Journal: Nature communications

Article Title: BMI1 regulates androgen receptor in prostate cancer independently of the polycomb repressive complex 1.

doi: 10.1038/s41467-018-02863-3

Figure Lengend Snippet: Fig. 5 BMI1-specific antagonist inhibits AR-signaling pathway. C4-2 (a) or 22Rv1 (b) was treated with PTC209 in indicated concentration for 48 h, BMI1 and AR were tested by western blot, and GAPDH served as loading control. c C4-2 cells were treated for 48 h as labeled, vehicle as control, and the concentration of each drug was PTC209 5 µM, MG132 20 µM, NH4Cl 10 mM, and chloroquine 200 µM. Total cell lysates were blotted for AR and BMI1, while GAPDH served as loading control. d C4-2 cells were treated with PTC209 (5 µM), and after 24 h, cells were infected with BMI1 lentivirus, BMI1-RING lentivirus, or BMI1ΔRING lentivirus as indicated in the presence of PTC209, and cells were lysed after another 24 h and probed by indicated antibodies. All experiments were biologically repeated at least three times. Representative images are shown

Article Snippet: Drug concentrations (unless otherwise indicated) were PTC209 (0, 1, 5, and 10 μM, HY-15888, MedChem Express), enzalutamide (IN034, Dieckmann), MDM2 inhibitor 373225 (25 μM, 50 μM, Millipore), MDM2 inhibitor 373226 (5 μM, 10 μM, Millipore), PRT4165 (100 μM 5047, TOCRIS), MG132 (CAS 133407-82-6 Calbiochem), ammonium chloride (NH4Cl, 12125-02-9, Sigma), chloroquine diphosphate salt (C6628, Sigma), and cycloheximide (CHX, C7698, Sigma), and all were commercially obtained.

Techniques: Concentration Assay, Western Blot, Control, Labeling, Infection

Journal: bioRxiv

Article Title: PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion

doi: 10.1101/2023.01.23.525196

Figure Lengend Snippet: Genetic and pharmacological disruption of polycomb proteins in BT-549 cells. (A) Expression levels for polycomb subunit-encoding genes (publicly available RNA-seq data from cBioPortal) is shown for TNBC-derived cell lines versus non-cancer mammary epithelial cells (HMEL). Mesenchymal: BT-549, MDA-MB-231, MDA-MB-436; basal: HCC2157, MDA-MB-468, HCC70, HCC1806; unclassified: BT-20, HCC1500. (B) Western blots of lysates from hTERT-HME1 (non-cancer) and BT-549 cells with antibodies against the indicated polycomb proteins or loading controls. (C) Overview of polycomb targeting strategies that were tested in BT-549 for comparison in this study. Genetic knock-down via siRNA against EZH2 and PCGF4 (BMI1) in BT-549 cells was confirmed by western blot. (D) PCA plot of normalized expression levels for 177 genes in all conditions (four independent treatments each) determined by a Nanostring assay. (E) Average baseline normalized expression of 177 genes (rows) for the control samples (DMSO and siCtrl) and log2 fold change expression values for all treatments. Genes symbols and numerical values are provided in Supplemental Table S1 . (F) H3K27me3 ChIP-seq data for BT-549 (DMSO-treated) from Lehmman et al. is shown in the transcription start site (TSS) plot, with rows corresponding to the order of rows in panel E.

Article Snippet: 24 hours after seeding, 50 μL of diluted compound was added to each cell sample to generate final concentrations of EZH2 inhibitors GSK126 (5.7 μM; Sigma, 5005800001) or GSK343 (9.5 μM; Sigma, SML0766), BMI1 inhibitors PTC209 (4.3 μM; Selleck Chemicals, S7372) or PTC596 (20.8 μM; Selleck Chemicals S8820), and CBX4/7 inhibitor UNC3866 (30 μM; Sigma, SML2408).

Techniques: Disruption, Expressing, RNA Sequencing, Derivative Assay, Western Blot, Comparison, Knockdown, Control, ChIP-sequencing

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Cells treated with PTC 209 and FMMC 419II cells stably transfected with Bmi1 shRNA plasmid show a decrease in Bmi1 mRNA expression. (B) Purified mRNA from the cells was reverse transcribed into cDNA and then analyzed for Bmi1 mRNA expression with quantitative PCR using TaqMan gene expression assays. The fold difference in expression between control samples and the PTC 209 treated of the Bmi1 shRNA transfected samples was calculated using the average of the Ct (threshold cycle) per group, relative to the expression of the internal control gene GAPDH . (C) FMMC cells were treated with PTC 209 (2 μM and 5 μM) for 24 hours and the expression of Bmi1 protein was detected with western blot. (D) PTC 209 treatment decreased Bmi1 protein expression in FMMC cells. Results are represented as mean ± S.E.M., *P <0.01, ***P <0.005.

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Expressing, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Control, Western Blot

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Untreated (control) FMMC 419II cells. (B) Cells treated with 2 μM PTC 209. (C) Cells treated with 5 μM PTC 209. (D) Results of PTC 209 treatment shown as bar graphs. There is a G0/G1 cell cycle arrest in FMMC 419II cells treated with PTC 209 when compared to untreated cells. Similarly, FMMC 419II cells that have been transfected with a Bmi1 shRNA show a G1 arrest. (E) Bar graphs of cell cycle profiles for FMMC 419II cells from control (F) , colony 2 (G) , colony 4 (H) , and colony 5 (I) . Cells stained with PI/RNAse staining buffer were run on a FACSAria flow cytometer and cell cycle progression was analyzed and quantified (D, E) using FlowJo.

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Control, Transfection, shRNA, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: Flow cytometry analysis of CD49f and CD24 expression for FMCC 419II cells treated with 2 μM PTC 209 versus Bmi1 shRNA transfected colonies 4 and 5 were carried out (A) , and the fluorescent intensities were quantitated (B) .

Article Snippet: For treatment with the Bmi1 small molecule inhibitor PTC 209 (Xcess Biosciences, San Diego, CA), cells were trypsinized, washed, and seeded into 6-well plates at around 30% confluency.

Techniques: Flow Cytometry, Expressing, shRNA, Transfection

Journal: ACS Medicinal Chemistry Letters

Article Title: Synthesis of Cyanoenone-Modified Diterpenoid Analogs as Novel Bmi-1-Mediated Antitumor Agents

doi: 10.1021/acsmedchemlett.8b00345

Figure Lengend Snippet: Western blot analysis of Bmi-1, H2AK119Ub (ub-H2A), H2A, and Ring1b levels in HCT116 cells. The expression of Bmi-1, ub-H2A, H2A, and Ring1b was detected by Western blot analysis in HCT116 after incubated with indicated concentrations of 33 or PTC-209 for 8 h.

Article Snippet: PTC-209 (belonging to PTC Therapeutics, ), which was identified by high-throughput screening of compounds using gene expression modulation by small molecules (GEMS) technology, has been reported as the first preclinical Bmi-1 inhibitor.

Techniques: Western Blot, Expressing, Incubation