skov3  (ATCC)


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    Structured Review

    ATCC skov3
    The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and <t>SKOV3</t> cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p
    Skov3, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy"

    Article Title: Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy

    Journal: Cancers

    doi: 10.3390/cancers13051141

    The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and SKOV3 cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p
    Figure Legend Snippet: The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and SKOV3 cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p

    Techniques Used: Animal Model, Injection, MTS Assay

    2) Product Images from "Elevated expression of pancreatic adenocarcinoma upregulated factor (PAUF) is associated with poor prognosis and chemoresistance in epithelial ovarian cancer"

    Article Title: Elevated expression of pancreatic adenocarcinoma upregulated factor (PAUF) is associated with poor prognosis and chemoresistance in epithelial ovarian cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30582-8

    Relationship between PAUF and TLR4 expression in ovarian cancer cell lines. ( A ) To probe surface and intracellular TLR4 expression level in SKOV3 and A2780 cells, PE-conjugated TLR4 antibody was confirmed and used in flow cytometric analysis. ( B,C ) PAUF expression level and secretion in ovarian cancer cells (SKOV3, A2780) were detected using western blot analysis, and PAUF in the culture supernatant of cancer cells was detected using ELISA. ( D ) The expression level of intracellular TLR4 was assessed by flow cytometric assays. TLR4 expression was decreased significantly in both cells after transfection of two TLR4-siRNAs in comparison to that of control-siRNA or control. (MFI: Mean Fluorescence Intensity) ( E ) To determine activation of MAPKs (ERK, P38 and JNK) and AKT by PAUF, starved cancer cells were treated with or without recombinant PAUF (5 μ g) and analyzed by western blotting. ( F ) To confirm MAPK activation in siRNA transfected A2780 or SKOV3 cells, cells were transfected with TLR4-siRNA or control siRNA and starved for 16 hours. Cells were treated with PAUF (5 μ g) for 20 min and analyzed by western blotting. The number below each western blot represents the ratio of the intensity of the band over the control intensity of scramble siRNA-treated cells. ( G ) For the cell proliferation assay, control, TLR4-, or PAUF-siRNA transfected A2780 or SKOV3 cells were cultured in 96-well white plates, and cell proliferation was detected using a Cell Titer-Glo luminescence assay kit. The data shown are the means ± s.e.m. for three independent experiments. β -actin was used as an internal reference. * p
    Figure Legend Snippet: Relationship between PAUF and TLR4 expression in ovarian cancer cell lines. ( A ) To probe surface and intracellular TLR4 expression level in SKOV3 and A2780 cells, PE-conjugated TLR4 antibody was confirmed and used in flow cytometric analysis. ( B,C ) PAUF expression level and secretion in ovarian cancer cells (SKOV3, A2780) were detected using western blot analysis, and PAUF in the culture supernatant of cancer cells was detected using ELISA. ( D ) The expression level of intracellular TLR4 was assessed by flow cytometric assays. TLR4 expression was decreased significantly in both cells after transfection of two TLR4-siRNAs in comparison to that of control-siRNA or control. (MFI: Mean Fluorescence Intensity) ( E ) To determine activation of MAPKs (ERK, P38 and JNK) and AKT by PAUF, starved cancer cells were treated with or without recombinant PAUF (5 μ g) and analyzed by western blotting. ( F ) To confirm MAPK activation in siRNA transfected A2780 or SKOV3 cells, cells were transfected with TLR4-siRNA or control siRNA and starved for 16 hours. Cells were treated with PAUF (5 μ g) for 20 min and analyzed by western blotting. The number below each western blot represents the ratio of the intensity of the band over the control intensity of scramble siRNA-treated cells. ( G ) For the cell proliferation assay, control, TLR4-, or PAUF-siRNA transfected A2780 or SKOV3 cells were cultured in 96-well white plates, and cell proliferation was detected using a Cell Titer-Glo luminescence assay kit. The data shown are the means ± s.e.m. for three independent experiments. β -actin was used as an internal reference. * p

    Techniques Used: Expressing, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Fluorescence, Activation Assay, Recombinant, Proliferation Assay, Cell Culture, Luminescence Assay

    3) Product Images from "Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion), Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion"

    Article Title: Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion), Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2015.03.003

    Modulation of NRF2 gene expression in normal and malignant ovarian epithelial cells by NRF2‐targeting ATFs. (A) Schematic overview of the NRF2 promoter region (transcript variant 1) containing the TSS (transcription start site, +1) and the target regions for the 6 engineered zinc finger proteins (ZFP OX1‐OX6). Histone modifications associated with ATF OX2 and OX5 were determined in ChIP region OX2‐ChIP6 and OX5‐ChIP5, respectively. CpGs are shown as vertical lines. (B) Relative NRF2 expression compared to empty vector control upon retroviral delivery of NRF2‐targeting ZFPs (OX1‐OX6) fused to the transcriptional repressor SKD in SKOV3, A2780 and OSE‐C2 cells. Relative NRF2 expression of the NRF2 cDNA control has only been determined in OSE‐C2 cells. Data is presented as mean ± SEM of at least three independent experiments. *p
    Figure Legend Snippet: Modulation of NRF2 gene expression in normal and malignant ovarian epithelial cells by NRF2‐targeting ATFs. (A) Schematic overview of the NRF2 promoter region (transcript variant 1) containing the TSS (transcription start site, +1) and the target regions for the 6 engineered zinc finger proteins (ZFP OX1‐OX6). Histone modifications associated with ATF OX2 and OX5 were determined in ChIP region OX2‐ChIP6 and OX5‐ChIP5, respectively. CpGs are shown as vertical lines. (B) Relative NRF2 expression compared to empty vector control upon retroviral delivery of NRF2‐targeting ZFPs (OX1‐OX6) fused to the transcriptional repressor SKD in SKOV3, A2780 and OSE‐C2 cells. Relative NRF2 expression of the NRF2 cDNA control has only been determined in OSE‐C2 cells. Data is presented as mean ± SEM of at least three independent experiments. *p

    Techniques Used: Expressing, Variant Assay, Chromatin Immunoprecipitation, Plasmid Preparation

    4) Product Images from "Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro"

    Article Title: Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.919089

    The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P
    Figure Legend Snippet: The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Techniques Used: In Vitro, Transfection, Migration, Cell Counting, CCK-8 Assay, Wound Healing Assay, Standard Deviation

    Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P
    Figure Legend Snippet: Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Techniques Used: Activation Assay, In Vitro, Expressing, Western Blot, Transfection, Plasmid Preparation, Cell Counting, CCK-8 Assay, Wound Healing Assay, Over Expression, Migration, Standard Deviation

    5) Product Images from "miRNA-574-3p inhibits metastasis and chemoresistance of epithelial ovarian cancer (EOC) by negatively regulating epidermal growth factor receptor (EGFR)"

    Article Title: miRNA-574-3p inhibits metastasis and chemoresistance of epithelial ovarian cancer (EOC) by negatively regulating epidermal growth factor receptor (EGFR)

    Journal: American Journal of Translational Research

    doi:

    Mir-574-3p enhances the drug sensitivity of EOC cells. A. SKOV3 and CAOV3 cells transfected with miR-574-3p, NC or anti-miR-574-3p lentivirus were treated with different concentrations of paclitaxel (0.0001, 0.0003, and 0.001 μg/ml). The cell inhibitory rate was quantified by the CCK-8 assay. B. SKOV3 and CAOV3 cells transfected with miR-574-3p, NC or anti-miR-574-3p lentivirus were treated with different concentrations of cisplatin (2.5, 5, and 10 μM). The cell inhibitory rate was quantified by the CCK-8 assay. C. MiR-574-3p-deficient SKOV3 and CAOV3 cells were transfected with or without EGFR siRNA and were treated with different concentrations of paclitaxel (0.0001, 0.0003, and 0.001 μg/ml). The cell inhibitory rate was quantified by the CCK-8 assay. D. MiR-574-3p-deficient SKOV3 and CAOV3 cells were transfected with or without EGFR siRNA and were treated with different concentrations of cisplatin (2.5, 5, and 10 μM). The cell inhibitory rate was quantified by the CCK-8 assay. *, P
    Figure Legend Snippet: Mir-574-3p enhances the drug sensitivity of EOC cells. A. SKOV3 and CAOV3 cells transfected with miR-574-3p, NC or anti-miR-574-3p lentivirus were treated with different concentrations of paclitaxel (0.0001, 0.0003, and 0.001 μg/ml). The cell inhibitory rate was quantified by the CCK-8 assay. B. SKOV3 and CAOV3 cells transfected with miR-574-3p, NC or anti-miR-574-3p lentivirus were treated with different concentrations of cisplatin (2.5, 5, and 10 μM). The cell inhibitory rate was quantified by the CCK-8 assay. C. MiR-574-3p-deficient SKOV3 and CAOV3 cells were transfected with or without EGFR siRNA and were treated with different concentrations of paclitaxel (0.0001, 0.0003, and 0.001 μg/ml). The cell inhibitory rate was quantified by the CCK-8 assay. D. MiR-574-3p-deficient SKOV3 and CAOV3 cells were transfected with or without EGFR siRNA and were treated with different concentrations of cisplatin (2.5, 5, and 10 μM). The cell inhibitory rate was quantified by the CCK-8 assay. *, P

    Techniques Used: Transfection, CCK-8 Assay

    EGFR knockdown reverses the promoting effect of anti-miR-574-3p on migration and invasion of EOC cells. In miR-574-3p-deficient SKOV3 (A) and CAOV3 (B) cells, the migration and invasion abilities were reversed after EGFR knockdown by siRNA. **, P
    Figure Legend Snippet: EGFR knockdown reverses the promoting effect of anti-miR-574-3p on migration and invasion of EOC cells. In miR-574-3p-deficient SKOV3 (A) and CAOV3 (B) cells, the migration and invasion abilities were reversed after EGFR knockdown by siRNA. **, P

    Techniques Used: Migration

    MiR-574-3p inhibits signaling pathways via targeting EGFR. A. EGFR siRNAs inhibited EGFR mRNA levels as confirmed by qRT-PCR. U6 was used as an internal control. B. EGFR siRNAs inhibited EGFR protein levels as confirmed by western blot analysis. GAPDH was used as an internal control. C. Protein levels of MMP-9, FAK, p-FAK, Src, p-Src, AKT, p-AKT and EGFR in anti-miR-574-3p-transfected SKOV3 cells with or without EGFR siRNA-3 co-transfection. GAPDH was used as a loading control for western blotting. D. Protein levels of MMP-9, FAK, p-FAK, Src, p-Src, AKT, p-AKT and EGFR in anti-miR-574-3p-transfected CAOV3 cells with or without EGFR siRNA-3 co-transfection. GAPDH was used as a loading control for western blotting. *, P
    Figure Legend Snippet: MiR-574-3p inhibits signaling pathways via targeting EGFR. A. EGFR siRNAs inhibited EGFR mRNA levels as confirmed by qRT-PCR. U6 was used as an internal control. B. EGFR siRNAs inhibited EGFR protein levels as confirmed by western blot analysis. GAPDH was used as an internal control. C. Protein levels of MMP-9, FAK, p-FAK, Src, p-Src, AKT, p-AKT and EGFR in anti-miR-574-3p-transfected SKOV3 cells with or without EGFR siRNA-3 co-transfection. GAPDH was used as a loading control for western blotting. D. Protein levels of MMP-9, FAK, p-FAK, Src, p-Src, AKT, p-AKT and EGFR in anti-miR-574-3p-transfected CAOV3 cells with or without EGFR siRNA-3 co-transfection. GAPDH was used as a loading control for western blotting. *, P

    Techniques Used: Quantitative RT-PCR, Western Blot, Transfection, Cotransfection

    MiR-574-3p inhibits multiple signaling pathways. A. Protein levels of Stat3, p-Stat3, AKT, p-AKT, ERK and p-ERK in transfected SKOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. B. Protein levels of Stat3, p-Stat3, AKT, p-AKT, ERK and p-ERK in transfected CAOV3 cells (NC, miR-574-3p or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. C. Protein levels of FAK, p-FAK, Src, p-Src, c-Jun and MMP-9 in transfected SKOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. D. Protein levels of FAK, p-FAK, Src, p-Src, c-Jun and MMP-9 in transfected CAOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. Ns, none significant, *, P
    Figure Legend Snippet: MiR-574-3p inhibits multiple signaling pathways. A. Protein levels of Stat3, p-Stat3, AKT, p-AKT, ERK and p-ERK in transfected SKOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. B. Protein levels of Stat3, p-Stat3, AKT, p-AKT, ERK and p-ERK in transfected CAOV3 cells (NC, miR-574-3p or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. C. Protein levels of FAK, p-FAK, Src, p-Src, c-Jun and MMP-9 in transfected SKOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. D. Protein levels of FAK, p-FAK, Src, p-Src, c-Jun and MMP-9 in transfected CAOV3 cells (NC, miR-574-3p, anti-NC or anti-miR-574-3p) cells. GAPDH was used as a loading control for western blotting. Ns, none significant, *, P

    Techniques Used: Transfection, Western Blot

    MiR-574-3p decreases EOC cell migration and invasion but does not affect cell viability. MiR-574-3p and anti-miR-574-3p decreased and promoted, respectively, cell migration and invasion in vitro in both SKOV3 (A) and CAOV3 cells (B). MiR-574-3p or anti-miR-574-3p had no effect on cell viability in both SKOV3 (C) and CAOV3 cells (D). *, P
    Figure Legend Snippet: MiR-574-3p decreases EOC cell migration and invasion but does not affect cell viability. MiR-574-3p and anti-miR-574-3p decreased and promoted, respectively, cell migration and invasion in vitro in both SKOV3 (A) and CAOV3 cells (B). MiR-574-3p or anti-miR-574-3p had no effect on cell viability in both SKOV3 (C) and CAOV3 cells (D). *, P

    Techniques Used: Migration, In Vitro

    MiR-574-3p decreases EGFR expression by directly targeting its 3’-UTR in EOC cells. According to the qRT-PCR results, miR-574-3p and anti-miR-574-3p lentiviruses significantly increased and decreased, respectively, miR-574-3p expression in both SKOV3 (A) and CAOV3 cells (B). C. Putative miR-574-3p-binding site in the EGFR 3’-UTR nucleotides. D. Luciferase activity assays with wild-type or mutant EGFR 3’-UTR were performed after co-transfection with miR-574-3p or negative control (NC) lentivirus in 293T, SKOV3 and CAOV3 cells. E. EGFR protein levels were determined by western blot analyses after transfection with miR-574-3p, anti-miR-574-3p or negative control lentivirus in SKOV3 and CAOV3 cells. GAPDH served as an internal control. Ns, none significant, *, P
    Figure Legend Snippet: MiR-574-3p decreases EGFR expression by directly targeting its 3’-UTR in EOC cells. According to the qRT-PCR results, miR-574-3p and anti-miR-574-3p lentiviruses significantly increased and decreased, respectively, miR-574-3p expression in both SKOV3 (A) and CAOV3 cells (B). C. Putative miR-574-3p-binding site in the EGFR 3’-UTR nucleotides. D. Luciferase activity assays with wild-type or mutant EGFR 3’-UTR were performed after co-transfection with miR-574-3p or negative control (NC) lentivirus in 293T, SKOV3 and CAOV3 cells. E. EGFR protein levels were determined by western blot analyses after transfection with miR-574-3p, anti-miR-574-3p or negative control lentivirus in SKOV3 and CAOV3 cells. GAPDH served as an internal control. Ns, none significant, *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Cotransfection, Negative Control, Western Blot, Transfection

    6) Product Images from "The lncRNA TUG1 promotes epithelial ovarian cancer cell proliferation and invasion via the WNT/β-catenin pathway"

    Article Title: The lncRNA TUG1 promotes epithelial ovarian cancer cell proliferation and invasion via the WNT/β-catenin pathway

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S167900

    Effect of TUG1 downregulation on EOC cell proliferation. Notes: ( A ) qRT-PCR analysis of the efficiency of TUG1 downregulation by treatment of HO8910 and SKOV3 EOC cells transfected with si-TUG1. MTT analysis 5 days after transfection in negative control-transfected and TUG1 shRNA-transfected HO8910 ( B ) and SKOV3 ( C ) cell lines (* P
    Figure Legend Snippet: Effect of TUG1 downregulation on EOC cell proliferation. Notes: ( A ) qRT-PCR analysis of the efficiency of TUG1 downregulation by treatment of HO8910 and SKOV3 EOC cells transfected with si-TUG1. MTT analysis 5 days after transfection in negative control-transfected and TUG1 shRNA-transfected HO8910 ( B ) and SKOV3 ( C ) cell lines (* P

    Techniques Used: Quantitative RT-PCR, Transfection, MTT Assay, Negative Control, shRNA

    Effect of TUG1 on EOC cell migration and invasion. Notes: ( A ) Cell migration and ( B ) cell invasion were significantly inhibited after overexpression of TUG1 in HO8910 and SKOV3 cells (* P
    Figure Legend Snippet: Effect of TUG1 on EOC cell migration and invasion. Notes: ( A ) Cell migration and ( B ) cell invasion were significantly inhibited after overexpression of TUG1 in HO8910 and SKOV3 cells (* P

    Techniques Used: Migration, Over Expression

    7) Product Images from "Functional analysis of 11q13.5 amplicon identifies Rsf-1 (HBXAP) as a gene involved in paclitaxel resistance in ovarian cancer"

    Article Title: Functional analysis of 11q13.5 amplicon identifies Rsf-1 (HBXAP) as a gene involved in paclitaxel resistance in ovarian cancer

    Journal:

    doi: 10.1158/0008-5472.CAN-08-3602

    Rsf-1 induction in SKOV3 cells increases paclitaxel resistance
    Figure Legend Snippet: Rsf-1 induction in SKOV3 cells increases paclitaxel resistance

    Techniques Used:

    8) Product Images from "miR-423-5p serves as a diagnostic indicator and inhibits the proliferation and invasion of ovarian cancer"

    Article Title: miR-423-5p serves as a diagnostic indicator and inhibits the proliferation and invasion of ovarian cancer

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6015

    miR-423-5p is downregulated in ovarian cancer. miR-423-5p expression was determined in (A) ovarian tumor tissues of 40 ovarian cancer patients and 20 ovarian endometriosis tissues, (B) plasma samples of 40 ovarian cancer patients and 20 plasma samples of healthy controls and in (C) the normal ovarian epithelial cell line HOEC and the ovarian cancer cell lines A2780s, A2780cp, SKOV3, CAOV3 and PA-1. Reverse transcription-quantitative polymerase chain reaction was employed and U6 was used as a loading control. **P
    Figure Legend Snippet: miR-423-5p is downregulated in ovarian cancer. miR-423-5p expression was determined in (A) ovarian tumor tissues of 40 ovarian cancer patients and 20 ovarian endometriosis tissues, (B) plasma samples of 40 ovarian cancer patients and 20 plasma samples of healthy controls and in (C) the normal ovarian epithelial cell line HOEC and the ovarian cancer cell lines A2780s, A2780cp, SKOV3, CAOV3 and PA-1. Reverse transcription-quantitative polymerase chain reaction was employed and U6 was used as a loading control. **P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    9) Product Images from "Acetate production from glucose and coupling to mitochondrial metabolism in mammals"

    Article Title: Acetate production from glucose and coupling to mitochondrial metabolism in mammals

    Journal: Cell

    doi: 10.1016/j.cell.2018.08.040

    ROS catalyzes the oxidative decarboxylation of pyruvate to generate acetate in mammalian cells (A) Schematic of the potential sources of elemental oxygen in acetate produced from different routes. (B) Experimental setup of 18 O 2 tracing assay as described in STAR Methods. (C) Incorporation of 18 O into metabolites in tryptophan metabolism. (D) Schematic of 18 O and 13 C incorporation into acetate produced from H 2 O 2 -mediated pyruvate decarboxylation, and representative chromatogram and tandem mass spectrum of [ 13 C 2 , 18 O 1 ]-acetate derivative. Blue open circles and red solid circles denote O and C, respectively. (E) Fraction of [ 13 C 2 , 18 O 1 ]-acetate out of the glucose-derived acetate pool. (F) Schematic of endogenous H 2 O 2 generated from superoxides and superoxide dismutase (SOD) and H 2 O 2 -mediated methionine oxidation and pyruvate decarboxylation. TTM, Ammonium tetrathiomolybdate, a SOD inhibitor. The effect of SOD inhibitor TTM on the relative levels of [ 18 O 1 ]-MetO (G), [ 13 C 2 , 18 O 1 ]-acetate (H) in SKOV3 cells cultured in [ 13 C 6 ]-glucose and 18 O 2 for 48 hrs. (I) Secretion of [ 13 C 2 ]-acetate from HCT116 cells in the presence or absence of TTM. (J) Release of [ 13 C 2 , 18 O 1 ]-acetate from HCT116 cells after addition of exogenous [ 18 O 2 ]-H 2 O 2 . (K) Relative abundance of [ 13 C 2 ]-acetate. The fractions in E used to represent the ROS contribution to acetate production were corrected for 18 O natural abundance and the 18 O 2 enrichment and. The data in J and K represent the acetate from the spent media collected at 10 min after addition of 0 or 300 μM [ 18 O 2 ]-H 2 O 2 (J) or H 2 O 2 (K) to HCT116 cells which were pre-incubated in [ 13 C 6 .
    Figure Legend Snippet: ROS catalyzes the oxidative decarboxylation of pyruvate to generate acetate in mammalian cells (A) Schematic of the potential sources of elemental oxygen in acetate produced from different routes. (B) Experimental setup of 18 O 2 tracing assay as described in STAR Methods. (C) Incorporation of 18 O into metabolites in tryptophan metabolism. (D) Schematic of 18 O and 13 C incorporation into acetate produced from H 2 O 2 -mediated pyruvate decarboxylation, and representative chromatogram and tandem mass spectrum of [ 13 C 2 , 18 O 1 ]-acetate derivative. Blue open circles and red solid circles denote O and C, respectively. (E) Fraction of [ 13 C 2 , 18 O 1 ]-acetate out of the glucose-derived acetate pool. (F) Schematic of endogenous H 2 O 2 generated from superoxides and superoxide dismutase (SOD) and H 2 O 2 -mediated methionine oxidation and pyruvate decarboxylation. TTM, Ammonium tetrathiomolybdate, a SOD inhibitor. The effect of SOD inhibitor TTM on the relative levels of [ 18 O 1 ]-MetO (G), [ 13 C 2 , 18 O 1 ]-acetate (H) in SKOV3 cells cultured in [ 13 C 6 ]-glucose and 18 O 2 for 48 hrs. (I) Secretion of [ 13 C 2 ]-acetate from HCT116 cells in the presence or absence of TTM. (J) Release of [ 13 C 2 , 18 O 1 ]-acetate from HCT116 cells after addition of exogenous [ 18 O 2 ]-H 2 O 2 . (K) Relative abundance of [ 13 C 2 ]-acetate. The fractions in E used to represent the ROS contribution to acetate production were corrected for 18 O natural abundance and the 18 O 2 enrichment and. The data in J and K represent the acetate from the spent media collected at 10 min after addition of 0 or 300 μM [ 18 O 2 ]-H 2 O 2 (J) or H 2 O 2 (K) to HCT116 cells which were pre-incubated in [ 13 C 6 .

    Techniques Used: Produced, Derivative Assay, Generated, Cell Culture, Incubation

    10) Product Images from "Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness"

    Article Title: Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24315-1

    Inverse tumorigenicity of ECM1 subtypes. a Detection of ECM1 mRNA levels in CLs of ovarian cancer cell lines (Hey, HeyA8, SKOV3, SKOV3ip1, OVCA433, OVCA429, and A2780) and a normal cell line (HOSE) by qRT-PCR. ACTB (encoding β-actin) was used to normalize the expression of ECM1. Data are presented as mean ± SD. n = 3 biologically independent repeats. Representative images of ECM1 staining performed by IHC in normal ovary ( b ), normal fallopian tube ( c ), and ovarian carcinoma (OC) ( d ) tissues. The arrows indicate typical tissues (normal or cancer epithelium) stained with ECM1. Bars with 200 or 100 μm indicate the magnification of images. Silencing efficiency of ECM1 with specific shRNAs detected by WB ( e ), 3D culture image showing spheroid formation ( f , bars = 400 µm) and numbers ( g , data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t -test), and xenograft tumor growth ( h , data are presented as mean ± SD, n = 8v8 mice, two-tailed t -test) in eight mice generated from cells expressing ECM1 shRNA or control shRNA. Functional examination of ECM1 subtypes in HeyA8-ECM1 shRNA cells (A8i) after transfection of ECM1a (A8i-A), ECM1b (A8i-B), or empty vector (A8i-V), as confirmed by WB analysis ( i ); 3D culture images ( j , bars = 400 µm) and spheroid number ( k , data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t -test); and assessment of xenograft tumor growth ( l , data are presented as mean ± SD, n = 8v8 mice, two-tailed t -test) and tumor tissues ( m ) in eight mice injected with cells expressing ECM1a or ECM1b. n Alterations in signaling molecules associated with AKT/FAK/Paxillin/Rac after silencing of ECM1 and overexpression of ECM1a and ECM1b. Detection of the expression of phosphorylated proteins in cells treated or not treated with an HA antibody ( o , A8i-A cells) or treated/not treated with bioactive ECM1 (ECM1a) ( p , OVCA429 cells). PBS and mouse IgG were used as controls. β-actin was used as a loading control for WB analysis.
    Figure Legend Snippet: Inverse tumorigenicity of ECM1 subtypes. a Detection of ECM1 mRNA levels in CLs of ovarian cancer cell lines (Hey, HeyA8, SKOV3, SKOV3ip1, OVCA433, OVCA429, and A2780) and a normal cell line (HOSE) by qRT-PCR. ACTB (encoding β-actin) was used to normalize the expression of ECM1. Data are presented as mean ± SD. n = 3 biologically independent repeats. Representative images of ECM1 staining performed by IHC in normal ovary ( b ), normal fallopian tube ( c ), and ovarian carcinoma (OC) ( d ) tissues. The arrows indicate typical tissues (normal or cancer epithelium) stained with ECM1. Bars with 200 or 100 μm indicate the magnification of images. Silencing efficiency of ECM1 with specific shRNAs detected by WB ( e ), 3D culture image showing spheroid formation ( f , bars = 400 µm) and numbers ( g , data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t -test), and xenograft tumor growth ( h , data are presented as mean ± SD, n = 8v8 mice, two-tailed t -test) in eight mice generated from cells expressing ECM1 shRNA or control shRNA. Functional examination of ECM1 subtypes in HeyA8-ECM1 shRNA cells (A8i) after transfection of ECM1a (A8i-A), ECM1b (A8i-B), or empty vector (A8i-V), as confirmed by WB analysis ( i ); 3D culture images ( j , bars = 400 µm) and spheroid number ( k , data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t -test); and assessment of xenograft tumor growth ( l , data are presented as mean ± SD, n = 8v8 mice, two-tailed t -test) and tumor tissues ( m ) in eight mice injected with cells expressing ECM1a or ECM1b. n Alterations in signaling molecules associated with AKT/FAK/Paxillin/Rac after silencing of ECM1 and overexpression of ECM1a and ECM1b. Detection of the expression of phosphorylated proteins in cells treated or not treated with an HA antibody ( o , A8i-A cells) or treated/not treated with bioactive ECM1 (ECM1a) ( p , OVCA429 cells). PBS and mouse IgG were used as controls. β-actin was used as a loading control for WB analysis.

    Techniques Used: Quantitative RT-PCR, Expressing, Staining, Immunohistochemistry, Western Blot, Two Tailed Test, Mouse Assay, Generated, shRNA, Functional Assay, Transfection, Plasmid Preparation, Injection, Over Expression

    11) Product Images from "Extracellular Tissue Transglutaminase Activates Noncanonical NF-?B Signaling and Promotes Metastasis in Ovarian Cancer 1Extracellular Tissue Transglutaminase Activates Noncanonical NF-?B Signaling and Promotes Metastasis in Ovarian Cancer 1 2"

    Article Title: Extracellular Tissue Transglutaminase Activates Noncanonical NF-?B Signaling and Promotes Metastasis in Ovarian Cancer 1Extracellular Tissue Transglutaminase Activates Noncanonical NF-?B Signaling and Promotes Metastasis in Ovarian Cancer 1 2

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Extracellular TG2 induces cell invasion and EMT. (A) Cell invasion in Matrigel using 2D cultures with A2780, OV90, and SKOV3/pcDNA3.1 (control) or SKOV3/ASTG2 cells treated with buffer (upper panels, left) or rTG2 (1 µ g/ml; lower panels, left)
    Figure Legend Snippet: Extracellular TG2 induces cell invasion and EMT. (A) Cell invasion in Matrigel using 2D cultures with A2780, OV90, and SKOV3/pcDNA3.1 (control) or SKOV3/ASTG2 cells treated with buffer (upper panels, left) or rTG2 (1 µ g/ml; lower panels, left)

    Techniques Used:

    Extracellular TG2 activates noncanonical NF-κB signaling in OC cells. Western blot analysis for Skp2 (A) or CD44 (B) in OV90, SKOV3, and A2780 cells treated with rTG2 (1 µ g/ml). Densitometry quantifies protein expression relative to GAPDH.
    Figure Legend Snippet: Extracellular TG2 activates noncanonical NF-κB signaling in OC cells. Western blot analysis for Skp2 (A) or CD44 (B) in OV90, SKOV3, and A2780 cells treated with rTG2 (1 µ g/ml). Densitometry quantifies protein expression relative to GAPDH.

    Techniques Used: Western Blot, Expressing

    12) Product Images from "Mitochondrial Dynamics Mediated by DRP1 and MFN2 Contributes to Cisplatin Chemoresistance in Human Ovarian Cancer SKOV3 cells"

    Article Title: Mitochondrial Dynamics Mediated by DRP1 and MFN2 Contributes to Cisplatin Chemoresistance in Human Ovarian Cancer SKOV3 cells

    Journal: Journal of Cancer

    doi: 10.7150/jca.61379

    Knockdown of DRP1 or MFN2 changed mitochondrial morphology and ATP level in SKOV3 or SKOV3/DDP cells. (A) Mitochondrial morphology in SKOV3 or SKOV3/DDP cells transfected with or without the related siRNA. pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells. The fluorescence signal of pDsRed2-Mito was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1', A2', A3' and A4' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (B) The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. (C) The intracellular ATP level in SKOV3 and SKOV3/DDP cells transfected with or without the targeting siRNA. At 48 h after transfection, the intracellular ATP level in two cell lines was detected using a commercial ATP assay kit under the microplate reader, and the ATP level was normalized to the protein content in each sample. * p
    Figure Legend Snippet: Knockdown of DRP1 or MFN2 changed mitochondrial morphology and ATP level in SKOV3 or SKOV3/DDP cells. (A) Mitochondrial morphology in SKOV3 or SKOV3/DDP cells transfected with or without the related siRNA. pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells. The fluorescence signal of pDsRed2-Mito was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1', A2', A3' and A4' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (B) The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. (C) The intracellular ATP level in SKOV3 and SKOV3/DDP cells transfected with or without the targeting siRNA. At 48 h after transfection, the intracellular ATP level in two cell lines was detected using a commercial ATP assay kit under the microplate reader, and the ATP level was normalized to the protein content in each sample. * p

    Techniques Used: Transfection, Fluorescence, Microscopy, ATP Assay

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the migratory capability of SKOV3 and SKOV3/DDP cells. (A, C) Migratory capability of SKOV3 and SKOV3/DDP cells was examined by wound healing assay. Cells in each group was photographed under a microscope at 0h, 24h, 48h after scratch, scale bar=400μm (A). The percent wound closure of each cell line at 24 h and 48 h after scratch was calculated from at least three fields in four independent experiments (C). (B) The migratory capability of SKOV3 and SKOV3/DDP was evaluated by Transwell assay. Scale bar=100μm. (D) The number of migrated cells in each group was calculated in three different fields from three independent experiments. * p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the migratory capability of SKOV3 and SKOV3/DDP cells. (A, C) Migratory capability of SKOV3 and SKOV3/DDP cells was examined by wound healing assay. Cells in each group was photographed under a microscope at 0h, 24h, 48h after scratch, scale bar=400μm (A). The percent wound closure of each cell line at 24 h and 48 h after scratch was calculated from at least three fields in four independent experiments (C). (B) The migratory capability of SKOV3 and SKOV3/DDP was evaluated by Transwell assay. Scale bar=100μm. (D) The number of migrated cells in each group was calculated in three different fields from three independent experiments. * p

    Techniques Used: Wound Healing Assay, Microscopy, Transwell Assay

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the intracellular ROS level in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) Effect of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intracellular ROS production in SKOV3 and SKOV3/DDP cells. After the indicated treatments, cells were stained with DCFH-DA, and then the intracellular ROS was measured by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of DCFH-DA in each group was measured from three independent experiments. *** p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the intracellular ROS level in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) Effect of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intracellular ROS production in SKOV3 and SKOV3/DDP cells. After the indicated treatments, cells were stained with DCFH-DA, and then the intracellular ROS was measured by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of DCFH-DA in each group was measured from three independent experiments. *** p

    Techniques Used: Staining, Flow Cytometry, Fluorescence

    Effects of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intrinsic apoptosis pathway in SKOV3 and SKOV3/DDP cells. (A, B) The expression of pro-apoptotic protein BAX and cleavage of Caspase-3/9 was examined by western blotting assay in SKOV3 and SKOV3/DDP cells. After the indicated treatments, the pro-apoptotic protein BAX and cleavage of Caspase-3/9 in each group were detected by immunoblotting, and β-actin was used as the endogenous reference. (C-H) The densitometric analysis of BAX and Cleaved Caspase-3/9 in (A) and (B) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: Effects of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intrinsic apoptosis pathway in SKOV3 and SKOV3/DDP cells. (A, B) The expression of pro-apoptotic protein BAX and cleavage of Caspase-3/9 was examined by western blotting assay in SKOV3 and SKOV3/DDP cells. After the indicated treatments, the pro-apoptotic protein BAX and cleavage of Caspase-3/9 in each group were detected by immunoblotting, and β-actin was used as the endogenous reference. (C-H) The densitometric analysis of BAX and Cleaved Caspase-3/9 in (A) and (B) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Western Blot

    Alteration in the expression of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. (A, C, E, G) The western immunoblotting of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. The whole cell lysates of SKOV3 cells treated with 1mg/L cisplatin for 0 d, 2 d, 4 d, and 6 d were separated by SDS-PAGE electrophoresis, and the expression of DRP1, MFN1, MFN2 and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F, H) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (A, C, E, G) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: Alteration in the expression of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. (A, C, E, G) The western immunoblotting of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. The whole cell lysates of SKOV3 cells treated with 1mg/L cisplatin for 0 d, 2 d, 4 d, and 6 d were separated by SDS-PAGE electrophoresis, and the expression of DRP1, MFN1, MFN2 and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F, H) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (A, C, E, G) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Western Blot, SDS Page, Electrophoresis

    The sensitivity of SKOV3 cells to DDP after knockdown of DRP1 or overexpression of MFN2. (A) The expression level of MFN2 in SKOV3 cells transfected with MFN2 plasmid was detected using western blotting assay. β-actin was used as the endogenous reference. (B) The densitometric analysis of MFN2 in (A) was performed from three independent experiments. The relative expression level of MFN2 is indicated as a normalization of the ratio of MFN2/β-actin in each sample to the control. (C) After transfected with DRP1 siRNA for 48 h or MFN2 plasmid for 24 h, SKOV3 cells were exposed to DDP at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (D) The IC50 of cisplatin against SKOV3 cells was calculated according to the cisplatin dose-response curve. * p
    Figure Legend Snippet: The sensitivity of SKOV3 cells to DDP after knockdown of DRP1 or overexpression of MFN2. (A) The expression level of MFN2 in SKOV3 cells transfected with MFN2 plasmid was detected using western blotting assay. β-actin was used as the endogenous reference. (B) The densitometric analysis of MFN2 in (A) was performed from three independent experiments. The relative expression level of MFN2 is indicated as a normalization of the ratio of MFN2/β-actin in each sample to the control. (C) After transfected with DRP1 siRNA for 48 h or MFN2 plasmid for 24 h, SKOV3 cells were exposed to DDP at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (D) The IC50 of cisplatin against SKOV3 cells was calculated according to the cisplatin dose-response curve. * p

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay

    The silencing efficiency of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells by siRNA. (A, C, E) The expression of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells transfected with DRP1 siRNA and MFN2 siRNA was detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F) The densitometric analysis of DRP1 and MFN2 in (A, C, E) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: The silencing efficiency of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells by siRNA. (A, C, E) The expression of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells transfected with DRP1 siRNA and MFN2 siRNA was detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F) The densitometric analysis of DRP1 and MFN2 in (A, C, E) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Transfection, Western Blot

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the level of mitochondrial membrane potential in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) The effect of DRP1 siRNA and MFN2 siRNA on DDP-induced disruption of mitochondrial membrane potential in SKOV3/DDP cells. After the indicated treatments, cells were stained with TMRE to label mitochondrial membrane potential, and then mitochondrial membrane potential was evaluated by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of TMRE in each group was measured from three independent experiments. *** p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the level of mitochondrial membrane potential in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) The effect of DRP1 siRNA and MFN2 siRNA on DDP-induced disruption of mitochondrial membrane potential in SKOV3/DDP cells. After the indicated treatments, cells were stained with TMRE to label mitochondrial membrane potential, and then mitochondrial membrane potential was evaluated by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of TMRE in each group was measured from three independent experiments. *** p

    Techniques Used: Staining, Flow Cytometry, Fluorescence

    The expression of mitochondrial dynamics related proteins in SKOV3 and SKOV3/DDP cells . (A) The cisplatin dose-response curve in SKOV3 and SKOV3/DDP cells. Two cell lines were exposed to DDP at different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (B) The IC50 of cisplatin against SKOV3 and SKOV3/DDP cells was calculated according to the cisplatin dose-response curve. (C) The western immunoblotting of DRP1, MFN1, 2 and OPA1 in SKOV3 and SKOV3/DDP cells. The total proteins in whole cell lysate of two cell lines were separated by SDS-PAGE electrophoresis, and the expression levels of DRP1, MFN1, MFN2, and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (D) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (C) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. (E) Mitochondrial morphology in SKOV3 and SKOV3/DDP cells. The pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells to label mitochondria. The red fluorescence signal of DsRed was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1' and A2' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (F) The difference in average length of mitochondria between SKOV3 and SKOV3/DDP cells. The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. * p
    Figure Legend Snippet: The expression of mitochondrial dynamics related proteins in SKOV3 and SKOV3/DDP cells . (A) The cisplatin dose-response curve in SKOV3 and SKOV3/DDP cells. Two cell lines were exposed to DDP at different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (B) The IC50 of cisplatin against SKOV3 and SKOV3/DDP cells was calculated according to the cisplatin dose-response curve. (C) The western immunoblotting of DRP1, MFN1, 2 and OPA1 in SKOV3 and SKOV3/DDP cells. The total proteins in whole cell lysate of two cell lines were separated by SDS-PAGE electrophoresis, and the expression levels of DRP1, MFN1, MFN2, and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (D) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (C) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. (E) Mitochondrial morphology in SKOV3 and SKOV3/DDP cells. The pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells to label mitochondria. The red fluorescence signal of DsRed was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1' and A2' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (F) The difference in average length of mitochondria between SKOV3 and SKOV3/DDP cells. The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. * p

    Techniques Used: Expressing, CCK-8 Assay, Western Blot, SDS Page, Electrophoresis, Fluorescence, Microscopy

    13) Product Images from "Mitochondrial Dynamics Mediated by DRP1 and MFN2 Contributes to Cisplatin Chemoresistance in Human Ovarian Cancer SKOV3 cells"

    Article Title: Mitochondrial Dynamics Mediated by DRP1 and MFN2 Contributes to Cisplatin Chemoresistance in Human Ovarian Cancer SKOV3 cells

    Journal: Journal of Cancer

    doi: 10.7150/jca.61379

    Knockdown of DRP1 or MFN2 changed mitochondrial morphology and ATP level in SKOV3 or SKOV3/DDP cells. (A) Mitochondrial morphology in SKOV3 or SKOV3/DDP cells transfected with or without the related siRNA. pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells. The fluorescence signal of pDsRed2-Mito was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1', A2', A3' and A4' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (B) The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. (C) The intracellular ATP level in SKOV3 and SKOV3/DDP cells transfected with or without the targeting siRNA. At 48 h after transfection, the intracellular ATP level in two cell lines was detected using a commercial ATP assay kit under the microplate reader, and the ATP level was normalized to the protein content in each sample. * p
    Figure Legend Snippet: Knockdown of DRP1 or MFN2 changed mitochondrial morphology and ATP level in SKOV3 or SKOV3/DDP cells. (A) Mitochondrial morphology in SKOV3 or SKOV3/DDP cells transfected with or without the related siRNA. pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells. The fluorescence signal of pDsRed2-Mito was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1', A2', A3' and A4' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (B) The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. (C) The intracellular ATP level in SKOV3 and SKOV3/DDP cells transfected with or without the targeting siRNA. At 48 h after transfection, the intracellular ATP level in two cell lines was detected using a commercial ATP assay kit under the microplate reader, and the ATP level was normalized to the protein content in each sample. * p

    Techniques Used: Transfection, Fluorescence, Microscopy, ATP Assay

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the migratory capability of SKOV3 and SKOV3/DDP cells. (A, C) Migratory capability of SKOV3 and SKOV3/DDP cells was examined by wound healing assay. Cells in each group was photographed under a microscope at 0h, 24h, 48h after scratch, scale bar=400μm (A). The percent wound closure of each cell line at 24 h and 48 h after scratch was calculated from at least three fields in four independent experiments (C). (B) The migratory capability of SKOV3 and SKOV3/DDP was evaluated by Transwell assay. Scale bar=100μm. (D) The number of migrated cells in each group was calculated in three different fields from three independent experiments. * p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the migratory capability of SKOV3 and SKOV3/DDP cells. (A, C) Migratory capability of SKOV3 and SKOV3/DDP cells was examined by wound healing assay. Cells in each group was photographed under a microscope at 0h, 24h, 48h after scratch, scale bar=400μm (A). The percent wound closure of each cell line at 24 h and 48 h after scratch was calculated from at least three fields in four independent experiments (C). (B) The migratory capability of SKOV3 and SKOV3/DDP was evaluated by Transwell assay. Scale bar=100μm. (D) The number of migrated cells in each group was calculated in three different fields from three independent experiments. * p

    Techniques Used: Wound Healing Assay, Microscopy, Transwell Assay

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the intracellular ROS level in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) Effect of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intracellular ROS production in SKOV3 and SKOV3/DDP cells. After the indicated treatments, cells were stained with DCFH-DA, and then the intracellular ROS was measured by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of DCFH-DA in each group was measured from three independent experiments. *** p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the intracellular ROS level in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) Effect of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intracellular ROS production in SKOV3 and SKOV3/DDP cells. After the indicated treatments, cells were stained with DCFH-DA, and then the intracellular ROS was measured by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of DCFH-DA in each group was measured from three independent experiments. *** p

    Techniques Used: Staining, Flow Cytometry, Fluorescence

    Effects of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intrinsic apoptosis pathway in SKOV3 and SKOV3/DDP cells. (A, B) The expression of pro-apoptotic protein BAX and cleavage of Caspase-3/9 was examined by western blotting assay in SKOV3 and SKOV3/DDP cells. After the indicated treatments, the pro-apoptotic protein BAX and cleavage of Caspase-3/9 in each group were detected by immunoblotting, and β-actin was used as the endogenous reference. (C-H) The densitometric analysis of BAX and Cleaved Caspase-3/9 in (A) and (B) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: Effects of mitochondrial dynamics mediated by DRP1 and MFN2 on DDP-induced intrinsic apoptosis pathway in SKOV3 and SKOV3/DDP cells. (A, B) The expression of pro-apoptotic protein BAX and cleavage of Caspase-3/9 was examined by western blotting assay in SKOV3 and SKOV3/DDP cells. After the indicated treatments, the pro-apoptotic protein BAX and cleavage of Caspase-3/9 in each group were detected by immunoblotting, and β-actin was used as the endogenous reference. (C-H) The densitometric analysis of BAX and Cleaved Caspase-3/9 in (A) and (B) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Western Blot

    Alteration in the expression of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. (A, C, E, G) The western immunoblotting of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. The whole cell lysates of SKOV3 cells treated with 1mg/L cisplatin for 0 d, 2 d, 4 d, and 6 d were separated by SDS-PAGE electrophoresis, and the expression of DRP1, MFN1, MFN2 and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F, H) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (A, C, E, G) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: Alteration in the expression of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. (A, C, E, G) The western immunoblotting of mitochondrial dynamics-related proteins in SKOV3 cells under cisplatin stress. The whole cell lysates of SKOV3 cells treated with 1mg/L cisplatin for 0 d, 2 d, 4 d, and 6 d were separated by SDS-PAGE electrophoresis, and the expression of DRP1, MFN1, MFN2 and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F, H) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (A, C, E, G) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Western Blot, SDS Page, Electrophoresis

    The sensitivity of SKOV3 cells to DDP after knockdown of DRP1 or overexpression of MFN2. (A) The expression level of MFN2 in SKOV3 cells transfected with MFN2 plasmid was detected using western blotting assay. β-actin was used as the endogenous reference. (B) The densitometric analysis of MFN2 in (A) was performed from three independent experiments. The relative expression level of MFN2 is indicated as a normalization of the ratio of MFN2/β-actin in each sample to the control. (C) After transfected with DRP1 siRNA for 48 h or MFN2 plasmid for 24 h, SKOV3 cells were exposed to DDP at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (D) The IC50 of cisplatin against SKOV3 cells was calculated according to the cisplatin dose-response curve. * p
    Figure Legend Snippet: The sensitivity of SKOV3 cells to DDP after knockdown of DRP1 or overexpression of MFN2. (A) The expression level of MFN2 in SKOV3 cells transfected with MFN2 plasmid was detected using western blotting assay. β-actin was used as the endogenous reference. (B) The densitometric analysis of MFN2 in (A) was performed from three independent experiments. The relative expression level of MFN2 is indicated as a normalization of the ratio of MFN2/β-actin in each sample to the control. (C) After transfected with DRP1 siRNA for 48 h or MFN2 plasmid for 24 h, SKOV3 cells were exposed to DDP at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (D) The IC50 of cisplatin against SKOV3 cells was calculated according to the cisplatin dose-response curve. * p

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay

    The silencing efficiency of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells by siRNA. (A, C, E) The expression of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells transfected with DRP1 siRNA and MFN2 siRNA was detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F) The densitometric analysis of DRP1 and MFN2 in (A, C, E) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p
    Figure Legend Snippet: The silencing efficiency of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells by siRNA. (A, C, E) The expression of DRP1 and MFN2 in SKOV3 and SKOV3/DDP cells transfected with DRP1 siRNA and MFN2 siRNA was detected using western blotting assay. β-actin was used as the endogenous reference. (B, D, F) The densitometric analysis of DRP1 and MFN2 in (A, C, E) was performed from at least three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of interest protein/β-actin in each sample to the control. * p

    Techniques Used: Expressing, Transfection, Western Blot

    Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the level of mitochondrial membrane potential in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) The effect of DRP1 siRNA and MFN2 siRNA on DDP-induced disruption of mitochondrial membrane potential in SKOV3/DDP cells. After the indicated treatments, cells were stained with TMRE to label mitochondrial membrane potential, and then mitochondrial membrane potential was evaluated by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of TMRE in each group was measured from three independent experiments. *** p
    Figure Legend Snippet: Mitochondrial dynamics mediated by DRP1 and MFN2 regulates the level of mitochondrial membrane potential in SKOV3 and SKOV3/DDP cells after cisplatin. (A, C) The effect of DRP1 siRNA and MFN2 siRNA on DDP-induced disruption of mitochondrial membrane potential in SKOV3/DDP cells. After the indicated treatments, cells were stained with TMRE to label mitochondrial membrane potential, and then mitochondrial membrane potential was evaluated by flow cytometry. (B, D) Ten thousand cells in each group were analyzed and the mean fluorescence intensity of TMRE in each group was measured from three independent experiments. *** p

    Techniques Used: Staining, Flow Cytometry, Fluorescence

    The expression of mitochondrial dynamics related proteins in SKOV3 and SKOV3/DDP cells . (A) The cisplatin dose-response curve in SKOV3 and SKOV3/DDP cells. Two cell lines were exposed to DDP at different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (B) The IC50 of cisplatin against SKOV3 and SKOV3/DDP cells was calculated according to the cisplatin dose-response curve. (C) The western immunoblotting of DRP1, MFN1, 2 and OPA1 in SKOV3 and SKOV3/DDP cells. The total proteins in whole cell lysate of two cell lines were separated by SDS-PAGE electrophoresis, and the expression levels of DRP1, MFN1, MFN2, and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (D) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (C) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. (E) Mitochondrial morphology in SKOV3 and SKOV3/DDP cells. The pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells to label mitochondria. The red fluorescence signal of DsRed was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1' and A2' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (F) The difference in average length of mitochondria between SKOV3 and SKOV3/DDP cells. The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. * p
    Figure Legend Snippet: The expression of mitochondrial dynamics related proteins in SKOV3 and SKOV3/DDP cells . (A) The cisplatin dose-response curve in SKOV3 and SKOV3/DDP cells. Two cell lines were exposed to DDP at different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10 mg/L) for 24 hours. Cell viability was determined by CCK-8 assay kit. (B) The IC50 of cisplatin against SKOV3 and SKOV3/DDP cells was calculated according to the cisplatin dose-response curve. (C) The western immunoblotting of DRP1, MFN1, 2 and OPA1 in SKOV3 and SKOV3/DDP cells. The total proteins in whole cell lysate of two cell lines were separated by SDS-PAGE electrophoresis, and the expression levels of DRP1, MFN1, MFN2, and OPA1 were detected using western blotting assay. β-actin was used as the endogenous reference. (D) The densitometric analysis of DRP1, MFN1, MFN2, and OPA1 in (C) was performed from three independent experiments. The relative expression level of each protein is indicated as a normalization of the ratio of mitochondrial dynamics-related protein/β-actin in each sample to the control. (E) Mitochondrial morphology in SKOV3 and SKOV3/DDP cells. The pDsRed2-Mito was transferred into SKOV3 and SKOV3/DDP cells to label mitochondria. The red fluorescence signal of DsRed was detected as mitochondrial morphology in cells under a confocal microscope, scale bar=20 μm. A1' and A2' show mitochondria with higher magnification in the inserted boxes, scale bar = 5 μm. (F) The difference in average length of mitochondria between SKOV3 and SKOV3/DDP cells. The length of 270 mitochondria in each group was measured and the average length was calculated from three independent experiments. * p

    Techniques Used: Expressing, CCK-8 Assay, Western Blot, SDS Page, Electrophoresis, Fluorescence, Microscopy

    14) Product Images from "N‐acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages"

    Article Title: N‐acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages

    Journal: EMBO Reports

    doi: 10.15252/embr.202051981

    SKOV3 metabolic reprogramming mimics that of OVCAR3‐sh GLUL cells Intracellular levels of (A) citrate, (B) aspartate (Asp) in SKOV3 cells compared with OVCAR3‐shNT and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). NAA levels in SKOV3 cells compared with SKOV3‐over‐ GLUL cells ( n = 6 biological replicates). Metabolite levels are all measured with LC‐MS/MS analysis after 84‐h incubation. Transduction of SKOV3 cells verified by the RFP fluorescent signal. The images were collected by CELENA ® S Digital Imaging System at 10× magnification ( n = 3 biological replicates). The scale bars indicate 25 μm. Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by one‐way ANOVA analyses with Tukey correction (A, B), unpaired t‐test (C) and defined as * P
    Figure Legend Snippet: SKOV3 metabolic reprogramming mimics that of OVCAR3‐sh GLUL cells Intracellular levels of (A) citrate, (B) aspartate (Asp) in SKOV3 cells compared with OVCAR3‐shNT and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). NAA levels in SKOV3 cells compared with SKOV3‐over‐ GLUL cells ( n = 6 biological replicates). Metabolite levels are all measured with LC‐MS/MS analysis after 84‐h incubation. Transduction of SKOV3 cells verified by the RFP fluorescent signal. The images were collected by CELENA ® S Digital Imaging System at 10× magnification ( n = 3 biological replicates). The scale bars indicate 25 μm. Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by one‐way ANOVA analyses with Tukey correction (A, B), unpaired t‐test (C) and defined as * P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation, Transduction, Imaging

    GS knockdown in OVCAR3 cells reprograms glutamine metabolism qRT–PCR quantification of GLUL mRNA transcript and GS protein expression in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL after 24 h of culture ( n = 3 biological replicates). qRT–PCR quantification of (B) glutaminase ( GLS1 ) and (C) glutamine transporters ( LAT1 and ASCT2 ) mRNA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL after 24 h of culture ( n = 3 biological replicates). Extracellular Gln levels measured at different indicated time points in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Effect of 24 h of glutamine deprivation on cell proliferation measured by occupied wound assay (E) ( n = 6 biological replicates) and clonogenic test (F) ( n = 3 biological replicates) in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells. Cell migration propensity was measured through a Matrigel‐coated micropore filter in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL ( n = 3 biological replicates). The migration of the cells, stained with Calcein‐AM, was analyzed by fluorescence microscopy. The scale bars indicate 100 μm. Cell invasion valuated by qRT–PCR quantification of (H) CDH1 and (I) MMP2 transcript levels in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL ( n = 3 biological replicates). Quantitative analysis of the total lengths of the endothelial capillary network formed by HUVEC cells cocultured with SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL for 6 h ( n = 6 biological replicates). Quantification of released VEGF by SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells in conditioned media ( n = 3 biological replicates). Angiogenesis propensity measured by qRT–PCR quantification of (L) NOS2 and (M) CDH5 mRNA levels in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by one‐way ANOVA analyses with Tukey correction (A–C, G–M), two‐way ANOVA analyses with Tukey correction (D–F) and defined as * P
    Figure Legend Snippet: GS knockdown in OVCAR3 cells reprograms glutamine metabolism qRT–PCR quantification of GLUL mRNA transcript and GS protein expression in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL after 24 h of culture ( n = 3 biological replicates). qRT–PCR quantification of (B) glutaminase ( GLS1 ) and (C) glutamine transporters ( LAT1 and ASCT2 ) mRNA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL after 24 h of culture ( n = 3 biological replicates). Extracellular Gln levels measured at different indicated time points in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Effect of 24 h of glutamine deprivation on cell proliferation measured by occupied wound assay (E) ( n = 6 biological replicates) and clonogenic test (F) ( n = 3 biological replicates) in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells. Cell migration propensity was measured through a Matrigel‐coated micropore filter in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL ( n = 3 biological replicates). The migration of the cells, stained with Calcein‐AM, was analyzed by fluorescence microscopy. The scale bars indicate 100 μm. Cell invasion valuated by qRT–PCR quantification of (H) CDH1 and (I) MMP2 transcript levels in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL ( n = 3 biological replicates). Quantitative analysis of the total lengths of the endothelial capillary network formed by HUVEC cells cocultured with SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL for 6 h ( n = 6 biological replicates). Quantification of released VEGF by SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells in conditioned media ( n = 3 biological replicates). Angiogenesis propensity measured by qRT–PCR quantification of (L) NOS2 and (M) CDH5 mRNA levels in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by one‐way ANOVA analyses with Tukey correction (A–C, G–M), two‐way ANOVA analyses with Tukey correction (D–F) and defined as * P

    Techniques Used: Quantitative RT-PCR, Expressing, Migration, Staining, Fluorescence, Microscopy

    OVCAR3‐sh GLUL cells enhance LPS/IFNγ macrophage polarization toward a M2‐like, GS‐high phenotype compared with OVCAR3 cells qRT–PCR quantification of (A) M1 and (B) M2 markers in resting (Mφ) and LPS/IFNγ macrophages after a 24 h of coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of GLUL transcript in resting (Mφ) and LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). GS protein expression quantification (with representative blot) in LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of (E) LAT1 and ASCT2 and (F) GLS1 transcript level in resting (Mφ) and LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Data information: Data are displayed as mean ± SEM. For all panels, statistical significance was calculated by one‐way ANOVA analyses with Tukey correction and defined as * P
    Figure Legend Snippet: OVCAR3‐sh GLUL cells enhance LPS/IFNγ macrophage polarization toward a M2‐like, GS‐high phenotype compared with OVCAR3 cells qRT–PCR quantification of (A) M1 and (B) M2 markers in resting (Mφ) and LPS/IFNγ macrophages after a 24 h of coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of GLUL transcript in resting (Mφ) and LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). GS protein expression quantification (with representative blot) in LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of (E) LAT1 and ASCT2 and (F) GLS1 transcript level in resting (Mφ) and LPS/IFNγ macrophages after coculture with or without SKOV3, OVCAR3‐shNT, or OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Data information: Data are displayed as mean ± SEM. For all panels, statistical significance was calculated by one‐way ANOVA analyses with Tukey correction and defined as * P

    Techniques Used: Quantitative RT-PCR, Expressing

    GLUL knockdown effect on release of IL‐10 and NAA and on consequent metabolic aspects Extracellular levels of (A) IL‐10, and (B) NAA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Extracellular levels of Asn (C) and cytosolic levels of citrate (D), Asp (E), and Glu (F) in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of (G) NAT8L and (H) ACLY mRNA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Ammonia production driven by glutaminolysis interferes with mitochondrial dehydrogenases, favoring citrate, AcCoA and OAA mitochondria accumulation. Up‐regulation of NAT8L along with Glu accumulation, driving OAA transamination to Asp, leads to NAA production. NAA is also synthesized by NAT8L in the cytoplasm using AcCoA derived from citrate via ACLY . In the absence of intracellularly synthesized Gln, Glu drives OAA transamination leading to Asp, which accumulates due to decreased ASNS activity. AcCoA: Acetyl‐CoA; ASNS: asparagine synthetase; AST: aspartate transaminase; ACLY : ATP‐citrate synthase; GLS1 : glutaminase; GS: glutamine synthetase; NAA: N ‐acetylaspartate; NAT8L : N ‐acetyltransferase 8 like; OAA: oxaloacetate; 2‐OG: 2‐oxoglutarate; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase. Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by two‐way ANOVA analyses with Tukey correction (A, B), one‐way ANOVA analyses with Tukey correction (C–H), and defined as * P
    Figure Legend Snippet: GLUL knockdown effect on release of IL‐10 and NAA and on consequent metabolic aspects Extracellular levels of (A) IL‐10, and (B) NAA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Extracellular levels of Asn (C) and cytosolic levels of citrate (D), Asp (E), and Glu (F) in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). qRT–PCR quantification of (G) NAT8L and (H) ACLY mRNA in SKOV3, OVCAR3‐shNT, and OVCAR3‐sh GLUL cells ( n = 3 biological replicates). Ammonia production driven by glutaminolysis interferes with mitochondrial dehydrogenases, favoring citrate, AcCoA and OAA mitochondria accumulation. Up‐regulation of NAT8L along with Glu accumulation, driving OAA transamination to Asp, leads to NAA production. NAA is also synthesized by NAT8L in the cytoplasm using AcCoA derived from citrate via ACLY . In the absence of intracellularly synthesized Gln, Glu drives OAA transamination leading to Asp, which accumulates due to decreased ASNS activity. AcCoA: Acetyl‐CoA; ASNS: asparagine synthetase; AST: aspartate transaminase; ACLY : ATP‐citrate synthase; GLS1 : glutaminase; GS: glutamine synthetase; NAA: N ‐acetylaspartate; NAT8L : N ‐acetyltransferase 8 like; OAA: oxaloacetate; 2‐OG: 2‐oxoglutarate; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase. Data information: Data are displayed as mean ± SEM. Statistical significance was calculated by two‐way ANOVA analyses with Tukey correction (A, B), one‐way ANOVA analyses with Tukey correction (C–H), and defined as * P

    Techniques Used: Quantitative RT-PCR, Synthesized, Derivative Assay, Activity Assay, AST Assay

    15) Product Images from "Evaluation of the Role of ITGBL1 in Ovarian Cancer"

    Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

    Journal: Cancers

    doi: 10.3390/cancers12092676

    The effect of ITGBL1 overexpression on the adhesiveness of OAW42 and SKOV3 cells. ( A , B ) comparison of cellular attachment to uncoated and fibronectin or collagen coated plastic surface (crystal violet assay), 5 min (OAW42) and 15 min (SKOV3) after seeding. Y -axis represents percentage of crystal violet released from fixed adherent cells (mean ± SD, n = 3, each in 12 technical repeats). The amount obtained from control cells was taken as the reference value (100%). Statistical significance was determined by one-way analysis of variance (ANOVA) with Scheffe’s adjustment for pairwise comparisons; ** indicates p
    Figure Legend Snippet: The effect of ITGBL1 overexpression on the adhesiveness of OAW42 and SKOV3 cells. ( A , B ) comparison of cellular attachment to uncoated and fibronectin or collagen coated plastic surface (crystal violet assay), 5 min (OAW42) and 15 min (SKOV3) after seeding. Y -axis represents percentage of crystal violet released from fixed adherent cells (mean ± SD, n = 3, each in 12 technical repeats). The amount obtained from control cells was taken as the reference value (100%). Statistical significance was determined by one-way analysis of variance (ANOVA) with Scheffe’s adjustment for pairwise comparisons; ** indicates p

    Techniques Used: Over Expression, Cell Attachment Assay, Crystal Violet Assay

    Construction and verification of cellular models. ( A ) RT-PCR amplified ITGBL1 CDS (variant 1 mRNA) was cloned into expression vector pLNCX2. ( B ) RT-PCR revealed that ITGBL1 mRNA (concurrent amplification of all isoforms) is detectable in the wild-type ES2 cell line (positive control) and in OAW42 and SKOV3 cell lines stably transduced with pLNCX1-ITGBL1 construct, while absent in wild-type and empty pLNCX2-transduced OAW42 and SKOV3 cells. ( Supplementary Material 3A ) No ITGBL1 signal could be detected by WB in the protein extracts from each analyzed cell line using Sigma-Aldrich HPA005676 antibody. ( C ) WB analysis with the same antibody confirmed presence of ITGBL1 in the culture media from indicated cell cultures (upper panel). Ponceau stained blot is shown to demonstrate protein loading uniformity (lower panel). Full blots from ( C ) are shown in Supplementary Material 3 . Full gel electrophoresis image from ( B ) is shown in Supplementary material 2 .
    Figure Legend Snippet: Construction and verification of cellular models. ( A ) RT-PCR amplified ITGBL1 CDS (variant 1 mRNA) was cloned into expression vector pLNCX2. ( B ) RT-PCR revealed that ITGBL1 mRNA (concurrent amplification of all isoforms) is detectable in the wild-type ES2 cell line (positive control) and in OAW42 and SKOV3 cell lines stably transduced with pLNCX1-ITGBL1 construct, while absent in wild-type and empty pLNCX2-transduced OAW42 and SKOV3 cells. ( Supplementary Material 3A ) No ITGBL1 signal could be detected by WB in the protein extracts from each analyzed cell line using Sigma-Aldrich HPA005676 antibody. ( C ) WB analysis with the same antibody confirmed presence of ITGBL1 in the culture media from indicated cell cultures (upper panel). Ponceau stained blot is shown to demonstrate protein loading uniformity (lower panel). Full blots from ( C ) are shown in Supplementary Material 3 . Full gel electrophoresis image from ( B ) is shown in Supplementary material 2 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay, Clone Assay, Expressing, Plasmid Preparation, Positive Control, Stable Transfection, Transduction, Construct, Western Blot, Staining, Nucleic Acid Electrophoresis

    Unsupervised analysis of gene expression profiles in the analyzed cell lines. ( A ) Principal Component Analysis performed on all samples. Samples tend to group by the cell line. This difference is most prominent (74% of variance) and is described by first principal component (PC1). ( B ) Hierarchical clustering of all samples according to PC1. Heat map demonstrates that genes from PC1 have distinct expression pattern in OAW42 and SKOV3 cells. ( C ) Hierarchical clustering of OAW42 samples according to PC2. PC2 differentiates ITGBL1 -overexpressing OAW42 samples from all control OAW42 samples (wild-type and with an empty pLNCX2). ( D ) Hierarchical clustering of OAW42 samples (only empty pLNCX2 controls and samples with ITGBL1 overexpression).
    Figure Legend Snippet: Unsupervised analysis of gene expression profiles in the analyzed cell lines. ( A ) Principal Component Analysis performed on all samples. Samples tend to group by the cell line. This difference is most prominent (74% of variance) and is described by first principal component (PC1). ( B ) Hierarchical clustering of all samples according to PC1. Heat map demonstrates that genes from PC1 have distinct expression pattern in OAW42 and SKOV3 cells. ( C ) Hierarchical clustering of OAW42 samples according to PC2. PC2 differentiates ITGBL1 -overexpressing OAW42 samples from all control OAW42 samples (wild-type and with an empty pLNCX2). ( D ) Hierarchical clustering of OAW42 samples (only empty pLNCX2 controls and samples with ITGBL1 overexpression).

    Techniques Used: Expressing, Over Expression

    Scratch assay. ( A ) Comparison of scratch area filling time required for OAW42-ITGBL1 and OAW42-PLNCX2 cells. ( C ) Comparison of scratch area filling time by SKOV3-ITGBL1 and SKOV3-PLNCX2 cells; X -axis represents observation time points, Y -axis represents size of the remaining scratch area (mean ± SD, n = 3, each in 10 technical repeats). The initial size of scratch area was assumed as 100%. Statistical significance was assessed with Student’s t -Test with Bonferroni correction for multiple testing; * indicates p
    Figure Legend Snippet: Scratch assay. ( A ) Comparison of scratch area filling time required for OAW42-ITGBL1 and OAW42-PLNCX2 cells. ( C ) Comparison of scratch area filling time by SKOV3-ITGBL1 and SKOV3-PLNCX2 cells; X -axis represents observation time points, Y -axis represents size of the remaining scratch area (mean ± SD, n = 3, each in 10 technical repeats). The initial size of scratch area was assumed as 100%. Statistical significance was assessed with Student’s t -Test with Bonferroni correction for multiple testing; * indicates p

    Techniques Used: Wound Healing Assay

    16) Product Images from "N-(phosphonacetyl)-L-aspartate induces TAp73-dependent apoptosis by modulating multiple Bcl-2 proteins: Potential for cancer therapy"

    Article Title: N-(phosphonacetyl)-L-aspartate induces TAp73-dependent apoptosis by modulating multiple Bcl-2 proteins: Potential for cancer therapy

    Journal: Oncogene

    doi: 10.1038/onc.2012.96

    p53 and TAp73 play opposing role in PALA-induced expression of Bim and Noxa: (A) 041 and derivatives; (B) PC3 and PC3-p53 and (C) SKOV3 cells were treated with 250 µM/L of PALA for the indicated times and total cell lysates were immunoblotted with anti-Bim and anti-Noxa. (D) O41 and its derivative cell lines and (E) SKOV3 and its derivatives expressing p53 and dominant negative p73 were treated with 250 µM/L of PALA for 48 or 72 h. Total RNA was used for the expression of Bim mRNA by real time PCR. For (D), p values for #, ##, * and ** are 0.0002, 0.0009, 0.0002 and 0.00002, respectively. For (E), p values for *, ** and *** are 0.0003, 0.0004 and 0.0008, respectively. (F, G) Cells were treated with PALA and expression levels of Noxa mRNA were examined by real time PCR. For (F), p values for * and ** are 0.004 and 0.012, respectively. For (G), p values for *, ** and *** are 0.001, 0.0004 and 0.007, respectively.
    Figure Legend Snippet: p53 and TAp73 play opposing role in PALA-induced expression of Bim and Noxa: (A) 041 and derivatives; (B) PC3 and PC3-p53 and (C) SKOV3 cells were treated with 250 µM/L of PALA for the indicated times and total cell lysates were immunoblotted with anti-Bim and anti-Noxa. (D) O41 and its derivative cell lines and (E) SKOV3 and its derivatives expressing p53 and dominant negative p73 were treated with 250 µM/L of PALA for 48 or 72 h. Total RNA was used for the expression of Bim mRNA by real time PCR. For (D), p values for #, ##, * and ** are 0.0002, 0.0009, 0.0002 and 0.00002, respectively. For (E), p values for *, ** and *** are 0.0003, 0.0004 and 0.0008, respectively. (F, G) Cells were treated with PALA and expression levels of Noxa mRNA were examined by real time PCR. For (F), p values for * and ** are 0.004 and 0.012, respectively. For (G), p values for *, ** and *** are 0.001, 0.0004 and 0.007, respectively.

    Techniques Used: Expressing, Dominant Negative Mutation, Real-time Polymerase Chain Reaction

    (A) Expression of Noxa was ablated in SKOV3 cells. Total cell lysates from PALA-treated and untreated cells expressing scrambled siRNA or Noxa shRNA were immunoblotted with Noxa. (B) SKOV3 cells transfected with shNOXA or control siRNA were treated with 250 µM/L of PALA for 6 days and apoptosis was measured by TUNEL assay. Experiments were repeated three times and average values for apoptosis with standard deviation as error bars are shown. p values were determined by Student’s T-test and values less than 0.05 are considered significant. (C) SKOV3 cells transfected with shNOXA or control siRNA were treated with 250 µM/L of PALA and release of cytochrome C into the cytosol was examined.
    Figure Legend Snippet: (A) Expression of Noxa was ablated in SKOV3 cells. Total cell lysates from PALA-treated and untreated cells expressing scrambled siRNA or Noxa shRNA were immunoblotted with Noxa. (B) SKOV3 cells transfected with shNOXA or control siRNA were treated with 250 µM/L of PALA for 6 days and apoptosis was measured by TUNEL assay. Experiments were repeated three times and average values for apoptosis with standard deviation as error bars are shown. p values were determined by Student’s T-test and values less than 0.05 are considered significant. (C) SKOV3 cells transfected with shNOXA or control siRNA were treated with 250 µM/L of PALA and release of cytochrome C into the cytosol was examined.

    Techniques Used: Expressing, shRNA, Transfection, TUNEL Assay, Standard Deviation

    Cleavage of PARP and caspase-3 by PALA: (A) 041 and its derivatives 041-p53 and 041-ddp73; (B) SKOV3 and its derivatives ddp73, #3 and ddp73, #5; (C) PC3 and PC3-p53; (D) A549-shGFP and A549-shp53 and (E) Tu686 cells were treated with 250 µM/L of PALA for the indicated times and total cell lysates were immunoblotted with PARP and caspase-3 antibodies that detect only the cleaved form of PARP and caspase-3. All experiments were repeated three times for reproducibility and representative data are shown.
    Figure Legend Snippet: Cleavage of PARP and caspase-3 by PALA: (A) 041 and its derivatives 041-p53 and 041-ddp73; (B) SKOV3 and its derivatives ddp73, #3 and ddp73, #5; (C) PC3 and PC3-p53; (D) A549-shGFP and A549-shp53 and (E) Tu686 cells were treated with 250 µM/L of PALA for the indicated times and total cell lysates were immunoblotted with PARP and caspase-3 antibodies that detect only the cleaved form of PARP and caspase-3. All experiments were repeated three times for reproducibility and representative data are shown.

    Techniques Used:

    Activation of p53 and TAp73 by PALA treatment. (A) 041 and derivatives; (B) PC3 and PC3-p53 and (C) SKOV3 cells were treated with 250 µM/L of PALA for the indicated times and expression of TAp73, ΔNp73 and p53 were examined by Western blotting. Expression of TAp73β was also examined in SKOV3 cells (C). Representative data from three independent experiments are presented. (D) 041 and derivatives expressing p53 and dominant negative p73; and (E) SKOV3 and its derivatives expressing dominant negative p73 were treated with 250 µM/L PALA for the indicated times and expression levels of p21 mRNA were examined by real time PCR. The experiments were done in triplicate and average results with standard deviations as error bars are shown. p values
    Figure Legend Snippet: Activation of p53 and TAp73 by PALA treatment. (A) 041 and derivatives; (B) PC3 and PC3-p53 and (C) SKOV3 cells were treated with 250 µM/L of PALA for the indicated times and expression of TAp73, ΔNp73 and p53 were examined by Western blotting. Expression of TAp73β was also examined in SKOV3 cells (C). Representative data from three independent experiments are presented. (D) 041 and derivatives expressing p53 and dominant negative p73; and (E) SKOV3 and its derivatives expressing dominant negative p73 were treated with 250 µM/L PALA for the indicated times and expression levels of p21 mRNA were examined by real time PCR. The experiments were done in triplicate and average results with standard deviations as error bars are shown. p values

    Techniques Used: Activation Assay, Expressing, Western Blot, Dominant Negative Mutation, Real-time Polymerase Chain Reaction

    PALA-induced apoptosis is mediated via Bim. (A) 041 cells were transduced with either scrambled siRNA (shControl) or shRNA against Bim (shBim) and clones were established by puromycin selection. Total cell lysates from PALA-treated and untreated cells were immunoblotted with anti-Bim. (B) Cells were treated with 250 µM/L of PALA for 6 days and apoptosis was measured by TUNEL. (C) Cells were treated with PALA for 72 h and cleavage of PARP and caspase-3 were examined by Western blotting. (D) Expression of Bim was ablated in SKOV3 cells. (E) SKOV3 cells with ablated Bim were treated with 250 µM/L of PALA for 7 days and apoptosis was measured by TUNEL staining. (F) Cells were treated with PALA for 72 h and cleavage of PARP and caspase-3 were examined by Western blotting. For B and E, average apoptosis from three independent experiments with standard deviation as error bars are shown. p values were determined by Student’s T-test and values
    Figure Legend Snippet: PALA-induced apoptosis is mediated via Bim. (A) 041 cells were transduced with either scrambled siRNA (shControl) or shRNA against Bim (shBim) and clones were established by puromycin selection. Total cell lysates from PALA-treated and untreated cells were immunoblotted with anti-Bim. (B) Cells were treated with 250 µM/L of PALA for 6 days and apoptosis was measured by TUNEL. (C) Cells were treated with PALA for 72 h and cleavage of PARP and caspase-3 were examined by Western blotting. (D) Expression of Bim was ablated in SKOV3 cells. (E) SKOV3 cells with ablated Bim were treated with 250 µM/L of PALA for 7 days and apoptosis was measured by TUNEL staining. (F) Cells were treated with PALA for 72 h and cleavage of PARP and caspase-3 were examined by Western blotting. For B and E, average apoptosis from three independent experiments with standard deviation as error bars are shown. p values were determined by Student’s T-test and values

    Techniques Used: Transduction, shRNA, Clone Assay, Selection, TUNEL Assay, Western Blot, Expressing, Staining, Standard Deviation

    p53 and TAp73 play opposing roles in PALA-induced apoptosis. (A) 041 and its derivatives expressing either wild-type p53 (041-p53) or dominant negative p73 (041-ddp73) were treated with 250 µM/L of PALA for 3 and 6 days and apoptosis was measured by TUNEL staining. p values for *, **, # and ## are 0.0008, 0,0011, 0.0018 and 0.0013, respectively. (B) Ovarian cancer cell line SKOV3 and two clones expressing dominant negative p73 (ddp73, #3 and ddp73, #5) were treated with 250 µM/L of PALA for 7 days and apoptosis was measured by TUNEL staining. p values for * and ** are 0.0011 and 0.0035, respectively. (C) Prostate cancer cell line PC3 and isogenic cells with wild-type p53 (PC3-p53) were treated with 250 µM/L of PALA for 3 and 5 days and the apoptotic population was measured by TUNEL staining. p values for * and ** are 0.0005 and 0.003, respectively. (D) PCI-13 and Tu686 cells expressing mutant p53 were treated with 250 µM/L of PALA for 3 and 5 days and apoptosis was measured. (E) A549 and its derivative with ablated p53 were treated with 250 µM/L of PALA for 2 and 3 days and apoptosis was measured. p values for * and ** are 0.0039 and 0.0001, respectively. (F) SKOV3 cells transfected with control siRNA and siRNA targeting TAp73 were treated with 250 µM/L of PALA for 3 days and apoptosis was measured. *=0.003. For A–F, average results from three independent experiments with standard deviations as error bars are shown. p values were determined by Student’s T-test and values less than 0.05 were considered statistically significant.
    Figure Legend Snippet: p53 and TAp73 play opposing roles in PALA-induced apoptosis. (A) 041 and its derivatives expressing either wild-type p53 (041-p53) or dominant negative p73 (041-ddp73) were treated with 250 µM/L of PALA for 3 and 6 days and apoptosis was measured by TUNEL staining. p values for *, **, # and ## are 0.0008, 0,0011, 0.0018 and 0.0013, respectively. (B) Ovarian cancer cell line SKOV3 and two clones expressing dominant negative p73 (ddp73, #3 and ddp73, #5) were treated with 250 µM/L of PALA for 7 days and apoptosis was measured by TUNEL staining. p values for * and ** are 0.0011 and 0.0035, respectively. (C) Prostate cancer cell line PC3 and isogenic cells with wild-type p53 (PC3-p53) were treated with 250 µM/L of PALA for 3 and 5 days and the apoptotic population was measured by TUNEL staining. p values for * and ** are 0.0005 and 0.003, respectively. (D) PCI-13 and Tu686 cells expressing mutant p53 were treated with 250 µM/L of PALA for 3 and 5 days and apoptosis was measured. (E) A549 and its derivative with ablated p53 were treated with 250 µM/L of PALA for 2 and 3 days and apoptosis was measured. p values for * and ** are 0.0039 and 0.0001, respectively. (F) SKOV3 cells transfected with control siRNA and siRNA targeting TAp73 were treated with 250 µM/L of PALA for 3 days and apoptosis was measured. *=0.003. For A–F, average results from three independent experiments with standard deviations as error bars are shown. p values were determined by Student’s T-test and values less than 0.05 were considered statistically significant.

    Techniques Used: Expressing, Dominant Negative Mutation, TUNEL Assay, Staining, Clone Assay, Mutagenesis, Transfection

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    The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and <t>SKOV3</t> cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p
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    The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and SKOV3 cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p

    Journal: Cancers

    Article Title: Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy

    doi: 10.3390/cancers13051141

    Figure Lengend Snippet: The influence of intracellular iron on the antitumor effect of PAM in cancer cells and normal cells. PAM was prepared with the same conditions as those used in the animal model. Cells were pre-treated with 200 μM DFO (Desferal for injection 500 mg, Novartis Pharma, Tokyo, Japan) before PAM treatment. ES2 and WL-38 cells were seeded at a density of 1 × 10 4 cells, and SKOV3 cells were seeded at a density of 3 × 10 3 cells in the wells of 96-well plates. Cell viability was assayed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at the corresponding PAM dilution ratio. Data are the mean ± SD. Three independent experiments were performed. The two-way ANOVA with Tukey’s post hoc test for equal variances was carried out between DFO-treated and -untreated cells. Statistics are shown as *** p

    Article Snippet: Cell Cultures The five human ovarian cancer cell lines ES2, SKOV3, OV90, OVCAR3, and CAOV3 and the normal human lung fibroblast cell line WI-38 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Animal Model, Injection, MTS Assay

    Relationship between PAUF and TLR4 expression in ovarian cancer cell lines. ( A ) To probe surface and intracellular TLR4 expression level in SKOV3 and A2780 cells, PE-conjugated TLR4 antibody was confirmed and used in flow cytometric analysis. ( B,C ) PAUF expression level and secretion in ovarian cancer cells (SKOV3, A2780) were detected using western blot analysis, and PAUF in the culture supernatant of cancer cells was detected using ELISA. ( D ) The expression level of intracellular TLR4 was assessed by flow cytometric assays. TLR4 expression was decreased significantly in both cells after transfection of two TLR4-siRNAs in comparison to that of control-siRNA or control. (MFI: Mean Fluorescence Intensity) ( E ) To determine activation of MAPKs (ERK, P38 and JNK) and AKT by PAUF, starved cancer cells were treated with or without recombinant PAUF (5 μ g) and analyzed by western blotting. ( F ) To confirm MAPK activation in siRNA transfected A2780 or SKOV3 cells, cells were transfected with TLR4-siRNA or control siRNA and starved for 16 hours. Cells were treated with PAUF (5 μ g) for 20 min and analyzed by western blotting. The number below each western blot represents the ratio of the intensity of the band over the control intensity of scramble siRNA-treated cells. ( G ) For the cell proliferation assay, control, TLR4-, or PAUF-siRNA transfected A2780 or SKOV3 cells were cultured in 96-well white plates, and cell proliferation was detected using a Cell Titer-Glo luminescence assay kit. The data shown are the means ± s.e.m. for three independent experiments. β -actin was used as an internal reference. * p

    Journal: Scientific Reports

    Article Title: Elevated expression of pancreatic adenocarcinoma upregulated factor (PAUF) is associated with poor prognosis and chemoresistance in epithelial ovarian cancer

    doi: 10.1038/s41598-018-30582-8

    Figure Lengend Snippet: Relationship between PAUF and TLR4 expression in ovarian cancer cell lines. ( A ) To probe surface and intracellular TLR4 expression level in SKOV3 and A2780 cells, PE-conjugated TLR4 antibody was confirmed and used in flow cytometric analysis. ( B,C ) PAUF expression level and secretion in ovarian cancer cells (SKOV3, A2780) were detected using western blot analysis, and PAUF in the culture supernatant of cancer cells was detected using ELISA. ( D ) The expression level of intracellular TLR4 was assessed by flow cytometric assays. TLR4 expression was decreased significantly in both cells after transfection of two TLR4-siRNAs in comparison to that of control-siRNA or control. (MFI: Mean Fluorescence Intensity) ( E ) To determine activation of MAPKs (ERK, P38 and JNK) and AKT by PAUF, starved cancer cells were treated with or without recombinant PAUF (5 μ g) and analyzed by western blotting. ( F ) To confirm MAPK activation in siRNA transfected A2780 or SKOV3 cells, cells were transfected with TLR4-siRNA or control siRNA and starved for 16 hours. Cells were treated with PAUF (5 μ g) for 20 min and analyzed by western blotting. The number below each western blot represents the ratio of the intensity of the band over the control intensity of scramble siRNA-treated cells. ( G ) For the cell proliferation assay, control, TLR4-, or PAUF-siRNA transfected A2780 or SKOV3 cells were cultured in 96-well white plates, and cell proliferation was detected using a Cell Titer-Glo luminescence assay kit. The data shown are the means ± s.e.m. for three independent experiments. β -actin was used as an internal reference. * p

    Article Snippet: SKOV3 and A2780 cells were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Fluorescence, Activation Assay, Recombinant, Proliferation Assay, Cell Culture, Luminescence Assay

    Modulation of NRF2 gene expression in normal and malignant ovarian epithelial cells by NRF2‐targeting ATFs. (A) Schematic overview of the NRF2 promoter region (transcript variant 1) containing the TSS (transcription start site, +1) and the target regions for the 6 engineered zinc finger proteins (ZFP OX1‐OX6). Histone modifications associated with ATF OX2 and OX5 were determined in ChIP region OX2‐ChIP6 and OX5‐ChIP5, respectively. CpGs are shown as vertical lines. (B) Relative NRF2 expression compared to empty vector control upon retroviral delivery of NRF2‐targeting ZFPs (OX1‐OX6) fused to the transcriptional repressor SKD in SKOV3, A2780 and OSE‐C2 cells. Relative NRF2 expression of the NRF2 cDNA control has only been determined in OSE‐C2 cells. Data is presented as mean ± SEM of at least three independent experiments. *p

    Journal: Molecular Oncology

    Article Title: Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion), Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion

    doi: 10.1016/j.molonc.2015.03.003

    Figure Lengend Snippet: Modulation of NRF2 gene expression in normal and malignant ovarian epithelial cells by NRF2‐targeting ATFs. (A) Schematic overview of the NRF2 promoter region (transcript variant 1) containing the TSS (transcription start site, +1) and the target regions for the 6 engineered zinc finger proteins (ZFP OX1‐OX6). Histone modifications associated with ATF OX2 and OX5 were determined in ChIP region OX2‐ChIP6 and OX5‐ChIP5, respectively. CpGs are shown as vertical lines. (B) Relative NRF2 expression compared to empty vector control upon retroviral delivery of NRF2‐targeting ZFPs (OX1‐OX6) fused to the transcriptional repressor SKD in SKOV3, A2780 and OSE‐C2 cells. Relative NRF2 expression of the NRF2 cDNA control has only been determined in OSE‐C2 cells. Data is presented as mean ± SEM of at least three independent experiments. *p

    Article Snippet: The human ovarian carcinoma cell lines A2780 and SKOV3, and the human embryonic kidney cell line HEK293T were obtained from the ATCC.

    Techniques: Expressing, Variant Assay, Chromatin Immunoprecipitation, Plasmid Preparation

    The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro

    doi: 10.12659/MSM.919089

    Figure Lengend Snippet: The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Article Snippet: Cell culture and transfection Two serous ovarian carcinoma cell lines, SKOV3 and OVCAR3, were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), Nontumorous ovarian surface epithelial (OSE) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

    Techniques: In Vitro, Transfection, Migration, Cell Counting, CCK-8 Assay, Wound Healing Assay, Standard Deviation

    Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro

    doi: 10.12659/MSM.919089

    Figure Lengend Snippet: Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

    Article Snippet: Cell culture and transfection Two serous ovarian carcinoma cell lines, SKOV3 and OVCAR3, were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), Nontumorous ovarian surface epithelial (OSE) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

    Techniques: Activation Assay, In Vitro, Expressing, Western Blot, Transfection, Plasmid Preparation, Cell Counting, CCK-8 Assay, Wound Healing Assay, Over Expression, Migration, Standard Deviation