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: EBioMedicine

Article Title: FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

doi: 10.1016/j.ebiom.2019.02.032

Figure Lengend Snippet: NFATC1 activates BMI1. (a) Serial deletion constructs containing the human BMI1 gene promoters, as well as BMI1-2259/+63bp-Luc plasmids with NFATC1 motif wildtype (wt), mutation (mut) or deletion (del) were transfected into MCF-7 cells for 24 h. Renilla plasmid was co-transfected as a transfection control. The mean luciferase activity of BMI1-2259/+63-Luc plasmids with NFATC1 motif wildtype was set as 1 (n = 6). Dual-luciferase reporter assay of NFATC1 motif at −1179 bp is required for BMI1 transcriptional activity. TSS, transcription start site. (b) MCF-7 cells were transfected with pcDNA-FUNDC1 or siRNA-NFATC1 for 24 h and then with BMI1-2259/+63-Luc plasmids with NFATC1 motif wild type or mutation for another 24 h. BMI1 promoter activities were measured by luciferase activity, normalized to that of Renilla (n = 6). (c) Quantitative chromatin immunoprecipitation (q-ChIP) assay showing that NFATC1 could bind to the fragment with NFATC1 motif at −1179 bp but not −1740 bp in MCF-7 cells; normal rabbit IgG was a control (n = 4). (d) q-ChIP analysis of enrichment of NFATC1 motif by overexpression of FUNDC1 and/or knockdown of NFATC1 (n = 4). (e,f) Increasing BMI1 mRNA and protein expression with overexpression of FUNDC1 was prevented by ML9 or 2APB pre-treatment of MCF-7 cells (n = 3). Data are mean ± SD. *P < 0.05.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the membrane was incubated overnight at 4 °C with antibody for FUNDC1 (1:500, Abcam: ab173226 ), BMI1 (1:1000, R&D System: MAB33342), NFATC1 (1:500, Abcam: ab2722), NFATC1 (phospho S54) (1:500, Abcam: ab200819 ), STIM1(1:500 Proteintech:11565-1-AP), ORAI1 (1:1000 Proteintech: 13130-1-AP), TRPC1 (1:500 Proteintech: 19482-1-AP) or mouse monoclonal antibody against beta ACTIN(1:2000, Abcam: ab8227) and GAPDH (1:1000; Santa Cruz Biotechnology: sc-47724.

Techniques: Construct, Mutagenesis, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Over Expression, Knockdown, Expressing

Journal: EBioMedicine

Article Title: FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

doi: 10.1016/j.ebiom.2019.02.032

Figure Lengend Snippet: BMI1 contributes to NFATC1-induced cell proliferation and migration. (a) Western blot analysis of downregulation of BMI1 at protein level on transfection with siRNA-FUNDC1 and successful recovery by co-transfection with p-cDNA BMI1 plasmid in SKBR3 cells for 48 h. GAPDH was the internal control (n = 3). (b–e) BMI1 overexpression on transfection with p-cDNA BMI1 induced SKBR3 cell proliferation and migration and rescued FUNDC1 knockdown-reduced cell proliferation and migration, detected by CCK8 assay (b, n = 6), cell counting (c, n = 6) and transwell assay (d,e, n = 5). (f) Positive correlation of FUNDC1 and BMI1 mRNA expression among 47 breast cell lines based on their RNA sequence from the CLLE database.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the membrane was incubated overnight at 4 °C with antibody for FUNDC1 (1:500, Abcam: ab173226 ), BMI1 (1:1000, R&D System: MAB33342), NFATC1 (1:500, Abcam: ab2722), NFATC1 (phospho S54) (1:500, Abcam: ab200819 ), STIM1(1:500 Proteintech:11565-1-AP), ORAI1 (1:1000 Proteintech: 13130-1-AP), TRPC1 (1:500 Proteintech: 19482-1-AP) or mouse monoclonal antibody against beta ACTIN(1:2000, Abcam: ab8227) and GAPDH (1:1000; Santa Cruz Biotechnology: sc-47724.

Techniques: Migration, Western Blot, Transfection, Cotransfection, Plasmid Preparation, Control, Over Expression, Knockdown, CCK-8 Assay, Cell Counting, Transwell Assay, Expressing, Sequencing

Journal: EBioMedicine

Article Title: FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

doi: 10.1016/j.ebiom.2019.02.032

Figure Lengend Snippet: BMI1 level correlates with FUNDC1 level in progression of clinical BC. (a–c) Correlation between FUNDC1 and BMI1 mRNA expression for clinical BC patient data from the indicated online databases. (d) Representative immunohistochemical co-staining for FUNDC1 and BMI1 protein in continuous slices of BC tissue. (e) Scatter diagram showing overall survival time for patients with co-expression of FUNDC1 and BMI1 protein. (d–e) Kaplan-Meier analysis of overall survival in patients with high level of FUNDC1 and BMI1 co-expression predicting worse progression of clinical BC.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the membrane was incubated overnight at 4 °C with antibody for FUNDC1 (1:500, Abcam: ab173226 ), BMI1 (1:1000, R&D System: MAB33342), NFATC1 (1:500, Abcam: ab2722), NFATC1 (phospho S54) (1:500, Abcam: ab200819 ), STIM1(1:500 Proteintech:11565-1-AP), ORAI1 (1:1000 Proteintech: 13130-1-AP), TRPC1 (1:500 Proteintech: 19482-1-AP) or mouse monoclonal antibody against beta ACTIN(1:2000, Abcam: ab8227) and GAPDH (1:1000; Santa Cruz Biotechnology: sc-47724.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: EBioMedicine

Article Title: FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

doi: 10.1016/j.ebiom.2019.02.032

Figure Lengend Snippet: Correlation of FUNDC1 and BMI1 protein levels in patients with breast cancer.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the membrane was incubated overnight at 4 °C with antibody for FUNDC1 (1:500, Abcam: ab173226 ), BMI1 (1:1000, R&D System: MAB33342), NFATC1 (1:500, Abcam: ab2722), NFATC1 (phospho S54) (1:500, Abcam: ab200819 ), STIM1(1:500 Proteintech:11565-1-AP), ORAI1 (1:1000 Proteintech: 13130-1-AP), TRPC1 (1:500 Proteintech: 19482-1-AP) or mouse monoclonal antibody against beta ACTIN(1:2000, Abcam: ab8227) and GAPDH (1:1000; Santa Cruz Biotechnology: sc-47724.

Techniques:

Journal: EBioMedicine

Article Title: FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

doi: 10.1016/j.ebiom.2019.02.032

Figure Lengend Snippet: The schematic model. Elevated FUNDC1 expression in MAMs location promotes Ca 2+ flux into cytoplasm, which de-phosphorylates NFATC1, transfers it to nucleus and activates BMI1 expression, further promotes the progression of BC.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the membrane was incubated overnight at 4 °C with antibody for FUNDC1 (1:500, Abcam: ab173226 ), BMI1 (1:1000, R&D System: MAB33342), NFATC1 (1:500, Abcam: ab2722), NFATC1 (phospho S54) (1:500, Abcam: ab200819 ), STIM1(1:500 Proteintech:11565-1-AP), ORAI1 (1:1000 Proteintech: 13130-1-AP), TRPC1 (1:500 Proteintech: 19482-1-AP) or mouse monoclonal antibody against beta ACTIN(1:2000, Abcam: ab8227) and GAPDH (1:1000; Santa Cruz Biotechnology: sc-47724.

Techniques: Expressing

Journal: Cells

Article Title: CD80-Mediated T-Cell Suppression by Cancer Stem-like Cells in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cells15030266

Figure Lengend Snippet: CD80 is related to cancer-cell stemness in PR and CR patients. ( A ) UMAP plots show re-clustering of epithelial cells in PR and CR groups. ( B ) Pseudotime analysis demonstrates the derivation of epithelial cells in PR and CR groups based on the derived trajectory, tissue origin, and monocle states. ( C ) The Heatmap shows the top genes expressed with the pseudotime trajectory of three fate cells. ( D ) The Boxplot shows the stemness scores of three state cells. ( E ) The PR and CR proportions in each state. ( F ) The Dot plot shows the expression levels of stemness-related genes in each cluster of epithelial. ( G ) The Boxplot shows stemness scores among different clusters of epithelial cells. ( H ) The state proportion in each cluster. ( I ) Pseudotime analysis shows the derivation of clusters 0, 1, 4, 7, 9, and 10 from epithelial cells based on monocle states and cluster origin. ( J ) Trajectory of the expression of CD80. ( K ) Analysis of the correlation between CD80 and stemness-related genes ALDH1A1 , SOX9 , MET, and MYC in epithelial cells from PR and CR groups. ( L ) Analysis of the correlation between CD80 and stemness-related genes CD44 , BMI1 , MET, and POU5F1 in HNSCC based on TCGA HNSCC datasets.

Article Snippet: The primary antibodies used were anti-BMI1 antibody (Proteintech, Wuhan, China, Cat#66161-1-Ig) and anti-Caspase3 antibody (Absin Bioscience Inc., Shanghai, China, Cat#abs119676).

Techniques: Derivative Assay, Expressing

Journal: Cells

Article Title: CD80-Mediated T-Cell Suppression by Cancer Stem-like Cells in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cells15030266

Figure Lengend Snippet: CD80 is highly expressed in CSCs. ( A ) Western blot shows protein expression levels of CD80 in 10 paired HNSCC tissues. ( B ) The expression level of CD80 in normal epithelial cell line (HaCaT) and HNSCC cell lines (HN6, CAL27, SCC9, and SCC25). Values are mean ± SD. ns, no significance; ** p < 0.01; *** p < 0.001; One-way ANOVA test; n = 3. ( C ) Flow cytometry isolates ALDH high CD44 + populations from HNSCC cell lines. The left panel shows the fluorescence minus one (FMO) control (the ALDH-only control). And RT-qPCR shows significantly higher CD80 expression in ALDH high CD44 + versus ALDH low CD44 − cancer cells. Values are mean ± SD. * p < 0.05; *** p < 0.001; Student’s t test, n = 3. ( D ) Flow cytometry isolates BMI1 + populations from HNSCC cell lines, and RT-qPCR shows significantly higher CD80 expression in BMI1 + versus BMI1 − cancer cells. Values are mean ± SD. * p < 0.05; *** p < 0.001; Student’s t test, n = 3. ( E ) RT-qPCR shows significantly higher CD80 expression in tumor sphere versus adherent cells in HNSCC cells. Values are mean ± SD. *** p < 0.001; Student’s t test, n = 3. ( F ) Representative images of flow-cytometry show CD80 + HNSCC cells. ( G ) Representative images of tumor spheres and quantification of the sphere number of CD80 − and CD80 + HNSCC cells. Scale bar, 200 μm. Values are mean ± SD. * p < 0.05; ** p < 0.01; Student’s t test, n = 3.

Article Snippet: The primary antibodies used were anti-BMI1 antibody (Proteintech, Wuhan, China, Cat#66161-1-Ig) and anti-Caspase3 antibody (Absin Bioscience Inc., Shanghai, China, Cat#abs119676).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Control, Quantitative RT-PCR