Journal: Scientific Reports

Article Title: A CXCR4 targeting peptide delivered by silica nanoparticles eliminates migrating cancer stem cells in pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-026-48584-2

Figure Lengend Snippet: BMI1 downstream CXCL12–CXCR4 regulates EMT and stemness. ( A ) Protein–protein interactions of CXCL12 and CXCR4 with relevant factors involved in metastasis (red), stemness (purple), sonic hedgehog signaling (blue), AKT signaling (grey) and NFκB pathway (yellow) (STRING). ( B ) BMI1 gene expression analysis and western blot analysis. GAPDH was used as a loading control. Intensity ratios (IR) calculated against control lane using ImageJ. Cropped blot for clarity. ( C ) Gene expression analysis with genes involved in EMT using qRT-PCR. ( D ) Immunofluorescence quantifications and representative micrographs for indicated cell lines with white arrowheads marking mesenchymal structures of actin filaments stained with Phalloidin (pink) and nucleus stained with DAPI (blue). ( E ) Migration assays towards serum containing media. ( F ) Experimental scheme to evaluate CD133 and CXCR4 surface expression using flow cytometry for Panc354 and MetPO1 (sh_ SCR , sh1_ BMI1 and sh2_ BMI1 ) with (or without) CXCL12. ( G ) Flow cytometry analysis of CD133 + cells and CD133 + CXCR4 + cells in MetPO1 (sh_ SCR , sh1_ BMI1 and sh2_ BMI1 ) treated with (or without) CXCL12. ( H ) Representative cytometry plots for MetPO1 cell line. Error bars represent the standard deviation. n = 3 for all experiments unless otherwise depicted in the datasets. * p < 0.05, ns = not significant.

Article Snippet: Recombinant BMI1 IgG1 (REA438) , APC , Miltenyi Biotec , 130-124-301.

Techniques: Protein-Protein interactions, Gene Expression, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Migration, Expressing, Flow Cytometry, Cytometry, Standard Deviation

Journal: Scientific Reports

Article Title: A CXCR4 targeting peptide delivered by silica nanoparticles eliminates migrating cancer stem cells in pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-026-48584-2

Figure Lengend Snippet: JM#21, most potent EPIX4 derivative to target miCSCs. ( A ) Migration assays towards CXCL12 using Panc354 for EPI-X4 and its derivatives at depicted concentrations. Pre-treatment with EPI-X4, WSCO2, JM#21 and the inactive peptide was applied for 30 min. ( B ) Representative micrographs (10x, DAPI staining) of transwell migration assays in Panc354 cells for the indicated conditions and concentrations. ( C ) Migration assays towards CXCL12 for MetPO1 using JM#21 and the inactive peptide at depicted concentrations. ( D ) Quantifications of percent mesenchymal structures after 15 min and 6 h of CXCL12 treatment in MetPO1 cells. JM#21 pre-treatment was applied for 30 min and representative micrographs with white arrowheads marking mesenchymal structures of actin filaments stained with Phalloidin (pink) and nuclear staining using DAPI (blue). ( E ) Gene expression analysis for indicated cell lines with genes involved in EMT and SHH pathway. ( F ) Gene expression analysis for indicated cell lines with genes involved in stemness. ( G ) Sphere formation assays for 1 st and 2 nd generation of sphere formation. ( H ) Western blot analysis of CADHERIN-1, VIMENTIN, CADHERIN-2, NANOG and BMI1 for indicated cell lines. GAPDH was used as a loading control. Intensity ratios (IR) calculated against control lane using ImageJ. Cropped blot for clarity. ( I ) Experimental design for combination therapy analyzing relapse using JM#21, gemcitabine (labelled as G) and paclitaxel (labelled as P). Quantification of cell viability and representative pictures for clonogenic assays after treatment with JM#21 (10 μM ), gemcitabine (Gem) for indicated concentrations as depicted in experimental design in MetPO1 cell line. ( J ) Flow cytometry for CD133 in Panc354 and MetPO1cells for the indicated treatments shown as fold change. Error bars represent the standard deviation. n = 3 for all experiments unless otherwise depicted in the datasets. * p < 0.05, ns = not significant.

Article Snippet: Recombinant BMI1 IgG1 (REA438) , APC , Miltenyi Biotec , 130-124-301.

Techniques: Migration, Staining, Gene Expression, Western Blot, Control, Flow Cytometry, Standard Deviation

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: Tumor pathologic classification after 4-NQO treatment for 4 weeks reveals more oncogenic changes in mice with ectopic BMI1 expression. A, Schematic of the DOX-inducible expression system (KrTB) used to overexpress ectopic BMI1 in murine tongue epithelial basal stem cells. B, Timeline outlining the different treatment groups for this experiment. Samples were collected at 10 or 16 weeks (4- or 10-week treatments). Samples from bolded groups [KrTB-DN (4w) and KrTB-DN (10w)] were also submitted for RNA-seq. C, Representative images of H&E-stained sections for normal tongue epithelium, hyperplasia, and dysplasia (100× magnification; scale bar, 100 μm). D, Graph of the distribution (%) of the most severe 4-NQO–induced lesion observed in each mouse by pathologic classification (at 4 weeks), with Kr-DN (4w) ( N = 7) and KrTB-DN (4w) ( N = 10) mice. E , Graph of the distribution (%) of the most severe 4-NQO–induced lesion observed in each mouse by pathologic classification (at 10 weeks), with Kr-DN (10w) ( N = 7) and KrTB-DN (10w) ( N = 10) mice. For D and E , statistical significance was determined with the χ 2 test. ****, P < 0.0001.

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Staining

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: Increases in proliferation and oxidative stress occur very early after 4-NQO addition upon BMI1 overexpression. A, BMI1, ( B ) Ki67, and ( C ) 4-HNE IHC stainings in Kr-DN (4w), KrTB-DN (4w), Kr-DN (10w), and KrTB-DN (10w) tongue epithelia (200×; scale bar, 100 μm; N = 3 mice/group, four fields/mouse; representative fields are shown). Ratios of the levels of these factors in all groups relative to levels in the Kr-DN (4w) group are also included. Data graphed denote the mean ± SD of the mean (SD). Statistical significance was determined using one-way ANOVA followed by Tukey test. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001; ****, P < 0.0001.

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: Over Expression

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: Increases in OSCC biomarkers occur early in 4-NQO–induced tumorigenesis upon BMI1 overexpression. A, HIF1α, ( B ) GLUT1, and ( C ) SOX9 IHC stainings in Kr-DN (4w), KrTB-DN (4w), Kr-DN (10w), and KrTB-DN (10w) tongue epithelia (200×; scale bar, 100 μm; N = 3 mice/group, four fields/mouse; representative fields are shown). Ratios of the levels of these factors in all groups relative to levels in the Kr-DN (4w) group are also included. Data graphed denotes the mean ± SD of the mean (SD). Statistical significance was determined using one-way ANOVA followed by Tukey test. *, 0.01 < P < 0.05; ***, 0.0001 < P < 0.001; ****, P < 0.0001.

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: Over Expression

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: Cells with ectopic BMI1 and increased HIF1α or GLUT1 expression are detected in 4-NQO–treated tongue epithelia at an early time point (4 weeks). A, BMI1 and HIF1α IF stainings in samples from Kr-DN (4w) and KrTB-DN (4w) tongue epithelia. B, BMI1 and GLUT1 IF stainings in samples from Kr-DN (4w) and KrTB-DN (4w) tongue epithelia. For A and B , we report ratios of integrated density of HIF1α or GLUT1 (measured by Fiji) over integrated density of Hoechst signal in the same area ( N = 3 mice/group, four fields/mouse). Representative fields are shown (200×; scale bar, 100 μm). All data graphed denote the mean ± SD of the mean (SD). Statistical significance was determined using Welch t test. **, 0.001 < P < 0.01; ****, P < 0.0001.

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: BMI1 KO in the human OSCC line SCC-25 decreases the expression of HIF1α and glycolysis-associated protein GLUT1. A, A scheme of Crispr/Cas9 technology strategy indicating the three gRNAs designed in exons 6 and 9 of the human BMI1 gene. Treatment with gRNA B (in red) resulted in the highest percentage of editing. B, Discordance plots detailing the level of alignment per base between the parental (control) and the edited (B#5 or B#6) SCC-25 cell lines in the interference windows, based on Sanger sequencing of fragments amplified via PCR from genomic DNA of treated cells. On the plots, green and orange lines are close together before the cutsite (dashed line) and remain far apart after this site. These plots were generated using the online tool provided by Synthego, ICE. Immunoblotting of the protein levels of ( C ) BMI1 and GAPDH, ( D ) HIF1α and GAPDH, and ( E ) GLUT1 and GAPDH in parental, B#5, and B#6 SCC-25 cells (triplicates). Quantifications of ( F ) HIF1α and ( G ) GLUT1 immunoblottings (relative to GAPDH) are included. Statistical significance was determined relative to parental SCC-25 cells using Welch t test (*, 0.01 < P < 0.05).

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: Expressing, CRISPR, Control, Sequencing, Amplification, Generated, Western Blot

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: Proliferation and oxidative stress are decreased in the human OSCC line SCC-25 upon BMI1 deletion. A, Proliferation in parental, B#5, and B#6 SCC25 cells using a cell counter ( N = 6 replicates/time point). Data graphed denotes the mean ± SD of the mean (SD). Statistical significance was determined using two-way RM ANOVA followed by Tukey multiple comparisons test. Significance values shown were calculated relative to the parental group. B, ROS in parental, B#5, and B#6 SCC25 cells, with and without H 2 O 2 induction. Representative images are shown for each of the six experimental groups and the three negative (no dye) controls. All images are 200×; scale bar, 100 μm. C, Quantification of ROS data from B , with N = 3 replicates/group, four fields/group. Data graphed denotes the mean ± SD of the mean (SD). Statistical significance was determined using one-way ANOVA followed by Tukey test. Significance was calculated relative to the parental (no H 2 O 2 ) group. For A and C , **, 0.001 < P < 0.01; ****, P < 0.0001.

Article Snippet: To prepare bead–antibody conjugates, M-270 Epoxy Dynabeads (Dynabeads Antibody Coupling Kit; Invitrogen; 14311D) were incubated at 37°C overnight with 10 μL per IP of rabbit monoclonal anti-Bmi1 antibody (Cell Signaling Technology; 6964S; lot 3; concentration: 87 μg/mL, RRID: AB_10828713) or 2.5 μL per IP of normal rabbit IgG (Santa Cruz; sc-2027; lot A3014; concentration: 400 μg/mL, RRID: AB_737197), following the manufacturer’s protocol.

Techniques: