bmi1 inhibitor ptc 209  (Tocris)

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Schema of BMI1-induced SREs (iSRE) establishment and culture from PBMCs. B Lentiviral transduction leads to an approximately four-fold increase in BMI1 transcript levels in iSREs compared to compared to untransduced (UT) SREs and empty vector transduced (EV) SREs at day 13 of expansion, which have, as expected, similar levels of BMI1. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on paired two-tailed Student's t -test. ***p < 0.0001. Source data are provided as a Source Data file. C Lentiviral transduction leads to an approximately four-fold increase in BMI1 protein levels compared to compared to UT and EV SREs at day 13 of expansion. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on paired two-tailed Student's t -test **p < 0.01. Source data are provided as a Source Data file. D iSREs have approximately a 10 4 -fold expansion, compared to untransduced (UT) SREs and empty vector transduced (EV) SREs, which have similar limited (200-300-fold) expansion. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. E FACS-based analysis of iSRE cultures reveals 3 subpopulations of cells. Population 1 is CD36+CD235 lo and consist of cells with an immature erythroblast morphology. Population 2 is CD36 + CD235+ and consists of cells with a slightly more mature morphology. Population 3 is CD36 lo CD235+ and consist of smaller hemoglobinizing cells with condensed nuclei. Cells are colored based on GFP expression, which is highest in Population 1 and lowest in Population 3. Size bar = 10um. A representative example of three independent experiments from three donors is shown. F Enrichment of immature erythroblasts by weekly fluorescence-activated cell sorting (FACS) leads to a 10 12 -fold expansion of iSREs, compared to untransduced (UT) and empty vector transduced (EV) control SREs. 3 independent iSRE cultures derived from 3 donors.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: Transduction, Plasmid Preparation, Derivative Assay, Two Tailed Test, Expressing, Fluorescence, FACS, Control

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Morphology of self-renewing iSREs at expansion day 34. Size bar = 10 um. Representative example of 6 independent cultures derived from 3 donors. B Surface expression of CD71, CD44, CD49d, and CD34 on iSREs at expansion day 30. Representative plots from one of three independent iSRE cultures. C Consistency of cell and nuclear area of iSREs on days 15, 30, and 55 of culture, as analyzed by imaging flow cytometry. Data from 3 independent cultures derived from 3 donors, mean ± SEM. D Karyotype of iSREs at day 45 of culture. Data from 1 of 3 independent cultures are shown (see Supplementary Fig. for additional two donors). E Principal Component Analysis (PCA) of global transcriptomes of CD34-derived erythroid populations (GEO GSE53983 and GSE128268 ) , and of iSREs (Supplementary Fig. ) indicate that iSREs are closest in gene expression identity to proerythroblasts (ProE). HSPC = hematopoietic stem and progenitor cells, BFU-E = burst forming units erythroid, CFU-E = colony forming units erythroid, EBasoE = early basophilic erythroblasts, LBasoE = late basophilic erythroblasts, PolyE = polychromatophilic erythroblasts, and OrthoE = orthochromatic erythroblasts. F BMI1 and EEF1A1 transcript levels during the differentiation trajectory of CD34+ HSPCs to OrthoE (GEO GSE53983 and GSE128268 ) , .

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: Derivative Assay, Expressing, Imaging, Flow Cytometry, Gene Expression

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Morphology of terminally maturing iSREs after 8 days of maturation culture. Representative example of 6 independent cultures derived from 3 donors. B Flow-based assay of enucleation of iSREs after 8 days of maturation. Representative example of 4 independent iSRE cultures derived from 3 donors. Percent gated is the mean ± SEM of the replicates. C Globin gene expression of iSREs matured for 3 days and quantified by qPCR. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. Source data are provided as a Source Data file. D BMI1 transcript levels rapidly decrease during in vitro maturation of iSREs. 4 independent iSRE cultures derived from 3 donors. Paired two-tailed Student's t -test * p = 0.027. Source data are provided as a Source Data file. E Flow-based surface expression of CD235a, Band3, Kell, Glycophorin C (GPC), and Rh on day 8 matured iSREs compared to FMO (fluorescence minus one) controls. F Gel column assay of 1.5 million terminally maturing iSREs after 7 days of maturation culture . Absence of antibody served as a negative control. G Gel column assay of expression of Rh antigens on D + C + c + E-e+ terminally maturing iSREs. H Gel column assay of Glycophorin B (S+, s-), Kell (K-, k+), and Duffy Fy(a-b+) blood groups on terminally maturing iSREs. I Gel column assay of type A terminally maturing iSREs using plasma from a type O and a type A donor. J Ficin-treated terminally maturing iSREs agglutinated with plasma from type A patients containing anti-D, anti-C, or anti-e, respectively.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: Derivative Assay, Gene Expression, In Vitro, Two Tailed Test, Expressing, Fluorescence, Negative Control, Clinical Proteomics

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A shRNA knockdown of BMI1 decreases BMI1 transcript levels compared to control luciferase shRNA culture. 3 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on one-tailed Student's t -test. ** p < 0.01. B shRNA knockdown of BMI1 leads to rapid collapse of iSRE cultures. Representative example shown, 3 independent iSRE cultures derived from 3 donors. C PTC-209 inhibition of BMI1 for 24 hours decreases BMI1 transcript levels compared to vehicle-treated controls. 3 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on a one-tailed Student's t -test. ** p = 0.010. *** p = 7.8e-6. D PTC-209 treatment leads to a dose-dependent inhibition of iSRE self-renewal. Representative example shown of 3 independent iSRE cultures, derived from 3 donors. E iSRE self-renewal remains dependent on the continued presence of exogenous erythropoietin (EPO), stem cell factor (SCF), and Dexamethasone (Dex). 6 independent iSRE cultures from 3 donors, mean ± SEM. All experiments were conducted with iSREs between expansion days 25 and 35. Source data are provided as a Source Data file.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: shRNA, Knockdown, Control, Luciferase, Derivative Assay, One-tailed Test, Inhibition

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: See Supplementary Fig. for samples used in CUT& RUN. A shRNA knockdown of RING1B leads to rapid collapse of iSRE cultures. p -value was calculated based on two-way ANOVA of all RING1B shRNA vs Luciferase. 2 independent iSRE cultures from 1 donor, mean ± range, expansion day 30. B Heatmaps of BMI1 enrichment in untransduced (UT) SREs and iSREs over union peaks (±2 kb). The color scale represents the RPKM of each sample using merged replicates. Ranked based on mean RPKMs. C Volcano plot showing differential occupancy of BMI1 in iSRE versus untransduced SREs. Significantly increased and decreased peaks are shown in red and blue, respectively, with FDR < 0.05. D Heatmaps of BMI1 occupancy, RING1B occupancy, H2A119ub, and H3K27me3 over BMI1 differentially increased (top) and decreased (bottom) peaks in untransduced SREs and in iSREs. The color scale represents RPKM of each sample using merged replicates. Ranking was sorted based on BMI1 binding in iSREs. Blue line- increased BMI1 peaks. Orange line- decreased BMI1 peaks. E Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over 2 clusters of BMI1 differentially increased peaks (kmeans=2, clustering based on H3K27me3 in iSREs) in untransduced SREs and in iSREs. The color scale represents RPKM of each sample using merged replicates. Ranking was based on BMI1 binding in iSREs. Blue line- Cluster 1. Orange line- Cluster 2. F Table of selected published gene sets that contain significant overlap with CUT&RUN gene sets (Enrichr). See Supplementary Data for details.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: shRNA, Knockdown, Luciferase, Binding Assay

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: See Supplementary Fig. for samples used in CUT&RUN and Supplementary Fig. for samples used in RNA-Seq. A Genomic annotations of BMI1 differentially increased peaks (left) and decreased peaks (right). B Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over genes (scaled TSS to TES ± 2 kb) associated with iSRE BMI1 peaks in untransduced SREs and iSREs. The color scale represents RPKM of each sample using merged replicates ranked based on the binding of BMI1 in iSREs. C Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over promoters (TSS ± 2 kb) of differentially expressed, upregulated (top) and downregulated (bottom), genes in untransduced SREs and in iSREs as determined by DeSeq2 analysis of RNA-Seq. The color scale represents RPKM of each sample using merged replicates ranked based on BMI1 binding in iSREs. Blue line- upregulated genes. Orange line- downregulated genes. D Quantitation of log2-fold change (iSRE/SRE) for BMI1, RING1B, H2A119ub, and H3K27me3 over promoters (TSS ± 500 bp) of the same differentially expressed genes shown in Fig. 6C. Box is Q1-Q3 with bar at the median and whiskers represent Q1-1.5x IQR and Q3 + 1.5x IQR. Statistics for log2 -old change is not equal to 0 based on a two-tailed Wilcoxon signed rank test with continuity correction. **** p < 2.2e−16, ns not significant. E Table of published gene sets with significant overlap with differentially upregulated and downregulated genes that have BMI1 associated with their promoters indicated on the left (Enrichr). See Supplementary Data for details.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: RNA Sequencing, Binding Assay, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Occupancy of indicated factors and presence of CpG islands (purple) in SRE (black) and iSRE (green) at the INK/ARF locus. Data represent merged replicates for all CUT&RUN studies. B The INK/ARF genes are expressed at lower levels in iSREs compared to SREs at day 13 of expansion. 6 independent cultures derived from 3 donors. C Relative expression of INK/ARF genes in day 25-35 iSRE following 24 hours of PTC-209 treatment relative to DMSO (Veh) control. N = 3 for p16. N = 5 for CDKN1B and CDKN2B, each derived from 2 donors. D Expression of BMI1 and p16 relative to luciferase (Luc) at 24 hours after siRNA treatment of expansion day 41-46 CD34-derived iSREs. N = 7 for Luc and BMI1-1 and N = 4 for BMI1-2, each derived from 2 donors. N = 3 for p16 with iSREs derived from 2 donors. E PTC-209 for 12 hours decreases the percentage of iSREs in S-phase in a dose-dependent manner compared to vehicle (Veh)-treated controls. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. F PTC-209 treatment of iSREs for 48 hours leads to dose-dependent increases of G0 cells. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. G PTC-209 treatment of iSRE for 12 hours leads to dose-dependent increases in early apoptotic cells. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. H Venn Diagram showing overlap in BMI1 occupied ribosomal protein genes (yellow) and ribosomal protein genes differentially upregulated in iSREs vs. SREs (blue). I Occupancy of indicated factors in SRE (black) and iSRE (green), and CpG islands (purple) at the Ribosomal Protein S19 ( RPS19 ) locus. Data represent merged replicates for all CUT&RUN studies. J Occupancy of indicated factors in SRE (black) and iSRE (green) and compared with RNA-seq reads at the BMI1 locus. Boxed region is not present in the viral overexpression construct and thus represents endogenous gene expression. All graphs plot mean ± SEM. p values were calculated using a two-tailed Student's t -test. * p < 0.05 ** p < 0.01 *** p < 0.001. Source data are provided for as a Source Data file.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: Derivative Assay, Expressing, Control, Luciferase, RNA Sequencing, Over Expression, Construct, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Cholesterol homeostasis depends on cholesterol synthesis, import, export and storage. B CUT&RUN studies reveal BMI1 and RING1B bind the HMGCR locus at higher levels in iSRE than SRE. Data represent merged replicates for all CUT&RUN studies. C The BMI1 inhibitor PTC-209 decreases the expression of BMI1, HMGCR, and HMGCS1 in iSREs at 25 to 35 days of expansion. In contrast, PTC-209 treatment increases expression of p16. 6 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors, mean. ± SEM. p -value was calculated using a two-tailed Student's t -test. * p < 0.05, ** p < 0.01. Source data are provided as a Source Data file. D iSREs contain cytoplasmic droplets that stain with filipin (cholesterol). Representative example of one of two cultures shown. E iSREs contain cytoplasmic droplets that stain with Nile red (lipids). Representative example of one of two cultures shown. F iSREs contain higher levels of cholesterol (filipin stain) compared to primary human proerythroblast (ProE). Staining of both cell populations normalized to primary orthochromatic erythroblasts (OrthoE). Human marrow samples derived from 3 donors. 4 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors, mean ± SEM. Two-tailed Student's t -test. ** p < 0.01. Source data are provided as a Source Data file. G iSREs contain higher levels of lipid rafts (CT-B staining) compared to primary human ProE. Staining of both cell populations normalized to primary OrthoE. Human marrow samples derived from 3 donors. 2 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors. Paired two-tailed Student's t -test. * p < 0.05 mean ± range. Source data are provided as a Source Data file. H While short-term removal of exogenous lipids does not alter iSRE proliferation, concomitant block of cholesterol synthesis with Ro 48 obviates iSRE proliferation, which can be partially rescued by addition of exogenous lipids. 3 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors. p -value was calculated based on two-tailed Student's t -test, mean ± SEM. ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: The BMI1 inhibitor PTC-209 (5191, Tocris Bioscience) was suspended in DMSO and added to iSRE cultures at final concentrations ranging from 0.5 – 12.5 μM.

Techniques: Expressing, Derivative Assay, Two Tailed Test, Staining, Blocking Assay

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a Association between BMI1 expression and overall survival in patients with hepatocellular carcinoma ( n = 194 patients, our cohort). b Correlation studies between BMI1 expression and tumor size in HCC patients ( n = 194 patients, our cohort). c Association between BMI1 expression and HCC cancer stages in TCGA (phs000178.v11.p8) ( n = 350 patients). d Kaplan–Meier overall survival study of HCC patients with or without BDTT ( n = 194 HCC patients, our cohort). e Correlation between BMI1 expression and overall survival in HCC patients with or without BDTT ( n = 194 HCC patients, our cohort). f In HCC patients, high BMI1 expression associated with increased incidence of BDTT. Representative pictures of H&E and BMI1 immunohistochemical staining in tumors derived from each group are given. Arrow indicates the position of bile duct. Magnified pictures from each group are given. g, h The relationship between TIC-related protein signature (including CD44/CD133/SOX9) and BMI1 expression in HCC patients. Dotted lines indicate the borders between tumor (T) and the non-tumor compartments (NTC). i, j Correlation studies between TIC-related protein signature expression and BDTT incidence/overall survival in HCC patients ( n = 194 patients, our cohort). k Association between TIC-related gene signature expression and HCC patient overall survival in TCGA (phs000178.v11.p8) ( n = 364 patients). l Elevated expression of the TIC-related protein signature along with BMI1 was associated with a more unfavorable patient survival outcome when contrasted with patients exhibiting low TIC-related gene signature and BMI1 expression. However, no distinction in overall survival emerged between HCC patients with either low TIC-related gene signature and high BMI1 expression or those displaying high TIC-related gene signature and low BMI1 expression ( n = 194 HCC patients, our cohort). m Kaplan–Meier survival study of TIC-related gene signature and BMI1 expression in HCC patients from TCGA (phs000178.v11.p8) ( n = 364 patients). a , d , e , j , k – m Log-rank (Mantel-Cox) test. b , c Unpaired two-tailed t test. h Two-sided Pearson correlation test. f , i Two-sided Chi-square test. Scale bars in ( a , f , g ) 100 μm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Expressing, Immunohistochemical staining, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a , b Western blot and RT-PCR analysis of BMI1 expression in WBF344/PLC cells stably transfected with either empty vector (WB Ctrl /PLC Ctrl ) or BMI1 overexpression plasmid (WB BMI1 /PLC BMI1 ). Molecular weight markers of the ladders used (in kDa) are given on the right-hand side. Bar chart shows the relative expression of BMI1 in WB BMI1 cells after normalization to WB Ctrl cells ( n = 3 independent experiments). c , d CCK8 proliferation assay of either WB Ctrl /WB BMI1 cells or PLC Ctrl /PLC BMI1 cells ( n = 4 independent experiments). e , f Colony formation assay of either WB Ctrl /WB BMI1 cells or PLC Ctrl /PLC BMI1 cells ( n = 3 independent experiments). g , h Transwell migration assays of either WB Ctrl /WB BMI1 cells or PLC Ctrl /PLC BMI1 cells ( n = 3 independent experiments). i , j Bar charts show the relative number of invaded cells in each group ( n = 3 independent experiments). k , l Bar charts show the number of tumor spheres in each group ( n = 3 independent experiments). m Schematic diagram of conditioned medium experiment in conjunction with transwell invasion assays. n , o Bar charts show the relative number of invaded cells in each group ( n = 3 independent experiments). p Nude mice were subcutaneously injected with either 5000/10,000 WB Ctrl or WB BMI1 cells. Pie charts show the probability of tumor formation in each group. Representative gross mouse image from each group is given. Dot lines indicate the positions of subcutaneous tumors ( n = 5 mice per group). q Bar charts represent the mean of tumor weight in each group ( n = 5 mice per group). Means ± S.E.M. a, b, e – o Unpaired two-tailed t test. q One-way ANOVA. c , d Two-way ANOVA. Scale bars in ( g – o ) represent 100 μm, ( q ) 1 cm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Over Expression, Molecular Weight, Proliferation Assay, Colony Assay, Migration, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a Schematic diagram of the procedure for orthotopic injection of WB Ctrl or WB BMI1 cells into the Glisson’s capsule of liver in a nude mouse. b Representative picture of the mice implanted with WB Ctrl or WB BMI1 cells 28 days post-injection. Black dotted lines indicate the mice with bilirubin thesaurismosis (jaundice). Pie charts indicate the incidence of jaundice from each group ( n = 5 mice per group). c Representative MRI pictures of the mice implanted with WB Ctrl or WB BMI1 cells 4-week post-injection are given. Green dotted lines indicate gallbladder and bile duct enlargement and white dotted lines for tumor positions ( n = 5 mice per group). d Representative gross images of livers derived from each group are given. Pie charts indicate the incidence of liver tumor formation and gallbladder and bile duct enlargement in each group ( n = 5 mice per group). e Representative pictures of H&E or CK7 and CK19 immunohistochemical staining in serial tissue sections derived from the livers of mice injected with either WB Ctrl or WB BMI1 cells are given. Arrow indicates the position of bile duct. Pie charts indicate the incidence of BDTT in each group ( n = 5 mice per group). f Schematic diagram of the procedure of intrasplenic injection in a nude mouse. g Representative pictures of the mice after intrasplenically injected with WB Ctrl or WB BMI1 cells are given. Pie charts indicate the incidence of jaundice in each group ( n = 6 mice per group). h Pie charts indicate the incidence of splenic tumor formation, liver metastasis and gallbladder and bile duct enlargement in each group ( n = 6 mice per group). i Representative pictures of H&E or CK7/CK19 IHC staining in serial tissue sections from indicated groups are given. Pie charts indicate the incidence of BDTT in each group ( n = 6 mice per group). Scale bars in ( c , d , h ) represents 1 cm, ( e , i ) 100 μm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Injection, Derivative Assay, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a , b Volcano plot and GO enrichment analysis of the proteomics data from WB Ctrl and WB BMI1 cells ( n = 3 independent experiments). Red color highlights CTSB related cellular components and pathways. BP stands for biological process, CC for cellular component, MF for molecular function. c , d Western blot and RT-PCR analysis of CTSB expression in either WB Ctrl and WB BMI1 cells or PLC Ctrl and PLC BMI1 cells ( n = 3 independent experiments). Bar charts represent means ± S.E.M. e – h CTSB ELISA and activity measurement analysis of the conditioned medium derived from each group ( n = 3 independent experiments). Bar charts represent means ± S.E.M. i – l Correlation between CTSB expression and overall survival/cancer progression/tumor size in HCC patients ( n = 194 HCC patients, our cohort). m , n Correlation between CTSB expression and BDTT incidence in HCC patients. Representative IHC pictures of CTSB staining in tumor sections derived from each group are given. Arrow indicates the position of bile duct. o ELISA assay analysis of the serum CTSB level in HCC patients with/without BDTT ( n = 36 HCC patients, our cohort). p High BMI1 and CTSB expression strongly correlated with poor overall survival in HCC patients as compared to patients with low BMI1 and CTSB expression ( n = 194 HCC patients, our cohort). q Correlation study between BMI1 and CTSB expression in HCC patients from our cohort ( n = 194 HCC patients). r High TIC-related protein signature and CTSB expression associated with poor overall survival in HCC patients when compared to patients with low TIC-related protein signature and CTSB expression ( n = 194 HCC patients, our cohort). s Correlation between TIC-related protein signature and CTSB expression in HCC patients ( n = 194 HCC patients). c – h , k , l , o Unpaired two-tailed t test. j , p , r Log-rank (Mantel-Cox) test. n Two-sided Chi-square test. q , s Two-sided Pearson correlation test. Scale bars in ( i , m ) represents 100 μm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Derivative Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a , b Transwell invasion assays of WB BMI1 or PLC BMI1 cells stably transfected with CTSB targeting siRNA-1/-2 or non-silencing control siRNA (siNSC) ( n = 3 independent experiments). c , d Transwell invasion assays of WB BMI1 or PLC BMI1 cells -/+ CTSB inhibitor CA-074 ( n = 3 independent experiments). e , f Conditioned medium experiment. Bar charts show the relative number of invaded cells in each group ( n = 3 independent experiments). g Schematic diagram of the trans-intrahepatic biliary epithelial migration assay. h – j Trans-intrahepatic biliary epithelial migration assays of fluorescently labeled PLC BMI1 /MHCC97H cells after either transfected with CTSB/BMI1 targeting siRNA/shRNA or non-silencing siRNA (siNSC)/scramble shRNA or treated with placebo/CTSB inhibitor at indicated doses. Representative images of fluorescently labeled migrated cells from each group are given ( n = 3 independent experiments). k , l Representative pictures of tumor bearing mice and gross tumor images from each group are given. Tumor growth curves show the tumor growth in each group. Bar chart indicates the mean tumor weight from each group ( n = 5 mice per group). Means ± S.E.M. m , n Representative pictures of tumor bearing mice, MRI and gross livers and spleens are given. Pie charts indicate the incidence of jaundice and elevated bilirubin level in each group ( n = 10 mice per group). o Pie charts indicate the incidence of liver tumor formation, gall bladder and bile duct enlargement and BDTT in each group ( n = 10 mice per group). p Bar charts indicate the mean of liver weight in each group ( n = 10 mice per group). Means ± S.E.M. q Kaplan–Meier survival study of WB BMI1 orthotopic liver tumor bearing mice after treated with placebo or CTSB inhibitor ( n = 10 mice per group). a – f , h – j One-way ANOVA. l Two-way ANOVA (line graph). l , p Unpaired two-tailed t test (Bar charts). q Log-rank (Mantel-Cox) test. Scale bars in ( a – f, h – j ) represent 100 μm, ( l , n ) 1 cm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Stable Transfection, Transfection, Control, Migration, Labeling, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: a Venn diagram showing the number of commonly predicted CTSB targeting miRNAs from three different online search tools, including TargetScan, miRmap and RNA22. b Heatmaps showed the top 20 altered CTSB targeting miRNAs in PLC BMI1 and MHCC97H-shBMI1 cells compared to PLC Ctrl and MHCC97H-scr cells respectively ( n = 3 independent experiments). c Venn diagram analysis revealed miR−218-1-3p as the sole miRNA correlated with BMI1 expression. d Schematic diagram of the predicated PRE sites in miR−218-1-3p promoter sequence. e ChIP assay examined BMI1 and H3K27me3 binding on miR−218-1-3p promoter’s predicted PRE sites in PLC BMI1 cells compared to PLC Ctrl cells ( n = 3 independent experiments). f Western blot analysis of WB BMI1 and PLC BMI1 cells after transfected with miR−218-1-3p mimics or NC mimics (negative control) ( n = 3 independent experiments). g ELISA assay of CTSB levels in conditioned medium harvested from WB BMI1 and PLC BMI1 cells after transfected with miR−218-1-3p mimics or NC mimics (negative control) ( n = 3 independent experiments). h Schematic diagram showing the seeding region matching between miR−218-1-3p and CTSB 3’UTR. i Luciferase reporter assays were performed in MHCC97H and PLC BMI1 cells, co-transfecting them with luciferase reporter vectors containing either the wild-type (WT-CTSB) or mutated (Mut-CTSB) CTSB 3’-UTR and pLK reference plasmid, along with miR-218-1-3p mimics or NC mimics ( n = 3 independent experiments). j Representative images of FISH analysis of miR-218-1-3p expression, negative (-ve) and positive (+ve) control probes in tumor sections from HCC patients. Correlation studies between miR-218-1-3p and BMI1/CTSB expression in HCC patient tumors ( n = 26 patients, our cohort). k , l WB BMI1 cells stably transfected with either NC mimics or miR-218-1-3p mimics were orthotopically implanted into the livers of nude mice ( n = 10 mice per group). m Pie charts display the occurrence of the specified conditions within each group ( n = 10 mice per group). n Bar charts show the mean of liver weight and tumor volume between groups ( n = 10 mice per group). e , g , n Unpaired two-tailed t test. i One-way ANOVA test. j Two-sided Pearson coefficient test. Scale bars in ( j ) represent 100 μm, ( l ) 1 cm. Source data are provided in a file.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Expressing, Sequencing, Binding Assay, Western Blot, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Control, Stable Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation

doi: 10.1038/s41467-023-42930-y

Figure Lengend Snippet: BMI1 overexpression malignantly transformed liver progenitor cells into BMI1 high TICs, which are capable of initiating hepatocarcinogenesis and BDTT. Mechanistically, BMI1 collaborates with EZH2 to repress miR-218-1-3p expression by increasing H3K27me3 level, which in turn epigenetically up-regulates CTSB secretion in BMI1 high TICs to enhance their invasion into bile ducts to form tumor thrombi.

Article Snippet: For the BMI1 inhibitor (i.e., PCT209 (Selleck-#S7539) or PRT4165 (Selleck-#S5315) experiments, the cells were treated with either DMSO, 1/10 μM PCT209 or 25/50 μM PRT4165 for 24 h and subjected to the experiments described above.

Techniques: Over Expression, Transformation Assay, Expressing

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: Cancers

Article Title: SPP1 Derived from Macrophages Is Associated with a Worse Clinical Course and Chemo-Resistance in Lung Adenocarcinoma

doi: 10.3390/cancers14184374

Figure Lengend Snippet: Gene expression analysis of cancer cells treated with the CM of macrophages or SPP1. ( A ) H23 cells were treated with the CM of macrophages and SPP1 for 24 h; then, the upregulated genes were detected by RNA-seq analysis (GEO: GSE212734). The upregulated genes in the cells treated with the CM of macrophages and SPP1. BMI1 expression was examined by qRT-PCR ( B ) and Western blot analysis ( C ). ( D ) H23 cells were treated with the indicated concentration of PTC-209 for 48 h, and the protein expression of BMI1 was determined by Western blot analysis. ( E ) H23 cells were treated with the indicated concentration of PTC-209 for 48 h, and cytotoxicity was examined by WST assay. ( F ) The standard curves were prepared by administering PTX to the H23 cells. The left side shows the standard curve without the administration of PTC-209, and the right side shows the standard curve under the administration of PTC-209 at a concentration of 0.5 µM. CT refers to H23 cells without the CM of macrophages and SPP1 added. *: statistically significant, p value < 0.05.

Article Snippet: PTC-209, the BMI1 inhibitor, was purchased from Selleck Bioteck (Tokyo, Japan), and dissolved according to the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, WST Assay

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Expression of BMI1 in endometrial cancer and paracancerous tissues. (a) The relative expression of BMI1 mRNA in endometrial cancer and paracancerous tissues. (b) IHC staining of BMI1 in endometrial cancer and paracancerous tissues.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 on proliferation of HEC-1B cells after ionizing radiation. ∗ P < 0.05, compared with control group; # P < 0.05, compared with BMI1 overexpression group.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Control, Over Expression

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 on migration of HEC-1B cells after ionizing radiation. (a, b) Effect of overexpression of BMI1 on migration of HEC-1B cells after ionizing radiation. ∗ P < 0.05, compared with control group; # P < 0.05, compared with BMI1 overexpression group.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Migration, Over Expression, Control

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 on invasion of HEC-1B cells after ionizing radiation. (a, b) Effect of overexpression of BMI1 on invasion of HEC-1B cells after ionizing radiation. ∗ P < 0.05, compared with control group; # P < 0.05, compared with BMI1 overexpression group.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Over Expression, Control

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 on apoptosis of HEC-1B cells after ionizing radiation. (a, b) Effect of overexpression of BMI1 on apoptosis of HEC-1B cells after ionizing radiation. ∗ P < 0.05, compared with control group; # P < 0.05, compared with BMI1 overexpression group.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Over Expression, Control

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 expression on invasion and migration related proteins. (a) Effect of overexpression of BMI1 on invasion-related proteins. (b) Effect of overexpression of BMI1 on migration-related proteins.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Expressing, Migration, Over Expression

Journal: Journal of Healthcare Engineering

Article Title: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation

doi: 10.1155/2022/7052066

Figure Lengend Snippet: Effect of BMI1 expression on P53, P21, and Bax protein.

Article Snippet: BMI1 antibody was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China), small molecule BMI1 inhibitor (PTC-209) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China); Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium was purchased from Wuhan Promoter Biological Co., Ltd. (Wuhan, China); cell counting kit-8 (CCK-8) kit was purchased from China Beyotime Biotech Co., Ltd. (Shanghai, China); P53, P21, and Bax primary antibodies were all purchased from German Miltenyi Biotec Company (San Jose, CA, USA); fetal bovine serum was purchased from Hangzhou Sijiqing Company (Hangzhou, China); ELX800 microplate reader was purchased from American Bio-Tek Company (BioTek Winooski, VT, USA); and CO 2 incubator was purchased from Nison Instrument (Shanghai) Limited (Shanghai, China).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A In human peripheral blood monocytes derived MΦs and MM-MΦs, transcriptional microarray analysis showed higher expression of BMI1 in MM-MΦs. B In murine bone marrow derived MΦ and MM-MΦ, RT-qPCR showed relative higher BMI1 mRNA expression in MM-MΦs. Statistical significance was determined by two-tailed Student t -test between MФs and MM-MФs, ** P < 0.01. C Western blot showed in both murine BM-derived and human PBMC-derived in vitro culture systems, MM-MΦs had higher BMI1 protein levels. D MM-MΦs from the 5T myeloma mice BM expressed higher BMI1 mRNA than MΦs from the healthy mice BM. E Left, flow cytometry histograms showed higher BMI1 levels in MΦs from myeloma mice BM. Right panel, statistic result of the mean fluorescence intensity (MFI) of BMI1 in MΦ and MM-MΦ. Statistical significance was determined by two-tailed Student t -test between MФs and MM-MФs, * P < 0.05. F Left, flow histograms showed the expression of BMI1 in BM CD14 + monocytes from 3 of 5 noncancer patients tested. Right, flow histograms showed the expression of BMI1 in BM MΦs from 3 of 5 MM patients tested. G Immunofluorescent staining of BM sections from 1 of 3 noncaner control donors and 2 of 5 MM patients showed more MΦs in the MM BM with higher BMI1 expression.

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Derivative Assay, Microarray, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, In Vitro, Flow Cytometry, Fluorescence, Staining, Control

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A Illustration of direct and trans-well co-culture of MΦs and MM cells. B Western blot showed in both direct and trans-well co-culture methods, the expression of BMI1 was elevated in MM-MΦs, relative to MΦs. C Western blot showed that protein levels of BMI1 were increased in MΦs upon rmSHH treatment. D Western blot showed that the GLI1/GLI2 transcriptional activity inhibitor GANT61 suppressed expression of BMI1 in both murine BM-derived and human PBMC-derived MM-MΦs. E Western blot showed that the Smo antagonist Cyclopamine reduced expression of BMI1 in both murine BM-derived and human PBMC-derived MM-MΦs. F Western blot showed c-Myc expression was increased in both murine and human derived MM-MΦs. G Western blot showed that the c-Myc inhibitor 10058-F4 reduced expression of BMI1 in both murine and human MM-MΦs. H ChIP-qPCR showed the relative occupancy of c-Myc and IgG control to BMI1 promoter region in MΦs and MM-MΦs derived from mouse and human. I Schematic of Hedgehog-c-Myc axis regulates BMI1 expression in MM-MΦs.

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Co-Culture Assay, Western Blot, Expressing, Activity Assay, Derivative Assay, ChIP-qPCR, Control

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A Left schematic shows the cross-breedings performed to generate BMI1 fl/fl Mx1-Cre mice and inducible Cre-mediated disruption of BMI1 in BMI1 fl/fl Mx1-Cre mice after poly I:C administration. BMI1 fl/fl mice received poly I:C were designated as wild-type (wt) controls. Western blot showed the disruption of BMI1 protein in MΦs from BMI1-KO mice after poly I:C administration (right panel). B Flow cytometry analyzed the expression of CD206 on the surface of cultured MΦs and MM-MΦs from wt control or BMI1-KO mice. MFI of CD206 indicated for each sample. C CFSE cell proliferation assay indicated the knockout of BMI1 in MM-MΦs impaired their proliferation capacity. D Flow cytometry analyzed BMI1 and Ki67 expression of MΦs from BM aspirates of 28 MM patients. Left, histograms of BMI1 and Ki67 expression of 2 representative patients with relative lower or higher BMI1 expression. Right panel, the co-expression pattern of BMI1 and Ki67 was showed in dot plots. The correlation was analyzed by linear regression with R square calculated. E The RNA-seq data of murine BM-derived MM-MΦs from wt and BMI1-KO mice were analyzed with GSEA, showing enrichment of Hallmark E2F targets and G2M checkpoint pathways in wt MM-MΦs. F Heatmap illustrated the RNA-seq data of CDKN2A, showing significant upregulation of CDKN2A in BMI1-KO MM-MΦs. G RT-qPCR showed the mRNA expression of P16 was significantly higher in BMI1-KO MM-MΦs, relative to wt MM-MΦs (left column). Flow cytometry showed the protein level of P16 was increased in BMI1-KO MM-MΦs (right panel). ** p < 0.01 (Student’s t -test, comparing 2 samples, error bars are SD).

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Disruption, Western Blot, Flow Cytometry, Expressing, Cell Culture, Control, Proliferation Assay, Knock-Out, RNA Sequencing, Derivative Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A Western blot showed the increased expression of MMP2 and VEGF in MM-MΦs derived from both mice and human, relative to MΦs. B Western blot showed that knockout of BMI1 impaired the expression of MMP2 and VEGF in MM-MΦs. C ELISA assay detected the concentration of VEGF in culture supernatant of murine BM-derived MФs and MM-MФs from wt or BMI1-KO mice. Statistical significance was determined by two-tailed Student t -test between wt and BMI1-KO MM-MФs, ** P < 0.01. D BMI1 knockout lowered the levels of NO in culture supernatant of MM-MФs. Statistical significance was determined by two-tailed Student t -test between wt and BMI1-KO MM-MФs, * P < 0.05. E GSEA of RNA-seq data showed the enrichment of genes negatively regulating blood vessel endothelial cell migration in BMI1-KO MM-MФs.

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Western Blot, Expressing, Derivative Assay, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay, Two Tailed Test, RNA Sequencing, Migration

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A The apoptotic rate of 5TMG1 cells treated with indicated drugs alone or with wt MФs or BMI1-KO MФs for 24 h. Statistical significance was determined by two-tailed Student t -test between drug treated 5TGM1 cells cultured alone and with wt MM-MФs, or between drug treated 5TGM1 cells cultured with wt MM-MФs and with BMI1-KO MM-MФs, * P < 0.05. B Western blot showed the activation status of apoptotic protein PARP and Capase-3 of 5TGM1 cells treated with indicated drugs alone or with wt or BMI1-KO MФs for 24 h. C Western blot showed the protein expression or phosphorylation changes of 5TGM1 cells cultured alone or with wt or BMI1-KO MФs. D Pathway enrichment of differential regulated genes from RNA-seq data of wt vs BMI1-KO MM-MФs, comparing with GO cellular component genesets. E RT-qPCR showed the mRNA expression of Galectin-3 was decreased in BMI1-KO MM-MФs, relative to wt MM-MΦs (left column). Western blot showed the protein level of Galectin-3 was lower in BMI1-KO MM-MФs (right panel). Statistical significance was determined by two-tailed Student t -test between wt and BMI1-KO MM-MФs, ** P < 0.01.

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Two Tailed Test, Cell Culture, Western Blot, Activation Assay, Expressing, Phospho-proteomics, RNA Sequencing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: BMI1 regulates multiple myeloma-associated macrophage’s pro-myeloma functions

doi: 10.1038/s41419-021-03748-y

Figure Lengend Snippet: A Schematic displayed that 5TGM1 cells were injected subcutaneously into the B-NDG mice alone or with BM-derived MΦs from wt or BMI1-KO mice. B Flow cytometry showed the proportion of MΦs in the subcutaneous tumor beds 7 days after inoculation (Left panel). Tumors with wt MΦs had the most MΦs in tumor beds (right column). Statistical significance was determined by two-tailed Student t -test between tumors with 5TGM1 alone and with wt MM-MФs, or between tumors with wt and BMI1-KO MM-MФs, * P < 0.05. C Left panel showed the growth curves of 5TGM1 xenografts, wt MΦs were the most potent in promoting tumor growth. Right panel showed monoclonal protein IgG2b concentrations in murine peripheral blood, mice bearing tumors with wt MΦs had the highest concentrations of IgG2b. Statistical significance was determined by two-tailed Student t -test between groups with wt and BMI1-KO MM-MФs, * P < 0.05. D Representative IHC staining images (left images) and quantification of positive cells (right column) of epithelial cell marker CD34. Statistical significance was determined by two-tailed Student t -test between tumors with 5TGM1 alone and with wt MM-MФs, or between tumors with wt and BMI1-KO MM-MФs, * P < 0.05.

Article Snippet: The BMI1 inhibitor PTC-209 was purchased from Cayman Chemical (MI, USA).

Techniques: Injection, Derivative Assay, Flow Cytometry, Two Tailed Test, Immunohistochemistry, Marker

Journal: Cancer cell

Article Title: The Polycomb Repressor Complex 1 Drives Double Negative Prostate Cancer Metastasis by Coordinating Stemness and Immune Suppression

doi: 10.1016/j.ccell.2019.06.009

Figure Lengend Snippet: (A) Control or RNF2- and BMI1-silenced PC3 cells were subjected to immunoblotting.

Article Snippet: The RNF2 inhibitor PRT4165 (5047), CCR2 antagonist RS504393 (2517), and BMI1 inhibitor PTC209 were from Tocris.

Techniques: Control, Western Blot

Journal: Cancer cell

Article Title: The Polycomb Repressor Complex 1 Drives Double Negative Prostate Cancer Metastasis by Coordinating Stemness and Immune Suppression

doi: 10.1016/j.ccell.2019.06.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The RNF2 inhibitor PRT4165 (5047), CCR2 antagonist RS504393 (2517), and BMI1 inhibitor PTC209 were from Tocris.

Techniques: Western Blot, Control, Microarray, Recombinant, Ubiquitin Proteomics, Software