Journal: Cell Reports Medicine

Article Title: Single-cell proteomics defines the cellular heterogeneity of localized prostate cancer

doi: 10.1016/j.xcrm.2022.100604

Figure Lengend Snippet:

Article Snippet: anti-human-EZH2 (SP129) , Spring Bioscience , Cat# 5246; RRID: AB_10694683.

Techniques: Recombinant, Blocking Assay, Electron Microscopy, Labeling, Mass Cytometry, Software

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: Glioblastoma multiforme tumors express BMI1 and EZH2. A, Formalin-fixed paraffin-embedded GBM samples were immunolabeled with antibodies against BMI1 or EZH2 (brown) and GFAP or Nestin (pink). Arrowheads point to double positive cells. B, Frozen GBM samples were immunolabeled with antibodies against BMI1 (red) and CD133 (green). DAPI (blue) stains nuclei. Arrowheads point to double positive cells. C, RT-PCR analysis performed on a freshly isolated GBM sample with primers against GAPDH, BMI1, MUSASHI, SOX2, and LHX2. D, Frozen GBM samples were immunolabeled with antibodies against BMI1 (red) and CD31 (green). DAPI (blue) stains nuclei. Arrowheads point to BMI1 cells surrounding a blood vessel. Dashed lines (in A and B) delineate the border of the tumor. Scale bars, 100 μm.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Formalin-fixed Paraffin-Embedded, Immunolabeling, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 and EZH2 are highly enriched in CD133+ cells. A, Frozen GBM neurospheres were immunolabeled with antibodies against BMI1 (top), or GFAP and MAP2 after plating in medium-induced differentiation (bottom). DAPI stains nuclei in blue. B, RT-PCR analysis performed on cultured neurospheres with primers against GAPDH, BMI1, MUSASHI, SOX2, and LHX2. C, Western blot analysis performed on freshly isolated GBM sample and neurospheres using anti-EZH2 and anti-β-actin antibodies. D, CD133− and CD133+ purified GBM cells with CD133-coupled magnetic beads were analyzed by real-time PCR for the expression of PROMININ (CD133), BMI1, and EZH2 transcripts. Data are expressed as fold change over gene expression in negative cells (CD133−), which was set at 1. Results are mean ± SD (n = 3; *p < 0.05; **p < 0.01). E, Dissociated neurospheres were stained with anti-BMI1 and anti-CD133 antibodies. The R1 gate delineates the cell population analyzed. Values are the percentages of cells in the corresponding regions. F, Gene copy number was assessed by quantitative real-time PCR in GBM samples. Data are expressed relative to healthy human retina genomic DNA and calibrated to β-actin used as internal standard. Another internal standard was used which consisted of PAX6. Scale bars, 60 μm.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Immunolabeling, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Western Blot, Isolation, Purification, Magnetic Beads, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Staining

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 or EZH2 knockdown reduces GBM cells clonogenic potential and leads to depletion of the CD133+ cell population. A, shRNA downregulation of BMI1 and EZH2 expression. Western blot analysis performed on shBMI1- or scramble-infected 293FT cells using anti-BMI1, anti-EZH2, and anti-β-actin antibodies. β-Actin was used as an internal standard to quantify BMI1 and EZH2 expression levels (left and middle). GBM cells were infected with one of two lentiviruses expressing shBMI1, or scramble virus, and were analyzed by real-time PCR for the expression of BMI1 transcripts. Data are expressed as fold change over BMI1 expression in the scramble-infected cells, which was set at 1. Results are mean ± SD (n = 3; **p < 0.01) (right). B, ShBMI1 or scramble-infected GBM cells were plated on Matrigel substrate. Forty-eight hours later, hygromycin selection was added and cells were allowed to grow for 10 d. Cultures were fixed and stained with cresyl violet, and phase contrast micrographs were taken. C, ShBMI1 or scramble-infected GBM cells were plated on non-adherent culture plates. Forty-eight hours later, hygromycin selection was added and cells were allowed to grow for 2 weeks. D, Cells cultured in C were followed for 5 passages. Colonies were passaged every 2 weeks. Results are cumulative cells number (log scale) over consecutive passages. Note that shBMI1-#1-infected cells were depleted after the first passage. E, GBM cells cultured as in C were analyzed for apoptosis using Annexin-V staining and FACS analysis. Data are expressed as the percentage of Annexin+/7-AAD− cells (mean ± SD; n = 3; *p < 0.05). F, ShBMI1 or scramble-infected GBM cells were plated on gelatin-coated chamber slides and allowed to differentiate for 7 d. Cultures were analyzed by immunofluorescence using antibodies against GFAP, or MAP2. Data are expressed as the percentages of positive cells over the total number of DAPI-stained nuclei (n = 3; *p < 0.05). G, GBM cells cultured as in C were analyzed by real-time PCR. Data are expressed as fold change over gene expression in the scramble-infected cells, which was set at 1. Results are mean ± SD (n = 3; *p < 0.05; **p < 0.01).

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Knockdown, shRNA, Expressing, Western Blot, Infection, Virus, Real-time Polymerase Chain Reaction, Selection, Staining, Cell Culture, Immunofluorescence, Gene Expression

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: High cell density prevents CD133+ cell depletion after BMI1 knockdown. A, Experimental scheme used to study differential gene expression (Microarray), BMI1 binding to candidate genes (ChIP), and BMI1 function in vivo. B, Scramble or BMI1 shRNA-infected neurospheres were dissociated and stained with anti-CD133 antibodies. The R1 gate delineates the cell population analyzed. All cells analyzed were GFP positive. The values are the percentages of CD133+ and CD133− in both cultures. The panel to the right shows CD133+ and CD133− cells as viewed under a fluorescence microscope. C, Size distribution of spheres obtained from scramble or shBMI1-infected CD133+ or CD133− sorted cells after 5 d of culture. Phase contrast pictures are shown.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Knockdown, Gene Expression, Microarray, Binding Assay, In Vivo, shRNA, Infection, Staining, Fluorescence, Microscopy

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 is required for intracranial GBM tumor formation. A, Kaplan–Meyer representation of the survival curves of mice injected with naive cells, cells infected with an empty virus, a scrambled or a ShBMI1 viruses. *Median survival for this mouse strain was previously reported (mean ± SD; 191 ± 11 d) (Christianson et al., 1997). B, Data summary from the Kaplan–Meyer curves shown in A. Statistical significance was assessed using the log rank test relative to scramble. C, Representative images of brains bearing secondary tumors following engraftment of scrambled virus-infected GBM cells. Tops Hemorrhagic tumor (dashed lines). Bottom, Nonhemorrhagic tumor. Cresyl violet coloration showed that GBM cells localize mainly in the grafted right hemisphere with some spreading in the contralateral hemisphere. D, Frozen sections of a brain injected with shBMI1-infected GBM cells were immunolabeled with antibodies to human nuclei (green). Nuclei were visualized by DAPI (blue). The grafted cells were confined to the injection site. The contralateral left hemisphere was used as a nongrafted control. Scale bar, 200 μm.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Injection, Infection, Virus, Immunolabeling, Control

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: Engrafted tumors resemble the tumor of origin and retain PcG proteins expression. Formalin-fixed paraffin-embedded sections of brains injected with scrambled virus-infected GBM cells were analyzed by immunohistochemistry. A, Sections were immunolabeled with antibodies against PCNA and GFAP. B, Sections were immunolabeled with antibodies against GFP (from the lentiviral transgene). C, Sections were immunolabeled with antibodies against human mitochondria and Ki67. D, Sections were immunolabeled with antibodies against BMI1, EZH2, GFAP, and Nestin. Dashed line delineates the border of the tumor. Scale bar, 100 μm.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Injection, Virus, Infection, Immunohistochemistry, Immunolabeling

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 knockdown results in glioma cell apoptosis in vivo. A, B, Formalin-fixed paraffin-embedded sections of brains injected with scrambled- or shBMI1-infected GBM cells were analyzed by immunohistochemistry 10 d postinjection. A, Sections were stained with hematoxylin and eosin. The tumor cell mass was consistently found in the right ventricle. B, Sections were immunolabeled with antibodies against GFP (carried by the transgene), or cleavage-activated caspase-3. GFP immunoreactivity revealed that tumor cells are in close association with the microvasculature. Caspase-3 immunoreactivity uncovered that BMI1 knockdown cells undergo massive apoptosis. Scale bars, 50 μm.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Knockdown, In Vivo, Formalin-fixed Paraffin-Embedded, Injection, Infection, Immunohistochemistry, Staining, Immunolabeling

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 knockdown induces an upregulation of critical cell death, growth arrest, and cell differentiation pathways

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Knockdown, Cell Differentiation, Immunopeptidomics, Membrane

Journal: The Journal of Neuroscience

Article Title: BMI1 Sustains Human Glioblastoma Multiforme Stem Cell Renewal

doi: 10.1523/JNEUROSCI.0968-09.2009

Figure Lengend Snippet: BMI1 promotes GBM tumor formation and stem cell renewal by blocking alternate tumor suppressor pathways. A, Schematic representation of the genomic DNA sequence of P21Cip1. The boxes represent the exons. The primers used were chosen in the promoter region. B, ChIP was performed with cultured GBM cell extracts. Immunoprecipitated DNA/protein complexes were analyzed by real-time PCR using primers directed against the P21 (5 sites), HOXC13 (2 sites), andβ- MAJOR promoters. Data are expressed as fold change relative to the input, which was set at 1. Results are mean ± SD (n = 5; *p < 0.05; **p < 0.01). C, Model of BMI1 function in gliomas. Crossed lines highlight the most common mutations and deletions found in GBM (CGAR, 2008). Dashed lines represent the classical targets of BMI1. Red lines represent possible new BMI1 targets, as reported in this study. Based on this finding, we propose that GBM tumor dependency over BMI1 is mediated through BMI1 repressive action on alternate tumor suppressor pathways that attempt to overcome INK4A/ARF/P53 inactivation and PI(3)K/AKT hyperactivity.

Article Snippet: Proteins were resolved in Laemmli buffer by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (Pall) that was exposed to the primary antibodies; mouse anti-mouse Bmi1, rabbit anti-human EZH2 (US Biological), and mouse-anti β-actin (Abcam) antibodies.

Techniques: Blocking Assay, Sequencing, Cell Culture, Immunoprecipitation, Real-time Polymerase Chain Reaction