with that in DMSO control. (e) Increased CD18 mRNA expression in primary MDS bone marrow cells treated with DZNep, compared with that in DMSO control. The primary bone marrow cells were from three cytogenetically normal MDS patients, which had marked trilineage dysplasia with varying numbers of blasts (case-1 with 10% blasts, case-2 with 4.6% blasts and case-3 with 11% blasts). The MDS bone marrow cells were treated with 1 μM DZNep or DMSO (control) for 24 h. The levels of PU.1 and CD18 mRNAs were measured by Q-RT-PCRs, while TBP mRNA was used for the internal normalization

Ex vivo growth of the SCLC PDX LX92 is significantly inhibited by the EZH2 inhibitors EPZ-5687, GSK343 and UNC1999 as measured by resazurin conversion (two-way analysis of variance, adjusted for multiple comparisons by the method of Dunnet).

Immunofluorescence staining of H3K4me2/3 (red) in the IVF, SCNT and MM-102-NT embryos. The nuclei (blue) were stained with DAPI. The merged images of H3K4me2/3 and DNA were purple. Scale bars: 50 μm.

(B) Effect of EPZ015666 on nuclear translocation of p65. Middle panel showed a representative photo of IF staining of p65 localization (green). Nuclei were stained with DAPI.

Treatment of HCC cells with 500 nmol/L GSK591 for 4 days led to significant loss of PRMT5-catalyzed methylarginine on H4 (H4R3me2s), the upregulation of BTG2, and the phosphorylation of ERK. Inhibition of ERK phosphorylation by PD184352 treatment reversed these effects. H4R3me2s was used to detect the PRMT5 activity. Histone 3 was used as nuclear loading control.

EZH2 inhibitors EI1 or GSK343 are applied in MDA-MB-231 breast cancer cells with NBAT1 over-expression. The invasion suppression effects of NBAT1 was reversed by concomitant EZH2 inhibitors.

LC3B‐II levels are increased following 120 h treatment of GSK503 (G, CRMM1 and CRMM2 at 10 μm, and CM2005.1 at 15 μm) or UNC1999 (U, 6 μm).

THP-1 cells were treated with MI-3 (6.25 μM, 6 days), GSK126 (5 μM, 6 days), or EPZ004777 (5 μM, 6 days), with orwithout PMA (50 ng/mL, 12 h), and surface expression of CD11b was determined by flow cytometric analysis. The data are representedas the mean ± SD, n = 3. THP-1 cells were collected for Wright-Giemsa staining.

(C) Four GCBDLBCL cells lines [1 wild-type (wt), 3 mutated (mut) EZH2] were treated with 500 nmol/l BAY 1238097 or with an EZH2 inhibitor (DZNep and GSK126, both used at the concentration of 500 nmol/l) as single agents and in combination for 72 h. The arrow indicates the EZH2 correct band. Dimethyl sulphoxide (DMSO) alone was added as negative control cells. Membranes were hybridized with antibodies against EZH2, H3K27me3, histone H3 and GAPDH.

Effects of microinjection of AMI-1 into the RVLM on cardiovascular activity in the Ang II-induced hypertensive rats. The representative original tracings (A)

(F) Real-time PCR analysis showed the VEGFA mRNA expression in the HOXC10-overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. (G) ChIP-qPCR enrichments of H3K4me3, polymerase II or IgG on VEGFA promoter region 3 in the HOXC10-overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. All the assays shown in the figure were independently replicated three times. Error bars, SD. * P < 0.05.

Treatment of c-Kit+ bone marrow (BM) cells from Setd2^f/f/Vav1-Cre mice with JQ1 500nM, EPZ-5676 1uM, BAY 1143572 400 nM for 24 h. Then cells were collected to determine the proteins levels of RNA pol II (Ser2P), pol II (Ser5P), Gata1, Gata3, Myc, and β-actin by immunoblotting.

Western blot analysis of day 7 lysates from LX92 lysates treated with various EZH2 inhibitors.

Representative confocal microscopy images of oocytes with DZNep or GSK343 treatment. The oocytes presented with a typical barrel-shaped spindle and well-aligned chromosomes on the metaphase plate not only in DZNep or GSK343 treated group but also in the control group. Spindle was recognized by α-tubulin (green) and DNA was recognized by PI (Propidium iodide, red). Scale bar = 4 μm.

The H3K9me2 inhibitor BIX01294 mimics the effects of BPA and DEHP on the expression of meiotic genes and Plzf in cultured testes. (A) representative double IF staining for Mvh (green) and Stra8 (red) and Scp3 (red) and (B) Plzf (red) in histological sections of control and BIX01294-treated testes. (C) RT-qPCR analysis of Stra8, Scp3, Rec8, Spo11 and plzf in testes from the control and BIX01294-treated groups. Data are represented as the mean ± SEM. ns, P > 0.05, *P < 0.05; **P < 0.01.

EZH2i-induced H3K27ac level change in cancer cells. Cells were treated with 1 mM EPZ-6438 and 1 mM GSK126 for 6 days, respectively. H3K27ac was detected by immunoblotting. Lysates from each cell line were blotted individually. EPZ: EPZ-6438.

H3K27me3 inhibitors inducing cell differentiation in the MDS-derived erythroid/myeloid cell line and primary MDS bone marrow cells via reducing H3K27me3 and increasing the expressions of PU.1 and its downstream genes. (a) ChIP experiments show a marked decrease in H3K27me3 at the PU.1 enhancer in the OCI-M2 cells treated with DZNep, compared with that in the cells treated with dimethyl sulfoxide (DMSO). (b) RT-PCRs show a significant increase in the expression of PU.1 mRNA in the OCI-M2 cells treated with DZNep in a dose-dependent manner. (c) Flow cytometric studies show that an increase in the expression of glycophorin A/B, a marker associated with erythroid differentiation, in the OCI-M2 cells treated with 0.25 μM of DZNep for 6 days. Red, isotype control; blue, no drug treatment (DMSO only); green, plus drug. (d) Increased PU.1 mRNA expression in primary MDS bone marrow cells treated with DZNep, compared with that in DMSO control. (e) Increased CD18 mRNA expression in primary MDS bone marrow cells treated with DZNep, compared with that in DMSO control. The primary bone marrow cells were from three cytogenetically normal MDS patients, which had marked trilineage dysplasia with varying numbers of blasts (case-1 with 10% blasts, case-2 with 4.6% blasts and case-3 with 11% blasts). The MDS bone marrow cells were treated with 1 μM DZNep or DMSO (control) for 24 h. The levels of PU.1 and CD18 mRNAs were measured by Q-RT-PCRs, while TBP mRNA was used for the internal normalization

Ex vivo growth of the SCLC PDX LX92 is significantly inhibited by the EZH2 inhibitors EPZ-5687, GSK343 and UNC1999 as measured by resazurin conversion (two-way analysis of variance, adjusted for multiple comparisons by the method of Dunnet).

Immunofluorescence staining of H3K4me2/3 (red) in the IVF, SCNT and MM-102-NT embryos. The nuclei (blue) were stained with DAPI. The merged images of H3K4me2/3 and DNA were purple. Scale bars: 50 μm.

(B) Effect of EPZ015666 on nuclear translocation of p65. Middle panel showed a representative photo of IF staining of p65 localization (green). Nuclei were stained with DAPI.

Treatment of HCC cells with 500 nmol/L GSK591 for 4 days led to significant loss of PRMT5-catalyzed methylarginine on H4 (H4R3me2s), the upregulation of BTG2, and the phosphorylation of ERK. Inhibition of ERK phosphorylation by PD184352 treatment reversed these effects. H4R3me2s was used to detect the PRMT5 activity. Histone 3 was used as nuclear loading control.

EZH2 inhibitors EI1 or GSK343 are applied in MDA-MB-231 breast cancer cells with NBAT1 over-expression. The invasion suppression effects of NBAT1 was reversed by concomitant EZH2 inhibitors.

LC3B‐II levels are increased following 120 h treatment of GSK503 (G, CRMM1 and CRMM2 at 10 μm, and CM2005.1 at 15 μm) or UNC1999 (U, 6 μm).

THP-1 cells were treated with MI-3 (6.25 μM, 6 days), GSK126 (5 μM, 6 days), or EPZ004777 (5 μM, 6 days), with orwithout PMA (50 ng/mL, 12 h), and surface expression of CD11b was determined by flow cytometric analysis. The data are representedas the mean ± SD, n = 3. THP-1 cells were collected for Wright-Giemsa staining.

(C) Four GCBDLBCL cells lines [1 wild-type (wt), 3 mutated (mut) EZH2] were treated with 500 nmol/l BAY 1238097 or with an EZH2 inhibitor (DZNep and GSK126, both used at the concentration of 500 nmol/l) as single agents and in combination for 72 h. The arrow indicates the EZH2 correct band. Dimethyl sulphoxide (DMSO) alone was added as negative control cells. Membranes were hybridized with antibodies against EZH2, H3K27me3, histone H3 and GAPDH.

Effects of microinjection of AMI-1 into the RVLM on cardiovascular activity in the Ang II-induced hypertensive rats. The representative original tracings (A)

(F) Real-time PCR analysis showed the VEGFA mRNA expression in the HOXC10-overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. (G) ChIP-qPCR enrichments of H3K4me3, polymerase II or IgG on VEGFA promoter region 3 in the HOXC10-overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. All the assays shown in the figure were independently replicated three times. Error bars, SD. * P < 0.05.