Journal: Oncotarget

Article Title: Oroxylin A modulates mitochondrial function and apoptosis in human colon cancer cells by inducing mitochondrial translocation of wild-type p53.

doi: 10.18632/oncotarget.7927

Figure Lengend Snippet: Figure 3: Oroxylin A-induced SOD2 activity inhibition and increased ROS were mediated by wt-p53 mitochondrial translocation in HCT-116 cells. (A) Western blot detection of SOD2 expression in p53 siRNA-transfected HCT-116 cells after treatment with 100 μM oroxylin A for 24 h. (B–C) Quantification of ROS production in p53 siRNA-transfected HCT-116 cells. (D) SOD2 activity in p53 siRNA-transfected HCT-116 cells. (E–F) Quantification of p53 siRNA-transfected HCT-116 apoptotic cells. (G) The subcellular distribution of p53 in Recql4 siRNA-transfected HCT-116 cells as detected by Western blot after treatment with 100 μM oroxylin A for 24 h. (H–I) Quantification of ROS production in Recql4 siRNA-transfected HCT-116 cells. (J) SO2 activity in Recql4 siRNA-transfected HCT-116 cells. (K) Western blot analysis of p53 expression in Recql4 siRNA-transfected HCT-116 cells after treatment with 100 μM oroxylin A for 24 h. (L) HCT-116 cells were incubated with the indicated concentrations of DMSO, pifithrin-μ (10 μM), oroxylin A (100 μM), and pifithrin-μ (10 μM) + oroxylin A (100 μM) for 24 h, and the subcellular distribution of p53 analyzed by Western blotting. (M–N) ROS production in p53 pifithrin-μ (10 μM)-treated HCT-116 cells was monitored as described above and ROS levels quantified. (O) SOD2 activity in pifithrin-μ (10 μM)-treated HCT-116 cells was evaluated as described above. (P–Q) HCT-116 cells were incubated with the indicated concentrations of DMSO, shRNA/pifithrin-μ (10 μM), oroxylin A (100 μM), and shRNA/pifithrin-μ (10 μM) + oroxylin A (100 μM) for 24 h. Confocal images of the cells show the fluorescence of p53 in blue, Mito in green, and the merged images in Column 3. Bar, SD. *P < 0.05 or **P < 0.01 versus the untreated control.

Article Snippet: The Recql4 shRNA plasmid (TL309882) was obtained from Origene (Rockville, MA, USA).

Techniques: Activity Assay, Inhibition, Translocation Assay, Western Blot, Expressing, Transfection, Incubation, shRNA, Fluorescence, Control

Journal: Oncotarget

Article Title: Oroxylin A modulates mitochondrial function and apoptosis in human colon cancer cells by inducing mitochondrial translocation of wild-type p53.

doi: 10.18632/oncotarget.7927

Figure Lengend Snippet: Figure 5: Oroxylin A induces cytosolic activation of p53 and recql4 mitochondrial translocation in HCT-116 cells. (A) HCT-116 cells were incubated with the indicated concentrations of DMSO or oroxylin A (100 μM). Confocal images of the cells show the fluorescence of p53 in blue, Mito in green, Recql4 in red, and the merge in Column 4. (B) Antibodies to p53, Recql4, and Bcl-xL were used for the immunoprecipitation. Western blotting was used to detect complex formation between proteins. (C) HCT-116 cells were incubated with the indicated concentrations of DMSO, TPA (20 ng), and oroxylin A (100 μM). Confocal images of the cells show the fluorescence of p53 in blue, Bcl-xL in green, and the merged images in Column 3. (D) After the mitochondria were isolated from HCT-116 cells, Western blotting was performed to analyze Recql4, p53, and Bcl-2.

Article Snippet: The Recql4 shRNA plasmid (TL309882) was obtained from Origene (Rockville, MA, USA).

Techniques: Activation Assay, Translocation Assay, Incubation, Fluorescence, Immunoprecipitation, Western Blot, Isolation

Journal: Oncotarget

Article Title: Oroxylin A modulates mitochondrial function and apoptosis in human colon cancer cells by inducing mitochondrial translocation of wild-type p53.

doi: 10.18632/oncotarget.7927

Figure Lengend Snippet: Figure 6: Oroxylin A induces Recql4-dependent mitochondrial translocation of p53 in vivo. (A–D) HCT-116 tumors were transplanted into BALB/C nude mice and 100 mg/kg of oroxylin A administrated by intraperitoneal injection. (E) Confocal images of the cells show the fluorescence of p53 in red, Mito in green, and the merged images in Column 3.

Article Snippet: The Recql4 shRNA plasmid (TL309882) was obtained from Origene (Rockville, MA, USA).

Techniques: Translocation Assay, In Vivo, Injection, Fluorescence

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Analysis of chromosome specific mBAND-FISH in the metaphase spreads of normal primary (HMEC), immortalized (MCF-10F) and tumorigenic breast cancer cell lines. Chromosomes and chromosome regions positive for the probe are shown in the inserts. (B) FISH analysis using spectrum orange labeled BAC (Bacterial Artificial Chromosome) probe proximal to 8q24.3 chromosome locus harboring the RecQL4 gene in MDA-MB453 and MDA-MB361 cells. The BAC probe was purchased from Open Biosystems (RP11–374B7, Huntsville, Alabama, USA). (C) Upper panel: Agarose gel electrophoresis showing the abundance of RecQL4 genomic DNA detected by PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used as an internal control; Lower panel: Abundance of RecQL4 genomic DNA detected by quantitative real time PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used for normalizing the values of RecQL4.

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Labeling, Agarose Gel Electrophoresis, Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Western blot analysis of RecQL4 expression in parental, control shRNA (ShControl) and RecQL4 specific shRNA transduced MDA-MB453 cells (ShRecQL4-C5 & C8). RecQL4 expression was highly reduced in ShRecQL4-C5 and C8 stably selected by puromycin antibiotics. (B) Clonogenic survival assay was performed on ShRecQL4-C5 and C8 cells relative to parental and ShControl cells. Data presented at each time point were the mean value of eight cultures from two independent experiments. Bars indicate mean±SD. (C) Analysis of in vivo tumorigenicity of MDA-MB453 cells after RecQL4 silencing. Parental, ShControl and ShRecQL4-C5 & C8 cells were subcutaneously injected into seven immunosuppressed nude mice and tumor growth was monitored for 4 weeks. Tumor growth as a function of time is shown in the left panel. Images of tumors resected from mice are shown in the right panel. Note that 4 of 7 mice injected with either ShRecQL4-C5 or C8 cells did not show any tumor growth. Bars indicate mean±SD.

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Control, shRNA, Stable Transfection, Clonogenic Cell Survival Assay, In Vivo, Injection

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 10 6 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 10 6 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2-survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(−), pFlag-RecQL4-HD(−) or pFlag-RecQL4-CT(−). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag-RecQL4 protein.

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Control, Western Blot, Construct, Co-Immunoprecipitation Assay, Transfection, Expressing

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A)Western blot analysis showed a reduced level of survivin expression and phospho-histone 3 (p-H3) in RecQL4 suppressed cells (ShRecQL4-C5 and C8) at 1 and 1.5 h post 0.25 mM H2O2 treatment. Bax protein did not show any alteration after oxidative DNA damage. β-Actin was used to verify equal loading of proteins. (B) Forced expression of RecQL4 in ShRecQL4-C5 and C8 cells led to an increased level of survivin expression at 48 h post transfection with pFlag-RecQL4 plasmid (lane 1) or plasmids (lane 2 & 3) containing nucleotide substitution at RecQL4 ShRNA-targeted area without changing the amino acid sequence.

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, shRNA, Sequencing

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Analysis of chromosome specific mBAND-FISH in the metaphase spreads of normal primary (HMEC), immortalized (MCF-10F) and tumorigenic breast cancer cell lines. Chromosomes and chromosome regions positive for the probe are shown in the inserts. (B) FISH analysis using spectrum orange labeled BAC (Bacterial Artificial Chromosome) probe proximal to 8q24.3 chromosome locus harboring the RecQL4 gene in MDA-MB453 and MDA-MB361 cells. The BAC probe was purchased from Open Biosystems (RP11–374B7, Huntsville, Alabama, USA). (C) Upper panel: Agarose gel electrophoresis showing the abundance of RecQL4 genomic DNA detected by PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used as an internal control; Lower panel: Abundance of RecQL4 genomic DNA detected by quantitative real time PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used for normalizing the values of RecQL4.

Article Snippet: Human control and RecQL4 specific ShRNA lentiviral particles were procured from Santa Cruz.

Techniques: Labeling, Agarose Gel Electrophoresis, Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.

Article Snippet: Human control and RecQL4 specific ShRNA lentiviral particles were procured from Santa Cruz.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Western blot analysis of RecQL4 expression in parental, control shRNA (ShControl) and RecQL4 specific shRNA transduced MDA-MB453 cells (ShRecQL4-C5 & C8). RecQL4 expression was highly reduced in ShRecQL4-C5 and C8 stably selected by puromycin antibiotics. (B) Clonogenic survival assay was performed on ShRecQL4-C5 and C8 cells relative to parental and ShControl cells. Data presented at each time point were the mean value of eight cultures from two independent experiments. Bars indicate mean±SD. (C) Analysis of in vivo tumorigenicity of MDA-MB453 cells after RecQL4 silencing. Parental, ShControl and ShRecQL4-C5 & C8 cells were subcutaneously injected into seven immunosuppressed nude mice and tumor growth was monitored for 4 weeks. Tumor growth as a function of time is shown in the left panel. Images of tumors resected from mice are shown in the right panel. Note that 4 of 7 mice injected with either ShRecQL4-C5 or C8 cells did not show any tumor growth. Bars indicate mean±SD.

Article Snippet: Human control and RecQL4 specific ShRNA lentiviral particles were procured from Santa Cruz.

Techniques: Western Blot, Expressing, Control, shRNA, Stable Transfection, Clonogenic Cell Survival Assay, In Vivo, Injection

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 10 6 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 10 6 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2-survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(−), pFlag-RecQL4-HD(−) or pFlag-RecQL4-CT(−). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag-RecQL4 protein.

Article Snippet: Human control and RecQL4 specific ShRNA lentiviral particles were procured from Santa Cruz.

Techniques: Immunoprecipitation, Control, Western Blot, Construct, Co-Immunoprecipitation Assay, Transfection, Expressing

Journal: PLoS ONE

Article Title: RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: (A)Western blot analysis showed a reduced level of survivin expression and phospho-histone 3 (p-H3) in RecQL4 suppressed cells (ShRecQL4-C5 and C8) at 1 and 1.5 h post 0.25 mM H2O2 treatment. Bax protein did not show any alteration after oxidative DNA damage. β-Actin was used to verify equal loading of proteins. (B) Forced expression of RecQL4 in ShRecQL4-C5 and C8 cells led to an increased level of survivin expression at 48 h post transfection with pFlag-RecQL4 plasmid (lane 1) or plasmids (lane 2 & 3) containing nucleotide substitution at RecQL4 ShRNA-targeted area without changing the amino acid sequence.

Article Snippet: Human control and RecQL4 specific ShRNA lentiviral particles were procured from Santa Cruz.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, shRNA, Sequencing