Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: Targeting CSCs by PTC209 is insufficient to eradicate cancers (A) Schematic diagram of PTC209 treatment schedule for PDX#3. (B) Tumor volume of PDX#3 treated with the vehicle or PTC209. Data are shown as the mean ± SD ( n = 12). ns, not significant, p > 0.05, ∗ p < 0.05, and ∗∗ p < 0.01 using an unpaired Student’s t test. (C) Tumor weight in PDX#3 treated with the vehicle or PTC209. Data are shown as the mean ± SD ( n = 12). ns, p > 0.05 using an unpaired Student’s t test. (D and E) ALDEFLUOR assay of PDX#3 treated with PTC209. ALDH + cell percentage was measured through flow cytometry. Data are shown as the mean ± SD ( n = 3). ns, p > 0.05, ∗ p < 0.05, and ∗∗ p < 0.01 using an unpaired Student’s t test.

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: Non-stem tumor cells compensate for the loss of CSCs and result in tumor relapse (A) Schematic image of treatment and lineage tracing for 4NQO-induced HNSCC in Bmi1 CreER ;Rosa tdTomato mice. Tamoxifen (Tam) was administered to tag Bmi1 + CSCs 1 day before sacrificing (Sac) the mice. (B) Representative images of tongue tumors from different treatment groups. Black dotted line circles indicate lesion areas. Scale bar, 2 mm. Lesion areas of tongue tumors from different treatment groups were quantified. Data are shown as the mean ± SD. n = 12. ∗∗ p < 0.01 using an unpaired Student’s t test. (C) Representative H&E-stained HNSCC tumor section from different treatment groups as indicated. The images in the left panels are of low magnification. Scale bar, 500 μm. The right panels show images with high magnification. Scale bar, 100 μm. (D and E) Quantification of tumor number (D) and area (E) from different treatment groups as indicated. Data are shown as the mean ± SD. n = 12. ∗ p < 0.05, and ∗∗ p < 0.01 using an unpaired Student’s t test. (F) Representative images of Bmi1 + Tomato + cells in HNSCC tumors from different treatment groups. The border between interstitial and tumor tissues is shown using a white dotted line. Scale bar, 20 μm. Percentage of Bmi1 + Tomato + CSCs in HNSCC tumors from different treatment groups is presented. Data are shown as the mean ± SD. n = 12, ∗∗ p < 0.01 using an unpaired Student’s t test. (G) Experimental design of Bmi1 + CSC lineage tracing in HNSCC after treatment with PTC209. After therapy, Tam was administered to the mice, and they were kept for 4 more weeks. (H) Representative images of tongue tumors from different treatment groups. The lesion areas are indicated using black dotted circles. Scale bar, 2 mm. Quantification of the lesion area of mice with different treatments is indicated. n = 12. ns, p > 0.05 using an unpaired Student’s t test. (I) Representative H&E-stained HNSCC sections from mice with different treatments as indicated. The images in the left panels are of low magnification. Scale bar, 500 μm. The right panels show images with high magnification. Scale bar, 100 μm. (J and K) Quantification of tumor number (J) and area (K) from different treatment groups. Data are shown as the mean ± SD. n = 12. ns, p > 0.05 using an unpaired Student’s t test. (L) Representative images of mice with Bmi1 + Tomato + CSCs in HNSCC tumors from different treatment groups. The white dotted line indicates the boundary between tumor and interstitial tissues. Scale bar, 20 μm. Percentage of Bmi1 + Tomato + CSCs in HNSCC from different treatment groups is shown. Data are shown as the mean ± SD. n = 12, ∗∗ p < 0.01 using an unpaired Student’s t test.

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: Staining

Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: Lineage tracing of K16 + non-stem tumor cells (A) Real-time qPCR analysis of epithelial differentiation marker expression in ALDH − and ALDH + tumor cells. Data are shown as the mean ± SD. ns, p > 0.05, ∗∗ p < 0.01 using an unpaired Student’s t test. (B) Western blot analysis of K16 expression in ALDH - and ALDH + tumor cells. (C and D) Immunostaining for K16 (green) in HNSCC tumors with Bmi1 + Tomato + lineage (red) after 1 day (C) and 1 week (D). DAPI was used to label the nuclei (blue). The white dashed lines demark tumor-stromal junction. Scale bar, 20 μm. (E) Expression scatterplots of HNSCC TCGA datasets ( n = 520) revealing correlation between BMI1/ALDH1 and K16. The p values are according to the Spearman coefficient test. (F) Experimental design for lineage tracing of K16 + Tomato + cells in HNSCC tumors after PTC209 treatment. After treatment, the mice were injected with Tam and maintained for 4 additional weeks. (G) Quantification of the lesion area, tumor number, and tumor area from mice receiving different treatments. Data are shown as the mean ± SD. n = 8. ns, p > 0.05 using an unpaired Student’s t test. (H) Representative images of BMI1 immunostaining (green) and K16 + Tomato + lineage tracing (red) over 4 weeks are indicated. Nuclei were stained with DAPI (blue). The white dashed lines indicate the tumor-stromal junction. Scale bar, 20 μm. (I) Schematic model. K16 + non-stem tumor cells rarely revert to CSCs at a steady state, whereas PTC209 treatment reverts K16 + non-stem tumor cells to CSCs. See also .

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: Marker, Expressing, Western Blot, Immunostaining, Injection, Staining

Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: CSCs ablation induces MYC expression in non-stem tumor cells (A) Scatterplots of RNA-seq expression values (in fragments per kilobase of transcript per million mapped reads, FPKM) in EpCAM + K16 + Tomato + cells treated with PTC209 or the vehicle. The plot has a log 2 -transformed scale. (B) Heatmap of RNA-seq data for expression of indicated genes in EpCAM + K16 + Tomato + cells treated with PTC209 or the vehicle. Fold change (FC) of PTC209 versus DMSO is listed on the right. (C) Real-time qPCR analysis of Myc expression in EpCAM + K16 + Tomato + cells treated with the vehicle or PTC209. Data are shown as the mean ± SD. ∗∗ p < 0.01 using an unpaired Student’s t test. Western blot analysis of MYC in EpCAM + K16 + Tomato + cells treated with the vehicle or PTC209 is shown. (D) Scatterplots of RNA-seq expression values (FPKM) in EpCAM + ALDH − cells treated with the vehicle or PTC209. The plot has a log 2 -transformed scale. (E) Heatmap of RNA-seq data for expression of indicated genes in EpCAM + ALDH − tumor cells treated with the vehicle or PTC209. FC of PTC209 versus DMSO is listed on the right. (F) Real-time qPCR analysis of MYC expression in EpCAM + ALDH − cells treated with the vehicle or PTC209. Data are shown as the mean ± SD. ∗∗ p < 0.01 using an unpaired Student’s t test. Western blot analysis of MYC in EpCAM + ALDH − cells treated with the vehicle or PTC209 is shown. (G) Strategy for genetic ablation of Myc in K16 + cells using K16 CreER ;Myc f/f mice. Tamoxifen (Tam) was topically applied in the oral cavity to knockout Myc . (H) Representative images of tongue tumors from the different treatment groups. The lesion areas are indicated by black dotted circles. Scale bar, 2 mm. (I) Quantification of lesion areas in mice from different treatment groups as indicated. Data are shown as the mean ± SD. n = 12. ∗ p < 0.05 and ∗∗ p < 0.01 using two-way ANOVA. (J) Representative images of H&E-stained HNSCC tumor sections from mice in different treatment groups. Scale bar, 500 μm. Lower panels show higher-magnification images. Scale bar, 100 μm. (K and L) Quantification of tumor numbers (K) and areas (L) in mice from different treatment groups. Data are shown as the mean ± SD. n = 12. ∗ p < 0.05 and ∗∗ p < 0.01 using two-way ANOVA. See also .

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: Expressing, RNA Sequencing, Transformation Assay, Western Blot, Knock-Out, Staining

Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: PTC209-induced DNA damage activates NF-κB signaling pathway in CSCs (A) Immunostaining and quantification of pH2A.X (red) in HNSCC cells treated with PTC209. DAPI stained the nuclei blue. Scale bar, 20 μm. (B) Percentage of pH2A.X in HNSCC cells from different treatment groups as indicated. Data are shown as the mean ± SD. ∗∗ p < 0.01 using an unpaired Student’s t test. (C) Western blot analysis of pH2A.X in HN6 and SCC15 cells treated with PTC209. (D and E) Histogram of the top 10 most-enriched GO (D) and KEGG (E) terms for upregulated genes (FC > 2) in ALDH + HN6 cells treated with PTC209. The red rectangles indicate genes related to NF-κB signaling. (F) Western blot analysis of pIKK-α/β, IKK-α, IKK-β, pIκB-α, IκB-α, p-P65, and P65 in HNSCC cells with PTC209 treatment. (G) Representative images of p65 immunostaining (green) in HN6 and SCC15 cells treated with PTC209. DAPI stained the nuclei (blue). Scale bar, 20 μm. See also .

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: Immunostaining, Staining, Western Blot

Journal: Cell Reports Medicine

Article Title: Co-targeting BMI1 and MYC to eliminate cancer stem cells in squamous cell carcinoma

doi: 10.1016/j.xcrm.2025.102077

Figure Lengend Snippet: IL-6 secreted from CSCs is responsible for non-stem tumor cells dedifferentiation (A) Heatmap of RNA-seq data for expression of genes encoding proinflammatory cytokines in ALDH + HN6 cells treated with PTC209. (B) Real-time qPCR analysis of IL-6 mRNA expression in HN6 and SCC15 cells treated with PTC209. Data are shown as the mean ± SD. ∗ p < 0.05 and ∗∗ p < 0.01 using an unpaired Student’s t test. (C) ELISA of protein levels of IL-6 secreted by HN6 and SCC15 cells treated with PTC209. Means ± SD are shown. ∗∗ p < 0.01 using an unpaired Student’s t test. (D) Real-time qPCR analysis of MYC in HN6 and SCC15 cells treated with IL-6. Data are shown as the mean ± SD. ∗∗ p < 0.01 using an unpaired Student’s t test. (E) Western blot analysis of MYC in HN6 and SCC15 cells treated with IL-6. (F) Experimental design used to label Bmi1 + CSCs after PTC209 plus IL-6 inhibitor treatment in mouse HNSCC tumors. (G) Representative images of mice with Bmi1 + Tomato + CSCs in HNSCC tumors from different treatment groups as indicated. The white dotted line indicates the boundary between tumor and interstitial tissues. Scale bar, 20 μm. (H) Percentage of Bmi1 + Tomato + CSCs in HNSCC tumors from different treatment groups as indicated. Data are shown as the mean ± SD. n = 5. ∗∗ p < 0.01 using one-way ANOVA. See also .

Article Snippet: To test the effects of MYC on PTC209 anti-tumor effects, a Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China; Cat# CK04) was used.

Techniques: RNA Sequencing, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

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