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: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Structure of EV-A71 inhibitors, PTC-209HBr and derivatives .

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques:

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Antiviral effect of PTC-209HBr and enviroxime in vitro . (A–H) PTC-209HBr or enviroxime significantly inhibited viral RNA level accumulation; viral protein expression and robustly impeded viral-caused CPE. Vero or RD cells were pretreated with EV-A71 at an MOI of 0.1 and then cells were incubated at 4 °C for 1 h, followed by treatment of the indicated concentration of PTC-209HBr or enviroxime for 24 h at 1 hpi. Relative viral RNA levels and protein levels in dose-dependent manner were analyzed by qRT-PCR (A, B) and Western blot (C, D), respectively. Further antiviral efficacy of PTC-209HBr against EV-A71 was tested by IF in Vero cells (E) and a CPE assay in RD cells (F). Further antiviral effects of enviroxime against EV-A71 were tested by IF in Vero cells (G) and CPE assay in RD cells (H).

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: In Vitro, Expressing, Incubation, Concentration Assay, Quantitative RT-PCR, Western Blot

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Antiviral effect of PTC-209HBr and enviroxime against the selected specieses of Enterovirus (genus). (A, B) Concentration-response profiles used for EC 50 determination are shown for enterovirus inhibitors (A) PTC-209HBr and (B) enviroxime. Data points are means of triplicates SEM, for curve-fitting, described in Materials and Methods. One representative dataset of at least two independent experiments is shown. (C) EC 50 s of PTC-209HBr and enviroxime for selected enterovirus species were determined in short-term treatment assay by CPE (Materials and Methods). Representative EC 50 values from 2 to 3 independent experiments are given.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Concentration Assay

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Selection of PTC-209HBr resistance and sensitivity to VP1 mutants. (A) The PTC-209HBr resistance profiling. (B) The sensitivity of PTC-209HBr to VP1 mutant viruses, which was determinated by a CPE assay. (C) EC 50 s of PTC-209HBr for VP1 mutant EV-A71 were determined in short-term treatment assay by CPE. (D) The sensitivity of PTC-209HBr to VP1 to PTC-209HBr at a concentration of 3.56 μM was determined by a plaque assay. For the plaque assay, RD cells were infected with EV-A71 or mutants at an MOI of 2 × 10 −4 . For the CPE assay, RD cells were infected with EV-A71 at an MOI of 0.1.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Selection, Mutagenesis, Concentration Assay, Plaque Assay, Infection

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Binding affinities of PTC-209HBr, derivatives, pirodavir and enviroxime among VP1 or VP1 mutants were determined by MST. (A) The change in fluorescence to thermophoresis at the increasing concentrations of PTC-209HBr (4.8–156000 nM) in the presence of 500 nM VP1-EGFP. (B) The binding affinities of PTC-209HBr, derivatives (12s, 12t), pirodavir and enviroxime among VP1 or VP1 mutants.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Binding Assay, Fluorescence

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: The action stage of PTC-209HBr, enviroxime and pirodavir was determined by a time-course assay. PTC-209HBr, enviroxime and pirodavir at 1.78 μM, 0.56 μM and 0.72 μM were added to the EV-A71-infected RD or Vero cells at the indicated time points. Samples were harvested at 12 hpi as described in the Materials and Methods. (A) Western blotting was carried out to assess the accumulation of VP1 expression in the presence of PTC-209HBr, enviroxime and pirodavir (A, B). The relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr, enviroxime and pirodavir were determined by qRT-PCR. One set of representative data out of at least two independent experiments with standard errors are showed.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: PTC-209HBr impedes EV-A71 binding and entry. The binding and entry assays were quantitated by qRT-PCR or Western blot. PTC-209HBr, enviroxime and pirodavir at specific concentration were added to the EV-A71-infected RD or Vero cells at the indicated time points. Samples were harvested at indicated time points as described in the Materials and Methods. (A) qRT-PCR was carried out to assess relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr (3.2 μM), enviroxime (0.32 μM) and pirodavir (0.64 μM) during the binding stage. (B) PTC-209HBr decreased EV-A71 infection in a dose-independent manner during the binding stage. (C) qRT-PCR was carried out to assess relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr (3.2 μM), enviroxime (0.32 μM) and pirodavir (0.64 μM) during the entry stage. (D) PTC-209HBr dose-dependently decreased EV-A71 infection during the entry stage. (E) The accumulation of VP1 expression in the presence of PTC-209HBr, enviroxime and pirodavir was analyzed by a Western blot assay during the binding stage. One set of representative data out of at least two independent experiments with standard errors are showed.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Infection, Expressing

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Colocalization of EV-A71 VP1 and hSCARB2 in the presence of PTC-209HBr, enviroxime and pirodavir. (A) The colocalization of EV-A71 VP1 and hSCARB2 in the presence of PTC-209HBr, enviroxime or pirodavir. The yellow square indicates the colocalization of EV-A71 VP1 and hSCARB2. EV-A71 was mixed with the indicated concentration of PTC-209HBr and then incubated at 37 °C for 1 h. Then, the mixtures were incubated with RD cells at 4 °C for 1 h, washed with cold PBS three times, fixed with 10% formaldehyde solution and further subjected to IF analysis. These data were acquired from at least two independent assays. (B) Inhibition of the interaction between VP1 and hSCARB2 by PTC-209HBr was analyzed by Co-IP. HEK293T cells were co-transfected with Flag-VP1 and HA-hSCARB2, followed by the treatment of PTC-209HBr or DMSO at 24h post transfection. Cell lysates were immunoprecipitated with an anti-Flag antibody and were then analyzed by a Western blot assay.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Concentration Assay, Incubation, Inhibition, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Colocalization of EV-A71 variants and hSCARB2 in the treatment of PTC-209HBr or DMSO. The yellow square indicates the colocalization of VP1 and hSCARB2. EV-A71 was mixed with the indicated concentration of PTC-209HBr and then incubated at 37 °C for 1 h, followed by incubation with RD cells at 37 °C for 1 h. The treated cells were washed with cold PBS five times and then incubated at 37 °C and 5% CO 2 for 6 h. Cells were fixed at 6 hpi and then analyzed by IF. These data were acquired from at least two independent assays.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Concentration Assay, Incubation

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: Proposed docking model of PTC-209HBr with EV-A71 VP1 protein. (A) Molecular docking model of the binding between PTC-209HBr and EV-A71 capsid protein VP1. (B) The predicted amino acid residues may bind with PTC-209HBr or be related to resistance to TC-209HBr. The hydrogen bond is showed as a red dotted line, and some hydrophobic amino acids within 5 Å are labeled. The oxygen atom (red), nitrogen atom (blue) and sulfur atom (yellow) are also shown.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Binding Assay, Labeling

Journal: Cell Insight

Article Title: Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

doi: 10.1016/j.cellin.2022.100016

Figure Lengend Snippet: The proposed model of PTC-209HBr inhibition of EV-A71. The model of the interaction between hSCARB2 and EV71 protomer as previously reported ( Zhou et al., 2019 ). VP1, VP2, VP3, VP4 and hSCARB2 are coloured in blue, green, red, yellow and orange, respectively.

Article Snippet: PTC-209HBr, pirodavir and enviroxime were purchased from Selleck (S7539), Targetmol (T1750) and MedKoo (319912), respectively.

Techniques: Inhibition

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 1. Schematic diagram of preparation and proposed mechanism of action of

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques:

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 2. Physicochemical characterization of the NPs and HA-NPs-PTC209. (A) FT-IR spectra of

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques: Zeta Potential Analyzer

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 4. In vitro stemness reversion of colon cancer cells. (A) Clone formation assay of CT26 can-

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques: In Vitro, Tube Formation Assay

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 5. Ex vivo tumor targeting activity of HA-NPs-PTC-209. (A) Ex vivo fluorescence images of

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques: Ex Vivo, Activity Assay, Fluorescence

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 6. In vivo antitumor activity of HA-NPs-PTC-209. (A) Experimental timeline for the drug

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques: In Vivo, Activity Assay

Journal: Biomaterials

Article Title: Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment.

doi: 10.1016/j.biomaterials.2019.119247

Figure Lengend Snippet: Figure 7. In vivo stemness reversion in the colon cancer tumor model. (A) Immunohistochemical

Article Snippet: Briefly, 10 mg NH2-PEG-PLGA and mPEG-PLGA (1:1) (75:25, MW-5000:15000, Xi’an Ruixi Biological Technology Co., Ltd), dissolved in 900 μL methylene chloride, and PTC209 (T2345-50 mg, TargetMol) dissolved in 100 μL DMSO/methanol (1:9) were transferred to a 10 mL centrifuge tube and then 200 μL water was also added.

Techniques: In Vivo, Immunohistochemical staining

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