Journal: Nature Communications

Article Title: Ezh2 programs T FH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

doi: 10.1038/s41467-018-07853-z

Figure Lengend Snippet: Ezh2 cooperates with Tcf1 to activate key T FH genes. a Venn diagram showing overlap between Ezh2 and Tcf1 binding peaks in T FH cells. b Distribution of Ezh2/Tcf1 co-occupied sites. Promoter is defined as +/–1 kb region flanking TSSs, and gene body refers to region covering +1 kb to transcription end site, with remainder as intergenic region. c De novo motif analysis of Ezh2/Tcf1 co-occupied sites. The motif logos and statistical significance are listed. d Ezh2, but not G9a, co-immunoprecipitates with Tcf1. FLAG-tagged Tcf1 was co-transfected with HA-tagged Ezh2 or G9a into 293T cells. The cell lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-HA. Data are representative of two experiments. e Ezh2 co-immunoprecipitates with Tcf1 in primary T FH cells. CXCR5 + SLAM lo T FH cells were sorted from WT mice infected with LCMV-Arm and immunoprecipitated with normal rabbit IgG or anti-Tcf1 followed by immunoblotting with anti-Ezh2. ‘m’ denotes marker lane. Data are representative of two experiments. f Ezh2 is recruited by Tcf1. WT or Tcf1 –/– Lef1 –/– mice were infected with VacV, and CXCR5 + SLAM lo T FH cells were sorted on 6 dpi and analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6 , Icos , Arf and a Cxcr5 upstream region was determined by ChIP-qPCR. Data are means ± s.d. from two experiments with each sample measured in duplicates. ns, not statistically significant; * p < 0.05; ** p < 0.01 by Student’s t -test. g Diagram of dual reporter retroviral construct. Red “x” denotes Tcf1 motif mutations in mutant Bcl6 reporter. h Thy1.1 reporter expression driven by the Bcl6 promoter. In vivo primed WT or Ezh2 –/– Smarta CD4 + T cells were infected with WT or mutant Bcl6 reporter retrovirus, followed by adoptive transfer and LCMV-Arm infection. On 4 dpi (equivalent to day 6 after initial priming), GFP + CD45.2 + T FH cells were detected in the recipient spleens and assessed for Thy1.1 expression. Cumulative data are means ± s.d. from experiments with ≥5 recipients analyzed for each genotype. *** p < 0.001 by Student’s t -test

Article Snippet: The cDNA coding N-terminus FLAG-tagged Tcf1 in the Mig-R1 retroviral vector was described , and the plasmids expressing HA-tagged human Ezh2 and G9a were from Addgene (Cat. No. #24230 and #33024, respectively), and the coding sequence was subcloned into Mig-R1 for expression.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Infection, Western Blot, Marker, Control, ChIP-qPCR, Retroviral, Construct, Mutagenesis, Expressing, In Vivo, Adoptive Transfer Assay

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: (A) Integrative Genomics Viewer (IGV) tracks showing E2F5, H3K4me3, and IgG CUT&RUN signal at the STAT6 promoter in HC11 mouse mammary epithelial cells. (B) Luciferase reporter assay in 293T cells showing that E2F5 represses STAT6 promoter activity. (C) STAT6 promoter-luciferase reporter assay of MCF7 cells treated with vehicle or R5020 and co-transfected with CMV-HA-E2F5 or CMV-Neo-Bam (empty vector).

Article Snippet: For the 293T reporter assay, HA-E2F5 (Addgene #24213) or vector control (Addgene #16440) was co-transfected with the empty luciferase reporter vector or the STAT6-promoter luciferase reporter using Lipofectamine3000 (Invitrogen #L3000008) following the manufacturers protocol.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 1: Overexpression of JMJD3 suppresses while silencing down of JMJD3 promotes stem cell-like characteristics in MDA-MB-231 cells. We established stable JMJD3-overexpressing and control MDA-MB-231 cell lines. (A) Efficiency of JMJD3 overexpression detected by western blotting. The quantification of relative protein level is shown at the right panel. (B) Flow cytometric analysis of ALDH activity in JMJD3-overexpressing and control cell lines. Statistical results are shown in the right panel. (C) Representative images of sphere formation assays. Statistical results are shown in the right panel. Scale bar = 100 μm. (D) ALDH expression was detected by western blotting in stable JMJD3-overexpressing and control MDA-MB-231 cells. The quantification of relative ALDH level is shown at the right panel. Then, we established stable JMJD3 knockdown and scramble control MDA-MB-231 cell lines. (E) Efficiency of JMJD3 knockdown detected by western blotting. The statistical result is shown at the right penal. (F and G) Results of ALDH activity and sphere formation assays. Data are from three independent experiments and are shown as the mean ± S.E.M.*P<0.05 and ***P<0.001. (H) ALDH expression was detected by western blotting in stable JMJD3 knockdown and scramble control MDA-MB-231 cells. The statistical results are shown at the right penal. (I) Limited dilutions of stable JMJD3-overexpressing and control MDA-MB-231 cells were subcutaneously injected into the fat pads of female BALB/C nude mice (n=5). Tumors were monitored every 2 days by manual palpation for 2 weeks. The tumorigenic capacity is shown in the table.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Over Expression, Control, Western Blot, Activity Assay, Expressing, Knockdown, Injection

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 2: Overexpression of JMJD3 suppresses while silencing down of JMJD3 promotes Oct4 expression in vitro and in vivo. We established JMJD3-overexpressing and control MDA-MB-231 or T47D cell lines. (A) mRNA expression of Oct4 in both cell lines detected by real-time PCR. (B) Expression level of Oct4 protein detected by western blotting. The result of quantification is shown in the right penal. Then, we established stable JMJD3 knockdown and scramble control MDA-MB-231 cell lines. (C) mRNA expression of Oct4 in stable cell lines detected by real-time PCR. (D) Expressional level of Oct4 protein detected by western blotting. The result of quantification is shown in the right penal. Data are shown as the mean ± S.E.M. of three independent experiments. *** P<0.001. Xenograft tumor models of breast cancer were established as described in Figure 1 using stable JMJD3-overexpressing and control MDA- MB-231 cell lines (n=5). (E) Representative western blots using antibodies against JMJD3, Oct4 and ALDH. Samples were homogenates of tumor tissues from various groups. Statistical results are shown in the right panel. Data are shown as the mean ± S.E.M.*P<0.05. (F) Representative images of immunohistochemical staining with antibodies against Oct4 and ALDH in tumor tissues. The right panel shows the statistical results. Scale bar =100 μm.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Over Expression, Expressing, In Vitro, In Vivo, Control, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Stable Transfection, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 3: Oct4 rescues the promotion of stem cell-like characteristics induced by knockdown of JMJD3. Stable JMJD3 knockdown or scramble control MDA-MB-231 cells were transiently transfected with Oct4 shRNA or control plasmid. (A) Oct4 expression was detected by western blotting (top) and the bottom panel shows the statistical result. (B) Flow cytometric analysis of ALDH activity in JMJD3 knockdown and control cell lines. Statistical results are shown in the right panel. **P<0.01. (C) Representative images of the sphere formation assay. Statistical results are shown in the right panel. Scale bar = 100 μm. Data are shown as the mean ± S.E.M. of three independent experiments. ** P<0.01.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Knockdown, Control, Transfection, shRNA, Plasmid Preparation, Expressing, Western Blot, Activity Assay, Tube Formation Assay

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 4: Effect of JMJD3 on the expression of Oct4 independently of its demethylase activity. MDA-MB-231 and T47D cells were transfected with JMJD3 overexpression or control plasmid. (A) Results of dual-luciferase reporter assay detecting the promoter activity of Oct4. Data are shown as the mean ± S.E.M. of three independent experiments. ** P<0.01. (B) MDA-MB-231 and T47D cells were treated with 10 μM GSK-J4 for 48 h. Western blot analysis was performed using antibodies against JMJD3, Oct4 or H3K27me3. Statistical results are shown in the right panel. (C and D) ChIP-PCR analysis and statistical results of H3K27me3 marks on the Oct4 promoter in JMJD3-overexpressing and control MDA-MB-231 cells. Mouse IgG was used as the negative control with the input as the loading control.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Expressing, Activity Assay, Transfection, Over Expression, Control, Plasmid Preparation, Luciferase, Reporter Assay, Western Blot, Negative Control

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 5: PHF20 mediates the regulatory effect of JMJD3 on the expression of Oct4. PHF20 expression at protein and mRNA levels detected by western blotting and RT-PCR in stable JMJD3-overexpressing and control MDA-MB-231/T47D cell lines (A and B). The right penal is the statistical result respectively. PHF20 expression at protein and mRNA levels detected by western blotting and RT- PCR in stable JMJD3 knockdown and scramble control MDA-MB-231 cells (C and D). The results of quantification are shown on the right panel. Stable JMJD3-overexpressing and control MDA-MB-231 cells were treated with 10 μM MG132 for 48 h. (E) PHF20 expression detected by western blotting and the quantification of protein level is shown on the right. PHF20 shRNA or the scrambled control were transfected into stable JMJD3 knockdown or scramble control MDA-MB-231 cells. (F) Results of dual-luciferase reporter assay detecting the promoter activity of Oct4. Data are shown as the mean ± S.E.M of three independent experiments. *P<0.05, ***P<0.001. (G) Western blot assay detecting the expression of PHF20 and Oct4. The right penal shows the statistical result of Oct4. (H) Schematic diagram shows the mechanism on the role of JMJD3 in regulation of stemness of breast cancer.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, shRNA, Transfection, Luciferase, Reporter Assay, Activity Assay

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 6: Vitamin D analogue paricalcitol induces JMJD3 and suppresses Oct4 and stem cell-like characteristics in MDA-MB-231 cells in vitro. MDA-MB-231 cells were treated with 0, 1 or 10 nM paricalcitol for 48 h. (A) mRNA expression (left) and the statistical result (right) of JMJD3 and UTX detected by RT-PCR. (B) Expression of JMJD3 and UTX detected by western blotting and the quantification results are presented on the right. (C and D) Results of ALDH activity measurements by flow cytometry and sphere formation assays of MDA-MB-231 cells treated with paricalcitol for 7 days. Data are shown as the mean ± S.E.M of three independent experiments. *P<0.05, **P<0.01. A nude mouse model of breast cancer was established with MDA-MB-231 cells. Paricalcitol or the vehicle control were administrated via intraperitoneal injection at a dose of 0.3 μg/kg body weight once every 2 days from the 3 days before tumor cell implantation (n=5). Tumor samples were obtained at 8 weeks after injection for the following analysis. (E and F) Representative western blots of JMJD3, Oct4 and ALDH in primary tumor tissues. Statistical results are shown in the right panel. Data are shown as the mean ± S.E.M.*P<0.05, **P<0.01. (G) A limited dilution assay was performed in vivo with MDA-MB-231 cells. An orthotopic mouse model was established according to method described in Figure 1. The tumorigenic capacity is shown in the table.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Flow Cytometry, Control, Injection, Dilution Assay, In Vivo

Journal: Oncotarget

Article Title: JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

doi: 10.18632/oncotarget.15747

Figure Lengend Snippet: Figure 7: JMJD3 is required for the effects of paricalcitol on the stem cell-like properties in MDA-MB-231 cells. Stable knockdown of JMJD3 or scramble control MDA-MB-231 cells were treated with 0, 1 or 10 nM paricalcitol for 48 h. (A) The analysis of ALDH activity by flow cytometry. (B) Result of sphere formation assay of stable JMJD3-knocking down and control MDA-MB-231 cells treated with paricalcitol for 7 days. Data are from three independent experiments and are shown as the mean ± S.E.M.*P<0.05 and ***P<0.001. (C) Expression of Oct4 detected by western blotting and the quantification results are presented in the right penal.

Article Snippet: Vector construction and establishment of stable cell lines The human JMJD3 expression plasmid pCMVHA-JMJD3 was purchased from Addgene (Cambridge MA).

Techniques: Knockdown, Control, Activity Assay, Flow Cytometry, Tube Formation Assay, Expressing, Western Blot