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: (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 avidin-biotin-peroxidase complex method.

Techniques: Cell Culture, Suspension, Expressing, 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: (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 avidin-biotin-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: (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 avidin-biotin-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: (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-494-3p low (494 low , the fluorescence intensity lower than 10 as similar to no beads control) or miR-494-3p high (494 high , 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 494 low or 494 high 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 avidin-biotin-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: BT-474 cells were firstly cultured into mammospheres and 1×10 5 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 mm 3 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 avidin-biotin-peroxidase complex method.

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