Journal: International Journal of Molecular Medicine

Article Title: Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a-RhoA signaling pathway to repair ischemic brain injury in rats

doi: 10.3892/ijmm.2025.5596

Figure Lengend Snippet: Effects of B lymphoma Mo-MLV insertion region 1 homolog and Wnt3a on the proliferation of MSCs. (A) Morphology of primary MSCs inoculated into culture flasks. Morphology of MSCs in primary culture for (B) 3 and (C) 7 days. (D) Morphological characterization of third-generation MSCs. Effects of different concentrations of (E) Wnt3a and (F) PTC209 on the proliferation of MSCs. ** P<0.01 vs. 0 μ M group. MSCs, mesenchymal stem cells.

Article Snippet: Wnt3a recombinant cytokine (cat. no. 96-315-20-10) was purchased from PeproTech; Thermo Fisher Scientific, Inc. and the small molecule inhibitor PTC209 (cat. n. S7372) was purchased from Selleck Chemicals.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a-RhoA signaling pathway to repair ischemic brain injury in rats

doi: 10.3892/ijmm.2025.5596

Figure Lengend Snippet: The mechanism underlying the effects of Bmi1 on MSC transplantation-induced ischemic brain injury repair. In vitro experiments used Wnt3a and PTC209 to treat MSCs to explore the roles and regulatory mechanisms of the Bmi1 and Wnt3a-Rhoa signaling pathways in the neural differentiation of MSCs. In vivo experiments were conducted to investigate the role of the Bmi1 and Wnt3a-RhoA signaling pathway in brain injury repair in MCAO rats and its mechanism. Different MSCs were transplanted into rat brain tissues after in vitro labeling, after which, the ischemic brain injury in each group was compared and the recovery of neurological function was assessed. Bmi1, B lymphoma Mo-MLV insertion region 1 homolog; H&E, hematoxylin and eosin; IF, immunofluorescence; MSCs, mesenchymal stem cells; NSS, Neurological Severity Score; WB, western blotting.

Article Snippet: Wnt3a recombinant cytokine (cat. no. 96-315-20-10) was purchased from PeproTech; Thermo Fisher Scientific, Inc. and the small molecule inhibitor PTC209 (cat. n. S7372) was purchased from Selleck Chemicals.

Techniques: Transplantation Assay, In Vitro, Protein-Protein interactions, In Vivo, Labeling, Immunofluorescence, Western Blot

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