Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, the protein levels of PODXL and BMI1 were determined with western blot analysis in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA 3.1 vector (VC, lane 2), cells stably transfected with PODXL (lane 3), cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I, lane 4), cells stably transfected with Bmi1 (lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODXL-shRNA (lane 7), cells stably transduced with BMI1-shRNA (lane 8), cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA, lane 9), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA, lane 10). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the Western blots was measured by densitometry, and the density of the PODXL and the Bmi1 blots was normalized against that of the GAPHD blot in the same sample to obtain a relative blot density to represent relative PODXL and Bmi1 content in each sample, respectively. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, mRNA levels of Bmi1 were determined with real-time RT-PCR in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Quantitative RT-PCR, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were transfected with human Bmi1 promoter/luciferase reporter plasmids and then cultured for 24 hours. Luciferase activities were determined in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The luciferase activity was expressed as fold changes to that of NC (designated as 1).

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Transfection, Luciferase, Cell Culture, Control, Stable Transfection, Plasmid Preparation, Transduction, shRNA, Activity Assay

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were pre-treated with transcription inhibitor actinomycin D (1 mg/mL) for 30 minutes, and then cultured for 1, 2 or 4 hours in medium containing actinomycin D (1 mg/mL). The Bmi1 mRNA level were then determined with real-time RT-PCR in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA).

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Cell Culture, Quantitative RT-PCR, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were treated with or without various concentrations of cisplatin for 48 hours. The half maximal inhibitory concentration (IC50) values were determined in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The IC50 dose-response curves were plotted with GraphPad Prism 5.0 (GraphPad Software). The dose-response curves for VC and SC in both SCC-4 and Tca8113 cells are presented in , because they mostly overlap with the dose-response curve of NC. IC50 values (mean±SD) are presented by histograms. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Concentration Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA, Software

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were treated with cisplatin (10 μM) for 12 and 24 hours. Apoptosis was measured with a microplate reader-based TiterTACS in situ apoptosis detection kit (R&D systems) in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). Cell apoptosis was shown as fold changes to that of untreated NC (at 0 hour of treatment; designated as 1). * p <0.05 vs. controls (NC, VC and SC).

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: In Situ, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, the FAK activity was determined with a Universal Tyrosine Kinase Assay kit (Takara Biomedical Technology) in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The FAK activity was shown as fold changes to that of NC (designated as 1). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: Human Bmi1 cDNA clone (SC116894) was purchased from Origene (Beijing, China) and the full length Bmi1 cDNA sequence was subcloned into the pcDNA 3.1 plasmid.

Techniques: Activity Assay, Universal Tyrosine Kinase Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: Biochimica et biophysica acta

Article Title: Bmi1 promotes prostate tumorigenesis via inhibiting p16(INK4A) and p14(ARF) expression.

doi: 10.1016/j.bbadis.2008.08.009

Figure Lengend Snippet: Fig. 1. Expression of Bmi1 in prostate cancer cells. (A) LNCaP, PC3, DU145, and MCF7 (breast cancer cells) cells were examined for Bmi1 expression by western blot. (B) IF staining of LNCaP, PC3, DU145, and MCF7 cells for Bmi1 protein. Nuclei were counter- stained with DAPI (blue). Scale bar represents 10 μM.

Article Snippet: Primary antibodies specific for Bmi1 (1:100, Santa Cruz Biotechnology), p16INK4A (1:100, Santa Cruz Biotechnology), and p14ARF (1:1000, Sigma) were incubated with the sections overnight at 4 °C.

Techniques: Expressing, Western Blot, Staining

Journal: Biochimica et biophysica acta

Article Title: Bmi1 promotes prostate tumorigenesis via inhibiting p16(INK4A) and p14(ARF) expression.

doi: 10.1016/j.bbadis.2008.08.009

Figure Lengend Snippet: Fig. 2. Expression of Bmi1 protein in primary prostate cancer tissues associates with reduction in p16INK4A and p14ARF. H and E (HE) and immunohistochemical (IHC) staining of normal prostatic gland (Normal), PIN, and carcinoma with Bmi1-, p16INK4A- and p14ARF specific antibodies or control IgG (IgG). All tissues were from the same patient/slide. Scale bar represents 40 μM. The inset areas are enlarged.

Article Snippet: Primary antibodies specific for Bmi1 (1:100, Santa Cruz Biotechnology), p16INK4A (1:100, Santa Cruz Biotechnology), and p14ARF (1:1000, Sigma) were incubated with the sections overnight at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Control

Journal: Biochimica et biophysica acta

Article Title: Bmi1 promotes prostate tumorigenesis via inhibiting p16(INK4A) and p14(ARF) expression.

doi: 10.1016/j.bbadis.2008.08.009

Figure Lengend Snippet: Fig. 3. Bmi1 inhibits the expression of p16INK4A and p14ARF and enhances hTERT expression in prostate cancer cells. (A) DU145, PC3, and LNCaP cells were stably transfected with empty retrovirus (pBabe) or Bmi1 retrovirus. Bmi1 expression was examined by western blot using anti-FLAG and anti-Bmi1 (αBmi1) antibodies. The expression of p16INK4A, p14ARF, and actin was also examined by western blot using specific antibodies. (B) 293T cells were transiently transfected with empty vector or Bmi1 (as indicated) with a hTERT promoter-driven luciferase construct plus a β-Gal construct for 48 h. Luciferase and β-Gal enzymatic activities were determined. Luciferase activities were normalized against β-Gal activities. Each transfection was carried out in triplicate and the experiment was repeated twice.

Article Snippet: Primary antibodies specific for Bmi1 (1:100, Santa Cruz Biotechnology), p16INK4A (1:100, Santa Cruz Biotechnology), and p14ARF (1:1000, Sigma) were incubated with the sections overnight at 4 °C.

Techniques: Expressing, Stable Transfection, Transfection, Western Blot, Plasmid Preparation, Luciferase, Construct

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, the protein levels of PODXL and BMI1 were determined with western blot analysis in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA 3.1 vector (VC, lane 2), cells stably transfected with PODXL (lane 3), cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I, lane 4), cells stably transfected with Bmi1 (lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODXL-shRNA (lane 7), cells stably transduced with BMI1-shRNA (lane 8), cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA, lane 9), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA, lane 10). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the Western blots was measured by densitometry, and the density of the PODXL and the Bmi1 blots was normalized against that of the GAPHD blot in the same sample to obtain a relative blot density to represent relative PODXL and Bmi1 content in each sample, respectively. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, mRNA levels of Bmi1 were determined with real-time RT-PCR in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Quantitative RT-PCR, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were transfected with human Bmi1 promoter/luciferase reporter plasmids and then cultured for 24 hours. Luciferase activities were determined in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The luciferase activity was expressed as fold changes to that of NC (designated as 1).

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Transfection, Luciferase, Cell Culture, Control, Stable Transfection, Plasmid Preparation, Transduction, shRNA, Activity Assay

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were pre-treated with transcription inhibitor actinomycin D (1 mg/mL) for 30 minutes, and then cultured for 1, 2 or 4 hours in medium containing actinomycin D (1 mg/mL). The Bmi1 mRNA level were then determined with real-time RT-PCR in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA).

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Cell Culture, Quantitative RT-PCR, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were treated with or without various concentrations of cisplatin for 48 hours. The half maximal inhibitory concentration (IC50) values were determined in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The IC50 dose-response curves were plotted with GraphPad Prism 5.0 (GraphPad Software). The dose-response curves for VC and SC in both SCC-4 and Tca8113 cells are presented in , because they mostly overlap with the dose-response curve of NC. IC50 values (mean±SD) are presented by histograms. a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Concentration Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA, Software

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: (A) SCC-4 and (B) Tca8113 OTSCC cells were treated with cisplatin (10 μM) for 12 and 24 hours. Apoptosis was measured with a microplate reader-based TiterTACS in situ apoptosis detection kit (R&D systems) in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). Cell apoptosis was shown as fold changes to that of untreated NC (at 0 hour of treatment; designated as 1). * p <0.05 vs. controls (NC, VC and SC).

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: In Situ, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: PLoS ONE

Article Title: Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0123208

Figure Lengend Snippet: In (A) SCC-4 and (B) Tca8113 OTSCC cells, the FAK activity was determined with a Universal Tyrosine Kinase Assay kit (Takara Biomedical Technology) in normal control cells (NC), cells stably transfected with the empty pcDNA 3.1 vector (VC), cells stably transfected with PODXL, cells stably transfected with PODXL and treated with focal adhesion kinase (FAK) inhibitor 14 (50 M) for 24 hours (PODXL+FAK-I), cells stably transfected with Bmi1, cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODXL-shRNA, cells stably transduced with BMI1-shRNA, cells stably transfected with PODXL and transduced with BMI1-shRNA (PODXL+BMI1-shRNA), and cells stably transfected with Bmi1 and transduced with PODXL-shRNA (Bmi1+PODXL-shRNA). The FAK activity was shown as fold changes to that of NC (designated as 1). a p <0.05 vs. controls (NC, VC and SC); b p <0.05 vs. PODXL; c p <0.05 vs. PODXL+FAK-I; d p <0.05 vs. Bmi1; e p <0.05 vs. PODXL-shRNA; f p <0.05 vs. Bmi1-shRNA; g p <0.05 vs. PODXL+Bmi1-shRNA.

Article Snippet: The PODXL PODXL (sc-44029-V) and Bmi1 (sc-29814-V) shRNA lentiviral particles purchd from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knockdown PODXL and Bmi1 gene expression, respectively.

Techniques: Activity Assay, Universal Tyrosine Kinase Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Transduction, shRNA

Journal: Oncotarget

Article Title: Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

doi: 10.18632/oncotarget.18648

Figure Lengend Snippet: Figure 2: Hinokitiol inhibits BMI1 protein expression, but not mRNA, in BCSCs. (A) AS-B145 or BT-474 cells were cultured into primary mammospheres and dissociated into single cell suspension by HyQTase treatment. Secondary mammosphere cells were then treated with hinokitiol as 1 or 10 μM for 48 h and harvested for analyzing BMI1 protein expression by western blot. BMI1 protein expression levels were normalized to GAPDH and compared with 0.1% EtOH treated group. *, P<0.05; **, P<0.01. (B) BT-474 cells were transfected with pCMV14-3X flag or pCMV-BMI1-flag for 48 hours and performed mammosphere cultivation under 0.1% ethanol (EtOH) or 10 μM hinokitiol treatment. The ALDH+ BCSCs were determined at Day 7 post treatment by ALDEFLUOR assay and FACS analysis. DEAB (N,N-diethylaminobenzaldehyde) was used for gating ALDH+ population of cells. V, pCMV14-3X flag; B, pCMV-BMI1-flag. (C) BMI1 mRNA expression in hinokitiol treated mammopsheres derived from AS-B145 or BT-474 cells was determined by SYBR Green based qRT-PCR. Data were expressed as the mean ± SD of two independent experiments.

Article Snippet: 5 μm sections were sliced and the expression of BMI1 or ALDH1A1 was detected by polyclonal rabbit anti-BMI1 antibody (Novus Biologicals, LLC) or polyclonal rabbit anti-ALDH1A1 antibody (GeneTex Inc.) followed by a standard avidinbiotin-peroxidase complex method.

Techniques: Expressing, Cell Culture, Suspension, Western Blot, Transfection, Derivative Assay, SYBR Green Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

doi: 10.18632/oncotarget.18648

Figure Lengend Snippet: Figure 3: miR-494-3p mediates the suppressive effect of hinokitiol in the self-renewal of BCSCs. (A) miR-494-3p expression in mammospheres derived from AS-B145 cells at Day 6 post hinokitiol treatment were determined by qRT-PCR. *, P<0.05; **, P<0.01. (B) AS-B145 mammosphere cells were transfected with 100nM of negative control inhibitor (NC inh) or miR-494-3p inhibitor (494 inh) for 24 hours and treated with 0.1% EtOH or 10 μM hinokitiol for further 48 hours. Cells were harvested for determination of BMI1 expression by western blot. (C) AS-B145 or BT-474 cells were firstly cultured into primary mammospheres, dissociated into single cell suspension, transfected with NC inh or 494 inh for 24 hours and performed secondary mammosphere cultivation under the treatment of 0.1% EtOH or 10 μM hinokitiol. Secondary mammosphere number was counted at Day 7 and data were expressed as the mean ± SD of triplicate determinations. White bar, EtOH treated group; gray bar, hinokitiol treated group. Scale bar= 100 μm. The experiments were repeated at least two times and data from one experiment were presented.

Article Snippet: 5 μm sections were sliced and the expression of BMI1 or ALDH1A1 was detected by polyclonal rabbit anti-BMI1 antibody (Novus Biologicals, LLC) or polyclonal rabbit anti-ALDH1A1 antibody (GeneTex Inc.) followed by a standard avidinbiotin-peroxidase complex method.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Cell Culture, Suspension

Journal: Oncotarget

Article Title: Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

doi: 10.18632/oncotarget.18648

Figure Lengend Snippet: Figure 4: BMI1 is a target of miR-494-3p. (A) The alignment of BMI1 3’-UTR and miR-494-3p was obtained from the website of MICRORNA. ORG (http://www.microrna.org). 293-T or AS-B145 cells were transfected with a negative control (NC) mimic or miR-494- 3p (494-3p) mimic at a concentration of 100 nM together with wildtype BMI1 3’-UTR (BMI1 3’-UTR WT) or mutant from (BMI1 3’UTR del 762-768) for 48 h and determined luciferase activities. Data were presented as mean±SD. *, P<0.05; **, P<0.01. (B) AS-B145 or BT-474 cells were firstly cultured into primary mammospheres, dissociated into single cell suspension and transfected with NC or 494-3p mimic at a concentration of 100 nM for 48 hours. BMI1 expression was then determined by western blot. Inset values indicate protein expression normalized to tubulin. The experiments were repeated at least two times and data from one experiment were presented.

Article Snippet: 5 μm sections were sliced and the expression of BMI1 or ALDH1A1 was detected by polyclonal rabbit anti-BMI1 antibody (Novus Biologicals, LLC) or polyclonal rabbit anti-ALDH1A1 antibody (GeneTex Inc.) followed by a standard avidinbiotin-peroxidase complex method.

Techniques: Transfection, Negative Control, Concentration Assay, Mutagenesis, Luciferase, Cell Culture, Suspension, Expressing, Western Blot

Journal: Oncotarget

Article Title: Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

doi: 10.18632/oncotarget.18648

Figure Lengend Snippet: Figure 5: miR-494-3p serves as an oncosuppressor in breast cancer. (A, B) The AS-B145 (A) or BT-474 (B) cells were transfected with negative control (NC) or miR-494-3p (494-3p) mimic at a concentrations of 100 nM for 24 hours and performed primary mammosphere cultivation. The number of formed primary mammospheres was counted at Day 7 and collected for second time transfection with NC or 494-3p mimic. After transfection for 24 hours, the cells were used for secondary mammosphere cultivation and counted the formed mammospheres at Day 7. *, P<0.05; **, P<0.01. Scale bar= 100 μm. (C, D) BT-474 cells were incubated with miR-494-3p Smartflare beads for 16 hours and sorted into miR-494-3plow (494low, the fluorescence intensity lower than 10 as similar to no beads control) or miR-494-3phigh (494high, the fluorescence intensity higher than 30) cells and detected the BMI1 expression by western blot (C). sh-Bmi1 transduced mammosphere cells from BT-474 were used as a control. The sorted 494low or 494high cells were then performed mammosphere cultivation and the number of formed mammosphere was pictured and counted at Day 7. **, p< 0.01. Scale bar= 50 μm. (E, F) BT-474 cells were transfected with NC or 494-3p mimic at a concentration of 100 nM for 24 hours and cells were harvested for xenograftment assay by injection into mammary fads of NOD/SCID mice (E). The formed tumors were taken and analyzed BMI1 expression by immunohistochemistry or western blot (F). Arrows indicated tumor cells with nuclear BMI1 expression.

Article Snippet: 5 μm sections were sliced and the expression of BMI1 or ALDH1A1 was detected by polyclonal rabbit anti-BMI1 antibody (Novus Biologicals, LLC) or polyclonal rabbit anti-ALDH1A1 antibody (GeneTex Inc.) followed by a standard avidinbiotin-peroxidase complex method.

Techniques: Transfection, Negative Control, Incubation, Fluorescence, Control, Expressing, Western Blot, Concentration Assay, Injection, Immunohistochemistry

Journal: Oncotarget

Article Title: Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

doi: 10.18632/oncotarget.18648

Figure Lengend Snippet: Figure 7: Hinokitiol reduces in vivo tumorigenicity of BCSCs. BT-474 cells were firstly cultured into mammospheres and 1×105 cells were injected into mammary fat pads of NOD/SCID mice for tumor growth. (A) The treatment of hinokitiol at a dose of 40mg/kg was performed when tumors reached 100 mm3 by twice/week until 18 weeks. (B) miR-494-3p expression in each formed tumor was determined by qRT-PCR. **, P< 0.01. (C) BMI1 expression in tumors was determined by western blot. H1 or H2 represented independent tumor samples from hinokitiol treated mice. GAPDH was used as protein loading control. The inserted numbers indicated the relative expression level of BMI1 when compared to the EtOH treated sample. (D) The expression of ALDH1A1 and BMI1 in formed tumors was determined by immunohistochemistry.

Article Snippet: 5 μm sections were sliced and the expression of BMI1 or ALDH1A1 was detected by polyclonal rabbit anti-BMI1 antibody (Novus Biologicals, LLC) or polyclonal rabbit anti-ALDH1A1 antibody (GeneTex Inc.) followed by a standard avidinbiotin-peroxidase complex method.

Techniques: In Vivo, Cell Culture, Injection, Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

doi: 10.3390/ijms17071092

Figure Lengend Snippet: Overexpression of miR-494-3p induces cellular senescence in SAS cells. SAS cells were transfected with 100 nM miR-494-3p mimic (494-3p) or negative control mimic (NC). ( A ) Cellular senescence was determined by senescence-associated β-galactosidase (SA-β-Gal) staining at day 7 post-transfection. The quantification results were collected by 3 random fields of each miRNA mimic transfected samples. Scale bar: 50 μm. **, p < 0.01; ( B ) The expression of p53, p21, p16 INK4a , retinoblastoma 1 (RB1), or B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) was determined by Western blot at day 2 post-transfection. β-actin was used as an internal control. The inserted numbers indicated relative expression levels as comparison with NC group.

Article Snippet: A firefly luciferase reporter plasmid with full length Bmi1 3′-UTR sequence was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Over Expression, Transfection, Negative Control, Staining, Expressing, Western Blot, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

doi: 10.3390/ijms17071092

Figure Lengend Snippet: Bmi1 was a target of miR-494-3p in SAS cells. ( A ) Negative control mimic (NC) or miR-494-3p mimic (494-3p) was transfected into SAS cells and total RNA were extracted at 48 h post-transfection. The mRNA expression of Bmi1 was determined by qRT-PCR. **, p < 0.01; ( B ) Schematic presentation of the constructed Bmi1 3′-untranslated region (UTR) reporter plasmids were used in this study. WT, wild type; Mut, mutant. SAS cells were transfected with negative control mimic (NC) or miR-494-3p mimic (494-3p) simultaneously with Bmi1 3′-UTR reporter plasmid for 48 h. The cells were then lysed with passive lysis buffer and the luciferase activity was determined. **, p < 0.01.

Article Snippet: A firefly luciferase reporter plasmid with full length Bmi1 3′-UTR sequence was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Negative Control, Transfection, Expressing, Quantitative RT-PCR, Construct, Mutagenesis, Plasmid Preparation, Lysis, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

doi: 10.3390/ijms17071092

Figure Lengend Snippet: Knockdown of Bmi1 increases radiosensitivity in SAS cells. ( A , B ) SAS cells were transduced with sh-Bmi1 or sh-LacZ carrying lentivirus and selected with 2 μg/mL puromycin for 3 days. The knockdown efficiency was determined by qPCR detection of Bmi1 mRNA expression ( A ). **, p < 0.01; Transduced cells were irradiated as 2, 4, or 8 Gy and cultured for further 72 h. Cell viability was determined by WST-1 reagent. Data were presented as survival fraction as comparison with non-irradiated cells ( B ). *, p < 0.05; ( C , D ) SAS cells were transfected with pcDNA3-Bmi1 vector with miR-494-3p mimic (494-3p). A negative control mimic (NC) was used as control. After 48 h, cells were harvested for determination of the Bmi1 expression by Western blot ( C ) and the radiation responses of SAS cells ( D ).

Article Snippet: A firefly luciferase reporter plasmid with full length Bmi1 3′-UTR sequence was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Transduction, Expressing, Irradiation, Cell Culture, Comparison, Transfection, Plasmid Preparation, Negative Control, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

doi: 10.3390/ijms17071092

Figure Lengend Snippet: Knockdown of Bmi1 activates senescence pathway in SAS cells. SAS cells were transduced with sh-Bmi1 or sh-LacZ carrying lentivirus and selected with 2 μg/mL puromycin for 3 days. ( A ) sh-LacZ or sh-Bmi1 transduced SAS cells were harvested by trypsin/EDTA and seeded into 12-well-plate at a density of 2 × 10 4 cells/well. The senescent cells were determined by SA-β-Gal staining at 72 h post seeding. Scale bar: 50 μm. **, p < 0.01; ( B ) The expression of p16 INK4a , RB1, or Bmi1 was determined by Western blot. β-actin was used as an internal control. The inserted numbers indicate relative expression levels as compared with sh-LacZ group.

Article Snippet: A firefly luciferase reporter plasmid with full length Bmi1 3′-UTR sequence was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Transduction, Staining, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells

doi: 10.3390/ijms17071092

Figure Lengend Snippet: The negative correlation between miR-494-3p and Bmi1 expression in HNC patients. Total RNA were extracted from tumor tissues of head and neck cancer (HNC) patients ( n = 35) and the expression of Bmi1 and miR-494-3p was determined by qRT-PCR methods and analyzed with Spearman rank correlation test.

Article Snippet: A firefly luciferase reporter plasmid with full length Bmi1 3′-UTR sequence was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Cells treated with PTC 209 and FMMC 419II cells stably transfected with Bmi1 shRNA plasmid show a decrease in Bmi1 mRNA expression. (B) Purified mRNA from the cells was reverse transcribed into cDNA and then analyzed for Bmi1 mRNA expression with quantitative PCR using TaqMan gene expression assays. The fold difference in expression between control samples and the PTC 209 treated of the Bmi1 shRNA transfected samples was calculated using the average of the Ct (threshold cycle) per group, relative to the expression of the internal control gene GAPDH . (C) FMMC cells were treated with PTC 209 (2 μM and 5 μM) for 24 hours and the expression of Bmi1 protein was detected with western blot. (D) PTC 209 treatment decreased Bmi1 protein expression in FMMC cells. Results are represented as mean ± S.E.M., *P <0.01, ***P <0.005.

Article Snippet: Mouse Bmi1 shRNA plasmid (Santa Cruz Biotechnology, Dallas, TX) consists of 3 target-specific lentivirus vector plasmids to knock down Bmi1 expression along with a puromycin selection gene.

Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Expressing, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Control, Western Blot

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: (A) Untreated (control) FMMC 419II cells. (B) Cells treated with 2 μM PTC 209. (C) Cells treated with 5 μM PTC 209. (D) Results of PTC 209 treatment shown as bar graphs. There is a G0/G1 cell cycle arrest in FMMC 419II cells treated with PTC 209 when compared to untreated cells. Similarly, FMMC 419II cells that have been transfected with a Bmi1 shRNA show a G1 arrest. (E) Bar graphs of cell cycle profiles for FMMC 419II cells from control (F) , colony 2 (G) , colony 4 (H) , and colony 5 (I) . Cells stained with PI/RNAse staining buffer were run on a FACSAria flow cytometer and cell cycle progression was analyzed and quantified (D, E) using FlowJo.

Article Snippet: Mouse Bmi1 shRNA plasmid (Santa Cruz Biotechnology, Dallas, TX) consists of 3 target-specific lentivirus vector plasmids to knock down Bmi1 expression along with a puromycin selection gene.

Techniques: Control, Transfection, shRNA, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation

doi: 10.18632/oncotarget.16317

Figure Lengend Snippet: Flow cytometry analysis of CD49f and CD24 expression for FMCC 419II cells treated with 2 μM PTC 209 versus Bmi1 shRNA transfected colonies 4 and 5 were carried out (A) , and the fluorescent intensities were quantitated (B) .

Article Snippet: Mouse Bmi1 shRNA plasmid (Santa Cruz Biotechnology, Dallas, TX) consists of 3 target-specific lentivirus vector plasmids to knock down Bmi1 expression along with a puromycin selection gene.

Techniques: Flow Cytometry, Expressing, shRNA, Transfection