Journal: Nature Communications

Article Title: CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice

doi: 10.1038/s41467-024-49908-4

Figure Lengend Snippet: a Experimental timeline: conditioning of adult C57BL/6 mice with total body irradiation (TBI), busulfan (BU) or treosulfan (TREO) combined or not with PLX3397 (PLX) by oral gavage 15 days post bone marrow transplant (BMT). Adult homozygous C57BL/6-CAG-GFP mice were used as bone marrow donors. The time points of the analysis are 3 months (3 M) or 7 months (7 M) post-BMT. b–f , Flow cytometry analyses. b Fraction of transplant-derived GFP+ microglia-like cells (MGLCs) measured in the brain 3 M post-BMT. c Fraction of transplant-derived GFP+ cells measured in the bone marrow (BM) 3 M post-BMT. a , b BU n = 4, BU + PLX n = 4, TBI n = 3, TBI + PLX n = 3, TREO n = 3, TREO + PLX n = 3. d Representative flow plots of CD45 + CD11b+ cells (left panel, gated on total CD45+ cells) and transplant-derived GFP+ MGLCs (right panel) measured in the brain 7 M post-BMT. e Fraction of transplant-derived GFP+ MGLCs in the brain 7 M post-BMT; U: Untreated mice. f Fractions of Ly6C + , CD3 + , and CD19+ cells in the brain of mice 7 M post-BMT, and in untreated mice. e-f BU n = 10, BU + PLX n = 7, U n = 6. g Sagittal section of the brain from a mouse treated with BU + PLX + BMT and analyzed 7 M post-BMT (image representative of n = 7 mice). h Representative images of transplant-derived GFP+ cells repopulating the olfactory bulb (OB), dentate gyrus (DG), choroid plexus (CP), and cortex (CTX) of mice conditioned with BU alone (top) or BU + PLX (bottom) and analyzed 7 M post-BMT; images representative of n = 4 mice/group. g – h Scale bars are depicted. b , c , e – f Data are Mean ± SD. Source data are provided as a Source Data file. Statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, the exact p-values of all comparisons are reported in the Source Data file; b , e , f One-way ANOVA with Tukey post-hoc; c Kruskal–Wallis test with Dunn’s. GFP: green fluorescent protein; DAPI: 4’,6-diamidino-2-phenylindole.

Article Snippet: Adult (8–12-week-old) C57BL/6 J mice (Jax strain #000664) were conditioned with busulfan (Sigma-Aldrich 14843), total body irradiation (TBI), or treosulfan (MedChemExpress HY-16503).

Techniques: Irradiation, Flow Cytometry, Derivative Assay

Journal: Oncotarget

Article Title: Stress and stem cells: adult Muse cells tolerate extensive genotoxic stimuli better than mesenchymal stromal cells

doi: 10.18632/oncotarget.25039

Figure Lengend Snippet: MSCs, non-Muse cells, and-Muse cells were treated with H202 or UV. ( A ) - The table shows the cell cycle analysis 1, 6, and 48 hr following treatments. Data are expressed in percentage (± SD, n = 3). In treated samples, the values in the ovals are statistically different ( p < 0.05) from corresponding controls. ( B ) - The graphs show results from the immunostaining analysis of proteins involved in DNA repair signaling 1, 6, and 48 hr following treatments. The mean percentages of ATM, RAD51, and DNA-PK positive cells are reported (± SD, n = 3, * p < 0.05, ** p < 0.05).

Article Snippet: To detect the different proteins involved in DNA repair, we incubated fixed cells with the following antibodies: ATM (ab36810, ABCAM, UK); g-H2AX (2577, Cell Signaling, MA, USA); RAD51 (ab88572, ABCAM, UK); and DNA-PK (sc390698, SantaCruz Biotech, CA, USA).

Techniques: Cell Cycle Assay, Immunostaining

Journal: Oncotarget

Article Title: Stress and stem cells: adult Muse cells tolerate extensive genotoxic stimuli better than mesenchymal stromal cells

doi: 10.18632/oncotarget.25039

Figure Lengend Snippet: MSCs, non-Muse cells, and-Muse cells were treated with H202 or UV. At 1, 6, and 48 hr following treatments, a flow cytometry analysis of γ-H2AX protein, combined with propidium iodide DNA staining, was performed to detect foci of damaged DNA in the different phases of the cycle. For every experimental condition, the percentages of γ-H2AX-positive cells are indicated in the table. Data are expressed with standard deviation ( n = 3). In treated samples, the values in the ovals are statistically different ( p < 0.05) from corresponding controls.

Article Snippet: To detect the different proteins involved in DNA repair, we incubated fixed cells with the following antibodies: ATM (ab36810, ABCAM, UK); g-H2AX (2577, Cell Signaling, MA, USA); RAD51 (ab88572, ABCAM, UK); and DNA-PK (sc390698, SantaCruz Biotech, CA, USA).

Techniques: Flow Cytometry, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of a Novel Noxo1 Mutant Increases Ros Production and Noxo1 Relocalisation

doi: 10.3390/ijms24054663

Figure Lengend Snippet: Antibodies references used during the study.

Article Snippet: Anti Citrate synthase , Mouse , SC390693; SantaCruz Biotechnologies , 1/400 , .

Techniques:

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