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  • 99
    Millipore cisplatin
    Confirmation that the DNA arc migrating behind normal dsDNA contained molecules with interstrand DNA crosslinks. ( A–D ) DNA in interstrand DNA arc was resistant to heat-denaturation. (A) The original undamaged MboI-digested human genomic DNA sample. (B) The original DNA after heat denaturation. (C) DNA after treatment with 1 μM <t>cisplatin</t> for 18 h. (D) DNA treated with cisplatin and heat denatured. ( E–G ) DNA in interstrand arc was refractory to amplification. Adaptor-ligated human genomic DNA (E) before and (F) after treatment with 1 μM cisplatin for 18 h was eluted from different labeled arcs of 2D-SDE gels. (G) PCR products of eluted DNA on an agarose gel. M: GeneRuler 100 bp Plus marker. Lanes 1–3 DNA amplified from the control (lane 1: dsDNA arc, lane 2: fraction behind dsDNA, lane 3: fraction in front of dsDNA). Lanes 4–6 DNA amplified from the cisplatin-treated sample (lane 4: dsDNA arc, lane 5: fraction behind dsDNA, lane 6: fraction in front of dsDNA. Lane 7: amplification in DNA-free gel, lane 8: positive control, adaptor-ligated DNA before gel-loading, lane 9: blank (H 2 O).
    Cisplatin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals cisplatin
    Confirmation that the DNA arc migrating behind normal dsDNA contained molecules with interstrand DNA crosslinks. ( A–D ) DNA in interstrand DNA arc was resistant to heat-denaturation. (A) The original undamaged MboI-digested human genomic DNA sample. (B) The original DNA after heat denaturation. (C) DNA after treatment with 1 μM <t>cisplatin</t> for 18 h. (D) DNA treated with cisplatin and heat denatured. ( E–G ) DNA in interstrand arc was refractory to amplification. Adaptor-ligated human genomic DNA (E) before and (F) after treatment with 1 μM cisplatin for 18 h was eluted from different labeled arcs of 2D-SDE gels. (G) PCR products of eluted DNA on an agarose gel. M: GeneRuler 100 bp Plus marker. Lanes 1–3 DNA amplified from the control (lane 1: dsDNA arc, lane 2: fraction behind dsDNA, lane 3: fraction in front of dsDNA). Lanes 4–6 DNA amplified from the cisplatin-treated sample (lane 4: dsDNA arc, lane 5: fraction behind dsDNA, lane 6: fraction in front of dsDNA. Lane 7: amplification in DNA-free gel, lane 8: positive control, adaptor-ligated DNA before gel-loading, lane 9: blank (H 2 O).
    Cisplatin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Teva cisplatin
    High-dose <t>cisplatin</t> cross-preserves hair cells exposed to toxic doses of gentamicin. (A) Mouse organ of Corti explant cultures exposed to 100 μM gentamicin (Gent) for 24 h results in significant hair cell loss (top panels). Additional supplementation of 500 μM cisplatin to the toxic concentration of gentamicin rescues the hair cells, albeit with severely reduced MYO7A immunoreactivity (bottom panels). (B) Outer hair cell counts per 100 μm (basal turn) after exposure to gentamicin with or without high dose cisplatin (Unpaired t -test, p -value:
    Cisplatin, supplied by Teva, used in various techniques. Bioz Stars score: 92/100, based on 450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Qilu Pharmaceutical cisplatin
    Pica induced by <t>cisplatin.</t> Rats had two distinct periods of eating kaolin in the cisplatin groups, and Ginger significantly decreased the weight of kaolin eaten induced by cisplatin during the 72 h observation period. Metoclopramide significantly decreased the weight of kaolin eaten during 24 h following cisplatin injection, but there was no significant decrease in the pica during 24–72 h. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). i.g., intragastrically.
    Cisplatin, supplied by Qilu Pharmaceutical, used in various techniques. Bioz Stars score: 92/100, based on 555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cisplatin cddp
    IGFBP5 expression vector reversed <t>cisplatin-resistance.</t> ( A ) Relative MTS activity of SLMT-1/CDDP1R, SLMT-1R-IGFBP5 and SLMT-1R-pcMV3 cells. Relative MTS activities were expressed as means ± standard error compared with the MTS activities at zero <t>CDDP</t> concentration and analyzed using one-way ANOVA. SLMT-1R-IGFBP5 was compared to SLMT-1R-pcMV3 with* p
    Cisplatin Cddp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bristol Myers cisplatin
    NPRL2 enhances the effect of <t>cisplatin</t> that can activate cell cycle checkpoints. H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.
    Cisplatin, supplied by Bristol Myers, used in various techniques. Bioz Stars score: 92/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Hospira cisplatin
    <t>Cisplatin</t> enhances the sensitivity of melanoma cells to PARP inhibitor olaparib. Five-day SRB growth assay on A375 melanoma cells in 96-well plates showing enhanced sensitivity to olaparib in the presence of 0.5 μM cisplatin. This cisplatin concentration is well below the IC50 value. In order to correct for the toxicity of cisplatin alone, for each curve growth is expressed as the percentage of the non-olaparib-treated control. Values plotted are mean % growth (±SEM) from three independent experiments
    Cisplatin, supplied by Hospira, used in various techniques. Bioz Stars score: 94/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem cisplatin
    Aldi-6 inhibits ALDH activity in HNSCC ( A ) ALDH3A1 activity in SCC4 xenograft tumor. Mice with SCC4 xenografts were treated intra-tumorally with Aldi-6 or vehicle control for three consecutive days, and the ALDH3A1 activity was measured using an isoelectric focusing assay (see Methods). Experiment was performed three times. ( B ) Representative FACS analyses of ALDH activity of SCC4 and PCI-13 cells. After a two-day treatment of <t>cisplatin</t> (0.88 μM) and/or Aldi-6 (30 μM), ALDH activity was measured in the surviving cells by Aldefluor assay. Grey line represents the DEAB-treated negative control for each treatment condition. Blue line represents the ALDH activity of each sample. ( C ) Changes in ALDH activity in (B) were quantified as a ratio of the shift of MFI between treated and untreated sample. The ratio in MFI shift was calculated by (MFI of treated sample-(MFI of treated sample+DEAB))/(MFI of untreated sample-(MFI of untreated sample+DEAB)). Results represent the means ± SEMs of 2–3 independent experiments with 10,000 cells each. (* p
    Cisplatin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM cisplatin
    Pemetrexed and <t>cisplatin</t> inhibit proliferation, whereas cSBL has a cytotoxic effect in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 0–72 h. The number of cells was estimated using a Muse ™ Count Viability Kit. ( A ) Cell growth rates were calculated as the ratio of the cell number at 72 h to the cell number at 0 h and presented as a fraction of the controls. ( B ) The number of live and dead cells every 24 h is shown. Statistically significant differences in the live cell number at 72 h were observed in the treatment groups compared to the control (***). Statistically significant differences in the dead cell number at 72 h were observed in cSBL-treated cells compared to cisplatin- or pemetrexed-treated cells ( †† ). * p
    Cisplatin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris cisplatin
    ( A ) Steps used in the clonogenic assay; ( B ) Toxicity and effect of <t>cisplatin</t> action in the absence and presence of GNPs. Data are means ± S.D. for n = 9 cell preparations over three independent experimental set-ups.
    Cisplatin, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    fluidigm cisplatin
    Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by <t>cisplatin</t> treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p
    Cisplatin, supplied by fluidigm, used in various techniques. Bioz Stars score: 92/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nippon Kayaku cisplatin
    Comparison of CCL2 and kidney injury molecule-1 (Kim-1) expression in the kidney ( a ) and ( b ) , Total RNA was extracted from isolated proximal tubules ( open circles ) and whole kidneys ( closed circles ) of rats treated with 5 mg/kg <t>cisplatin.</t> This RNA was reverse-transcribed to yield cDNA. Real-time polymerase chain reaction analysis of CCL2 ( a ) and Kim-1 ( b ) was performed using these cDNAs. GAPDH mRNA was used as an internal control. Ratio to day 0 was calculated, and data are expressed as means ± S.E. of 3–5 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P
    Cisplatin, supplied by Nippon Kayaku, used in various techniques. Bioz Stars score: 92/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cis diammineplatinum ii dichloride
    Comparison of CCL2 and kidney injury molecule-1 (Kim-1) expression in the kidney ( a ) and ( b ) , Total RNA was extracted from isolated proximal tubules ( open circles ) and whole kidneys ( closed circles ) of rats treated with 5 mg/kg <t>cisplatin.</t> This RNA was reverse-transcribed to yield cDNA. Real-time polymerase chain reaction analysis of CCL2 ( a ) and Kim-1 ( b ) was performed using these cDNAs. GAPDH mRNA was used as an internal control. Ratio to day 0 was calculated, and data are expressed as means ± S.E. of 3–5 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P
    Cis Diammineplatinum Ii Dichloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    APP Pharmaceuticals cisplatin
    Figure 1. Effect of GHRH antagonist JMR-132, 5-Fluorouracil (5-FU), irinotecan and <t>cisplatin</t> ( A ) or their combination ( B ) on the proliferation of HCT-116 human colon cancer cells in vitro. Cultured cells were treated for 72 h with indicated concentrations
    Cisplatin, supplied by APP Pharmaceuticals, used in various techniques. Bioz Stars score: 92/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Beyotime cisplatin
    PARP1 is a target of miR-770-5p in <t>cisplatin-resistant</t> ovarian cancer cells. ( A ) StarBase online predicted the binding sites of miR-770-5p and PARP1. ( B and C ) Luciferase activity was analyzed in A2780/DDP and SKOV3/DDP cells co-transfected with PARP1 3ʹUTR-WT or PARP1 3ʹUTR-MUT and miR-NC or miR-770-5p. ( D and E ) The mRNA and protein levels of PARP1 were measured in A2780/DDP and SKOV3/DDP cells transfected with miR-NC or miR-770-5p by qRT-PCR and Western blot. ( F ) The expression of PARP1 mRNA was detected in cisplatin-sensitive (n=18) and cisplatin-resistant ovarian cancer tissues (n=19) by qRT-PCR. ( G ) The linear correlation between the expression of PARP1 mRNA and miR-770-5p in ovarian cancer tissues was analyzed. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P
    Cisplatin, supplied by Beyotime, used in various techniques. Bioz Stars score: 99/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher cisplatin
    The time profile of platinum remaining in the tumor after intratumoral injection of <t>cisplatin/polymer</t> formulation (1% w/w, 50 μ L). Values are expressed as mean ± STD ( n = 6).
    Cisplatin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cayman Chemical cisplatin
    Sox2 inhibits <t>cisplatin-induced</t> apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P
    Cisplatin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fresenius Kabi cisplatin
    CSE recovers CD11b levels in <t>cisplatin-treated</t> mice. Cisplatin was administered at 5 mg/kg/day for 4 doses in the absence or presence of CSE (9.6 mL/kg/day) for 7 days. Immunohistochemical analysis was conducted with anti-CD11b to analyze spleen and observed by a microscope at 40x (a–c) and 200x (d–f) magnification; bar, 50 μ m. Shown are representative images ( n = 5‐8 per group).
    Cisplatin, supplied by Fresenius Kabi, used in various techniques. Bioz Stars score: 92/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Collaborative Drug Discovery Inc cisplatin dose
    Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the whole cohort. CCD, cumulative <t>cisplatin</t> dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.
    Cisplatin Dose, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ebewe Pharma cisplatin
    Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by <t>cisplatin</t> treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p
    Cisplatin, supplied by Ebewe Pharma, used in various techniques. Bioz Stars score: 92/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mayne Pharma cisplatin
    TRAIL treatment stimulates phosphorylation of H2AX. The right Y-axes and gray lines show the proportion of MEF cells ( a , c , e ) or LN18 cells ( b , d , f ) bearing phosphorylated H2AX after 1 or 5 h exposure to cross-linked TRAIL ( a , b ), soluble TRAIL ( c , d ) or <t>cisplatin</t> ( e , f ). The left Y-axes and black lines represent the percentage of viable cells after same drug exposures. Error bars indicate s.e.m. from three independent experiments.
    Cisplatin, supplied by Mayne Pharma, used in various techniques. Bioz Stars score: 93/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Confirmation that the DNA arc migrating behind normal dsDNA contained molecules with interstrand DNA crosslinks. ( A–D ) DNA in interstrand DNA arc was resistant to heat-denaturation. (A) The original undamaged MboI-digested human genomic DNA sample. (B) The original DNA after heat denaturation. (C) DNA after treatment with 1 μM cisplatin for 18 h. (D) DNA treated with cisplatin and heat denatured. ( E–G ) DNA in interstrand arc was refractory to amplification. Adaptor-ligated human genomic DNA (E) before and (F) after treatment with 1 μM cisplatin for 18 h was eluted from different labeled arcs of 2D-SDE gels. (G) PCR products of eluted DNA on an agarose gel. M: GeneRuler 100 bp Plus marker. Lanes 1–3 DNA amplified from the control (lane 1: dsDNA arc, lane 2: fraction behind dsDNA, lane 3: fraction in front of dsDNA). Lanes 4–6 DNA amplified from the cisplatin-treated sample (lane 4: dsDNA arc, lane 5: fraction behind dsDNA, lane 6: fraction in front of dsDNA. Lane 7: amplification in DNA-free gel, lane 8: positive control, adaptor-ligated DNA before gel-loading, lane 9: blank (H 2 O).

    Journal: Nucleic Acids Research

    Article Title: Northern lights assay: a versatile method for comprehensive detection of DNA damage

    doi: 10.1093/nar/gky645

    Figure Lengend Snippet: Confirmation that the DNA arc migrating behind normal dsDNA contained molecules with interstrand DNA crosslinks. ( A–D ) DNA in interstrand DNA arc was resistant to heat-denaturation. (A) The original undamaged MboI-digested human genomic DNA sample. (B) The original DNA after heat denaturation. (C) DNA after treatment with 1 μM cisplatin for 18 h. (D) DNA treated with cisplatin and heat denatured. ( E–G ) DNA in interstrand arc was refractory to amplification. Adaptor-ligated human genomic DNA (E) before and (F) after treatment with 1 μM cisplatin for 18 h was eluted from different labeled arcs of 2D-SDE gels. (G) PCR products of eluted DNA on an agarose gel. M: GeneRuler 100 bp Plus marker. Lanes 1–3 DNA amplified from the control (lane 1: dsDNA arc, lane 2: fraction behind dsDNA, lane 3: fraction in front of dsDNA). Lanes 4–6 DNA amplified from the cisplatin-treated sample (lane 4: dsDNA arc, lane 5: fraction behind dsDNA, lane 6: fraction in front of dsDNA. Lane 7: amplification in DNA-free gel, lane 8: positive control, adaptor-ligated DNA before gel-loading, lane 9: blank (H 2 O).

    Article Snippet: In a total volume of 100 μl, 1 μg DNA was incubated in 25 mM Tris–HCl, pH 7.8 with 1–15 μM of cisplatin (Sigma-Aldrich, cat no. 479306), at 37°C for 18 h in the dark.

    Techniques: Amplification, Labeling, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Positive Control

    Cisplatin crosslinks in DNA detected with NLA. ( A ) Analysis of DNA crosslinks in a complex DNA sample. (a) Untreated but Mbo I-digested human genomic DNA (green smear) migrated with the double-stranded Cy5-labeled marker. (b) The same DNA after incubation at 37°C for 16 h showing slight heat-induced damage. (c–e) DNA samples treated with the indicated concentration of cisplatin. Quantification of fractions of duplicate experiments with standard deviations (SD) is shown. A significant increase in DNA migrating behind and in front of undamaged DNA was detected after treatment with all concentrations of cisplatin ( P

    Journal: Nucleic Acids Research

    Article Title: Northern lights assay: a versatile method for comprehensive detection of DNA damage

    doi: 10.1093/nar/gky645

    Figure Lengend Snippet: Cisplatin crosslinks in DNA detected with NLA. ( A ) Analysis of DNA crosslinks in a complex DNA sample. (a) Untreated but Mbo I-digested human genomic DNA (green smear) migrated with the double-stranded Cy5-labeled marker. (b) The same DNA after incubation at 37°C for 16 h showing slight heat-induced damage. (c–e) DNA samples treated with the indicated concentration of cisplatin. Quantification of fractions of duplicate experiments with standard deviations (SD) is shown. A significant increase in DNA migrating behind and in front of undamaged DNA was detected after treatment with all concentrations of cisplatin ( P

    Article Snippet: In a total volume of 100 μl, 1 μg DNA was incubated in 25 mM Tris–HCl, pH 7.8 with 1–15 μM of cisplatin (Sigma-Aldrich, cat no. 479306), at 37°C for 18 h in the dark.

    Techniques: Labeling, Marker, Incubation, Concentration Assay

    Neutralizing endotrophin activity with monoclonal antibodies sensitizes tumours to cisplatin treatment A. Pieces of tumours from PyMT mice were implanted into wild-type hosts. Tumour-bearing mice were given cisplatin (1 mg/kg, ip., every 5 days) or PBS, combined with either TZD (20 mg/kg) or anti-endotrophin monoclonal antibodies (100 µg/mouse, once a week) for tumour progression. Tumour volumes were determined by caliper measurements. Data represent mean ± SD ( n = 5/group). * p

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: Neutralizing endotrophin activity with monoclonal antibodies sensitizes tumours to cisplatin treatment A. Pieces of tumours from PyMT mice were implanted into wild-type hosts. Tumour-bearing mice were given cisplatin (1 mg/kg, ip., every 5 days) or PBS, combined with either TZD (20 mg/kg) or anti-endotrophin monoclonal antibodies (100 µg/mouse, once a week) for tumour progression. Tumour volumes were determined by caliper measurements. Data represent mean ± SD ( n = 5/group). * p

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Activity Assay, Mouse Assay

    Bypassing endotrophin-downstream pathways with COL6 −/− mice abolishes beneficial effects of TZD on cisplatin sensitivity A. PyMT/COL6 −/− mice were given TZD (20 mg/kg) or ND at 8 weeks of age. Cisplatin (1 mg/kg, ip., 2 times/week) or PBS treatment was initiated in 10 week old mice. Tumour growth was determined by caliper measurements, and PyMT littermates given TZD were represented as a control. Data represent mean ± SD ( n = 6–8/group). B–D. Histological analysis. H E staining and necrotic area quantification (B), showing no necrotic area in PyMT/COL6 −/− mice following TZD combination with cisplatin treatment. EMT was determined by immunostaining for E-Cadherin (C) and Vimentin (D), showing no significant effects in TZD/CIS groups comparable to non-treated groups (PBS). Data represent mean ± SD (multiple images from n = 5–6/group). p = n.s (no significant) versus PyMT/COL6 −/− /TZD by unpaired Student's t -test. Scales: 200 µm.

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: Bypassing endotrophin-downstream pathways with COL6 −/− mice abolishes beneficial effects of TZD on cisplatin sensitivity A. PyMT/COL6 −/− mice were given TZD (20 mg/kg) or ND at 8 weeks of age. Cisplatin (1 mg/kg, ip., 2 times/week) or PBS treatment was initiated in 10 week old mice. Tumour growth was determined by caliper measurements, and PyMT littermates given TZD were represented as a control. Data represent mean ± SD ( n = 6–8/group). B–D. Histological analysis. H E staining and necrotic area quantification (B), showing no necrotic area in PyMT/COL6 −/− mice following TZD combination with cisplatin treatment. EMT was determined by immunostaining for E-Cadherin (C) and Vimentin (D), showing no significant effects in TZD/CIS groups comparable to non-treated groups (PBS). Data represent mean ± SD (multiple images from n = 5–6/group). p = n.s (no significant) versus PyMT/COL6 −/− /TZD by unpaired Student's t -test. Scales: 200 µm.

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Mouse Assay, Staining, Immunostaining

    TZD augments cisplatin sensitivity and correlates with the COL6A3 levels A. FP635/PyMT mice were given TZD containing chow (supply approx. 20 mg/kg/day, rosiglitazone) or normal-diet (ND) starting at 8-weeks of age, and cisplatin (1 mg/kg) or PBS treatment was initiated at 10 weeks of age (ip., 3 times/week) over the course of tumour progression. Tumour burden was monitored with a fluorescence scanner (IVIS, Caliper life science). Quantified results are represented as mean ± SD ( n = 8–9/group). * p = 0.04, ND/CIS versus TZD/CIS by unpaired Student's t -test. Metastatic burden was determined by fluorescence signals in lung tissues. B. Primary cancer cells isolated from tumours in PyMT mice were implanted into WT mice. TZD were given 5 days prior to cisplatin treatment (1 mg/kg, every 5 days). Tumour volumes were determined by caliper measurement and represented as mean ± SD ( n = 5–6/group). * p

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: TZD augments cisplatin sensitivity and correlates with the COL6A3 levels A. FP635/PyMT mice were given TZD containing chow (supply approx. 20 mg/kg/day, rosiglitazone) or normal-diet (ND) starting at 8-weeks of age, and cisplatin (1 mg/kg) or PBS treatment was initiated at 10 weeks of age (ip., 3 times/week) over the course of tumour progression. Tumour burden was monitored with a fluorescence scanner (IVIS, Caliper life science). Quantified results are represented as mean ± SD ( n = 8–9/group). * p = 0.04, ND/CIS versus TZD/CIS by unpaired Student's t -test. Metastatic burden was determined by fluorescence signals in lung tissues. B. Primary cancer cells isolated from tumours in PyMT mice were implanted into WT mice. TZD were given 5 days prior to cisplatin treatment (1 mg/kg, every 5 days). Tumour volumes were determined by caliper measurement and represented as mean ± SD ( n = 5–6/group). * p

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Mouse Assay, Fluorescence, Isolation

    TZD enhances cisplatin sensitivity through suppression of endotrophin-mediated EMT, fibrosis and angiogenesis A,B. Schematic diagram for allografts (A), indicating cancer cells were isolated from tumours in PyMT (Ctrl) and PyMT/endotrophin (ETP) mice and implanted into wild-type hosts (0.5 × 10 6 cells/mouse). Host mice were given TZD (20 mg/kg) or ND diet at 10 days before implantation for tumour progression. Cisplatin (1 mg/kg, ip., every 5 days) was administered at 3-weeks post-implantation. Quantification of tumour volume (B), showing TZD suppressed tumour growth in endotrophin + -tumours. Data represent mean ± SD ( n = 8–9/group). * p = 0.05, ** p = 0.01 and *** p = 0.001 Ctrl/ND versus ETP/ND; ## p = 0.01 and ### p = 0.001 ETP/ND versus ETP/TZD by unpaired Student's t- test. C–F. Histological analysis of tumours in allografts after cisplatin treatment. H E staining and necrotic area quantification (C), showing significantly increased chemo-sensitivity in endotrophin + tumours upon combination of TZD with cisplatin. Necrotic area (*). *** p

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: TZD enhances cisplatin sensitivity through suppression of endotrophin-mediated EMT, fibrosis and angiogenesis A,B. Schematic diagram for allografts (A), indicating cancer cells were isolated from tumours in PyMT (Ctrl) and PyMT/endotrophin (ETP) mice and implanted into wild-type hosts (0.5 × 10 6 cells/mouse). Host mice were given TZD (20 mg/kg) or ND diet at 10 days before implantation for tumour progression. Cisplatin (1 mg/kg, ip., every 5 days) was administered at 3-weeks post-implantation. Quantification of tumour volume (B), showing TZD suppressed tumour growth in endotrophin + -tumours. Data represent mean ± SD ( n = 8–9/group). * p = 0.05, ** p = 0.01 and *** p = 0.001 Ctrl/ND versus ETP/ND; ## p = 0.01 and ### p = 0.001 ETP/ND versus ETP/TZD by unpaired Student's t- test. C–F. Histological analysis of tumours in allografts after cisplatin treatment. H E staining and necrotic area quantification (C), showing significantly increased chemo-sensitivity in endotrophin + tumours upon combination of TZD with cisplatin. Necrotic area (*). *** p

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Isolation, Mouse Assay, Staining

    Histological analysis of tumours in PyMT mice with different levels of endotrophin after chemotherapy Endotrophin staining and quantification, showing increased endotrophin levels upon cisplatin treatment which was further augmented in PyMT/endotrophin mice, whereas it was barely detectable in PyMT/TZD and PyMT/COL6 −/− mice. ** p = 0.0174, ## p = 0.009, # p = 0.024 and ### p = 0.0004. H E staining and necrotic lesion area quantification on tumours, showing increased cell death after cisplatin treatment in all groups, and further augmented sensitivity in PyMT/TZD. * p = 0.0463. E-cadherin staining and quantification, showing decreased membrane integrity of epithelial cancer cells after cisplatin treatment in PyMT mice. TZD reverses cisplatin-induced loss of E-cadherin levels. ** p = 0.0045 and # p = 0.0285. Vimentin staining and quantification, showing increased EMT in PyMT/endotrophin mice, whereas it was decreased in PyMT/TZD and PyMT/COL6 −/− mice. ** p = 0.0085 and # p = 0.0111. Quantified results represent mean ± SD (multiple images from n = 5–6/group). Statistics (*PBS vs. CIS; # CIS vs. ETP/CIS, TZD/CIS, or COL6 −/− /CIS) were analysed by unpaired Student's t -test. Scales: 50 µm (A), 200 µm (B) and 100 µm (C–D). Hypothetical modeling of cisplatin responsiveness in PyMT mice relying on the endotrophin levels and EMT status. Arrow indicates TZD augments chemo-sensitivity through suppression of endotrophin levels and EMT.

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: Histological analysis of tumours in PyMT mice with different levels of endotrophin after chemotherapy Endotrophin staining and quantification, showing increased endotrophin levels upon cisplatin treatment which was further augmented in PyMT/endotrophin mice, whereas it was barely detectable in PyMT/TZD and PyMT/COL6 −/− mice. ** p = 0.0174, ## p = 0.009, # p = 0.024 and ### p = 0.0004. H E staining and necrotic lesion area quantification on tumours, showing increased cell death after cisplatin treatment in all groups, and further augmented sensitivity in PyMT/TZD. * p = 0.0463. E-cadherin staining and quantification, showing decreased membrane integrity of epithelial cancer cells after cisplatin treatment in PyMT mice. TZD reverses cisplatin-induced loss of E-cadherin levels. ** p = 0.0045 and # p = 0.0285. Vimentin staining and quantification, showing increased EMT in PyMT/endotrophin mice, whereas it was decreased in PyMT/TZD and PyMT/COL6 −/− mice. ** p = 0.0085 and # p = 0.0111. Quantified results represent mean ± SD (multiple images from n = 5–6/group). Statistics (*PBS vs. CIS; # CIS vs. ETP/CIS, TZD/CIS, or COL6 −/− /CIS) were analysed by unpaired Student's t -test. Scales: 50 µm (A), 200 µm (B) and 100 µm (C–D). Hypothetical modeling of cisplatin responsiveness in PyMT mice relying on the endotrophin levels and EMT status. Arrow indicates TZD augments chemo-sensitivity through suppression of endotrophin levels and EMT.

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Mouse Assay, Staining

    Endotrophin overexpression confers cisplatin resistance in PyMT mice A. Ten week old PyMT and PyMT/endotrophin (PyMT/ETP) mice were given high dosage of cisplatin (2.5 mg/kg, ip., 2 times/week). Tumour growth was determined by caliper measurements. Data represent mean ± SD ( n = 7–10/group). *** p

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: Endotrophin overexpression confers cisplatin resistance in PyMT mice A. Ten week old PyMT and PyMT/endotrophin (PyMT/ETP) mice were given high dosage of cisplatin (2.5 mg/kg, ip., 2 times/week). Tumour growth was determined by caliper measurements. Data represent mean ± SD ( n = 7–10/group). *** p

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Over Expression, Mouse Assay

    The absence of COL6 in PyMT mice sensitizes tumours to cisplatin treatment A. Eleven week old FP635/PyMT and FP635/PyMT/COL6 −/− mice were given cisplatin (1 mg/kg, ip., 2 times/week) or PBS over the course of tumour progression. Tumour burden at a whole body level was monitored with a fluorescence scanner (IVIS) once a week. Representative images and quantification showing increased cisplatin sensitivity in PyMT/COL6 −/− mice. Tumour burden at the end point was determined and represented as mean ± SD ( n = 5/group). * p = 0.0294 versus FP635/PyMT/CIS by Mann–Whitney t -test. B. Total RNA was prepared from the tumour tissues from PyMT, PyMT/COL6 −/− and PyMT/COL6 −/− /ETP mice. mRNA levels for the COL1A1, COL6A1, -A2, -A3-N (amino-terminus of COL6A3) and ETP were determined by qRT-PCR. Values were normalized with 36B4 and represented as mean ± SD ( n = 4/group). Relative values of each gene are represented as fold increase over PyMT. *** p

    Journal: EMBO Molecular Medicine

    Article Title: Inhibition of endotrophin, a cleavage product of collagen VI, confers cisplatin sensitivity to tumours

    doi: 10.1002/emmm.201202006

    Figure Lengend Snippet: The absence of COL6 in PyMT mice sensitizes tumours to cisplatin treatment A. Eleven week old FP635/PyMT and FP635/PyMT/COL6 −/− mice were given cisplatin (1 mg/kg, ip., 2 times/week) or PBS over the course of tumour progression. Tumour burden at a whole body level was monitored with a fluorescence scanner (IVIS) once a week. Representative images and quantification showing increased cisplatin sensitivity in PyMT/COL6 −/− mice. Tumour burden at the end point was determined and represented as mean ± SD ( n = 5/group). * p = 0.0294 versus FP635/PyMT/CIS by Mann–Whitney t -test. B. Total RNA was prepared from the tumour tissues from PyMT, PyMT/COL6 −/− and PyMT/COL6 −/− /ETP mice. mRNA levels for the COL1A1, COL6A1, -A2, -A3-N (amino-terminus of COL6A3) and ETP were determined by qRT-PCR. Values were normalized with 36B4 and represented as mean ± SD ( n = 4/group). Relative values of each gene are represented as fold increase over PyMT. *** p

    Article Snippet: Reagents Cisplatin (Sigma, 479306) was diluted to 1 mg/ml in PBS and was sonicated briefly before injection.

    Techniques: Mouse Assay, Fluorescence, MANN-WHITNEY, Quantitative RT-PCR

    High-dose cisplatin cross-preserves hair cells exposed to toxic doses of gentamicin. (A) Mouse organ of Corti explant cultures exposed to 100 μM gentamicin (Gent) for 24 h results in significant hair cell loss (top panels). Additional supplementation of 500 μM cisplatin to the toxic concentration of gentamicin rescues the hair cells, albeit with severely reduced MYO7A immunoreactivity (bottom panels). (B) Outer hair cell counts per 100 μm (basal turn) after exposure to gentamicin with or without high dose cisplatin (Unpaired t -test, p -value:

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: High-dose cisplatin cross-preserves hair cells exposed to toxic doses of gentamicin. (A) Mouse organ of Corti explant cultures exposed to 100 μM gentamicin (Gent) for 24 h results in significant hair cell loss (top panels). Additional supplementation of 500 μM cisplatin to the toxic concentration of gentamicin rescues the hair cells, albeit with severely reduced MYO7A immunoreactivity (bottom panels). (B) Outer hair cell counts per 100 μm (basal turn) after exposure to gentamicin with or without high dose cisplatin (Unpaired t -test, p -value:

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Concentration Assay

    Effects of timing of treatment with sorafenib and insulin on hair cell protection from cisplatin-induced cell death. (A–C) Cisplatin exposure results in marked immunoreactivity of caspase-3 and loss of outer hair cells. There is significant protection of outer hair cells and limited immunoreactivity of caspase-3 with either pre- (1 h treatment prior to 200 μM cisplatin) or post-treatment (added after 1 h exposure to cisplatin) with 500 nM sorafenib and 100 nM insulin. Differences between pre- and post-treatment paradigms were not significant, though each were significantly different from cisplatin alone. ANOVA with post hoc test (Tukey) was performed for statistical analysis. Asterisks in plots indicate p -values (***

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Effects of timing of treatment with sorafenib and insulin on hair cell protection from cisplatin-induced cell death. (A–C) Cisplatin exposure results in marked immunoreactivity of caspase-3 and loss of outer hair cells. There is significant protection of outer hair cells and limited immunoreactivity of caspase-3 with either pre- (1 h treatment prior to 200 μM cisplatin) or post-treatment (added after 1 h exposure to cisplatin) with 500 nM sorafenib and 100 nM insulin. Differences between pre- and post-treatment paradigms were not significant, though each were significantly different from cisplatin alone. ANOVA with post hoc test (Tukey) was performed for statistical analysis. Asterisks in plots indicate p -values (***

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques:

    Cisplatin exposure activates both the c-Jun N-terminal kinase (JNK) and mammalian target of rapamycin (mTOR) pathways in sensory hair cells. (A) Cisplatin exposure resulted in a coordinated increase in phosphorylated JNK (p-JNK) and phosphorylated ribosomal protein S6 (p-rpS6) immunoreactivity, indicating an activation of the JNK and mTOR pathways. When explant cultures were exposed to both cisplatin and the multikinase inhibitor sorafenib (500 nM), the activation of JNK and mTOR pathways were inhibited. Rapamycin exposure did not alter the cisplatin-induced activation of JNK, but prevented activation of mTOR. When cultures were incubated with 100 nM insulin for 4 h, it resulted in robust activation of mTOR but not the JNK pathway (bottom panel). (B) AHA-biotin immunoblot showing that when administered with cisplatin, insulin lead to a 34% increase ( p -value 0.004) in overall cellular protein synthesis, when compared to cisplatin alone. (C) Immunoblot confirming cisplatin-induced activation of the JNK pathway (as measured by p-c-Jun) and mTOR (as measured by p-rpS6), and the modulation of this response by sorafenib, rapamycin and insulin. Scale bar 20 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Cisplatin exposure activates both the c-Jun N-terminal kinase (JNK) and mammalian target of rapamycin (mTOR) pathways in sensory hair cells. (A) Cisplatin exposure resulted in a coordinated increase in phosphorylated JNK (p-JNK) and phosphorylated ribosomal protein S6 (p-rpS6) immunoreactivity, indicating an activation of the JNK and mTOR pathways. When explant cultures were exposed to both cisplatin and the multikinase inhibitor sorafenib (500 nM), the activation of JNK and mTOR pathways were inhibited. Rapamycin exposure did not alter the cisplatin-induced activation of JNK, but prevented activation of mTOR. When cultures were incubated with 100 nM insulin for 4 h, it resulted in robust activation of mTOR but not the JNK pathway (bottom panel). (B) AHA-biotin immunoblot showing that when administered with cisplatin, insulin lead to a 34% increase ( p -value 0.004) in overall cellular protein synthesis, when compared to cisplatin alone. (C) Immunoblot confirming cisplatin-induced activation of the JNK pathway (as measured by p-c-Jun) and mTOR (as measured by p-rpS6), and the modulation of this response by sorafenib, rapamycin and insulin. Scale bar 20 μm.

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Activation Assay, Incubation

    Cisplatin-induced hair cell death and dose-response curve. (A–C) Immunoreactivity of MYO7A and cleaved caspase-3 in organ of Corti explants with varying concentrations of cisplatin. Increasing cisplatin (cis) concentrations of up to 250 μM lead to an increase in hair cell death and caspase-3 positive hair cells. At very high concentrations ( > 500 μM) of cisplatin, however, there is a near complete protection of hair cells and an absence of cleaved caspase-3 staining in hair cells. Supporting cells exhibited strong caspase-3 immunoreactivity at higher concentrations. Outer hair cell (OHC) numbers were counted over a length of 100 μm in the basal turn. Caspase-3 positive cells were counted over a length of 200 μm of the basal turn. Error bars indicate SEM (standard error of the mean). n = 4 organs per experimental group. (D) Both moderate (100 μM) and high (500 μM) dose cisplatin activate JNK in hair cells (with different kinetics), suggesting that even at high doses, cisplatin uptake is not inhibited. Scale bar 20 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Cisplatin-induced hair cell death and dose-response curve. (A–C) Immunoreactivity of MYO7A and cleaved caspase-3 in organ of Corti explants with varying concentrations of cisplatin. Increasing cisplatin (cis) concentrations of up to 250 μM lead to an increase in hair cell death and caspase-3 positive hair cells. At very high concentrations ( > 500 μM) of cisplatin, however, there is a near complete protection of hair cells and an absence of cleaved caspase-3 staining in hair cells. Supporting cells exhibited strong caspase-3 immunoreactivity at higher concentrations. Outer hair cell (OHC) numbers were counted over a length of 100 μm in the basal turn. Caspase-3 positive cells were counted over a length of 200 μm of the basal turn. Error bars indicate SEM (standard error of the mean). n = 4 organs per experimental group. (D) Both moderate (100 μM) and high (500 μM) dose cisplatin activate JNK in hair cells (with different kinetics), suggesting that even at high doses, cisplatin uptake is not inhibited. Scale bar 20 μm.

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Staining

    Analysis of hair cell integrity after exposure to high cisplatin concentrations. Explants were treated as follows: 24 h in 100 μM cisplatin (A,B) , 24 h in 500 μM cisplatin (C,D) or 24 h in 500 μM, followed by 24 h in normal growth medium (E,F) . Organs were then co-stained for F-actin (phalloidin, green), MYO7A (gray), cleaved casp-3 (red) and Hoechst (blue), colors are used in merged images only. In the merged images of (C,D) , the MYO7A (gray) signal, for which a high image gain was used for better visibility, was omitted. (A,C,E) are optical sections at the level of hair bundles (top section) and outer hair cell nuclei (bottom section). (B,D,F) are side views (generated by Reslice function in ImageJ), with the yellow dotted lines indicating the level of optical sections used in (A,C,E) . The protected hair cells at high cisplatin concentrations (500 μM) display normal nuclear morphology. Continuing the culture for 24 h in normal growth medium leads to near complete recovery of MYO7A immunoreactivity in hair cells initially exposed to 500 μM cisplatin. Scale bar 20 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Analysis of hair cell integrity after exposure to high cisplatin concentrations. Explants were treated as follows: 24 h in 100 μM cisplatin (A,B) , 24 h in 500 μM cisplatin (C,D) or 24 h in 500 μM, followed by 24 h in normal growth medium (E,F) . Organs were then co-stained for F-actin (phalloidin, green), MYO7A (gray), cleaved casp-3 (red) and Hoechst (blue), colors are used in merged images only. In the merged images of (C,D) , the MYO7A (gray) signal, for which a high image gain was used for better visibility, was omitted. (A,C,E) are optical sections at the level of hair bundles (top section) and outer hair cell nuclei (bottom section). (B,D,F) are side views (generated by Reslice function in ImageJ), with the yellow dotted lines indicating the level of optical sections used in (A,C,E) . The protected hair cells at high cisplatin concentrations (500 μM) display normal nuclear morphology. Continuing the culture for 24 h in normal growth medium leads to near complete recovery of MYO7A immunoreactivity in hair cells initially exposed to 500 μM cisplatin. Scale bar 20 μm.

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Staining, Generated

    Inhibition of JNK activation with simultaneous stimulation of protein synthesis results in protection from cisplatin ototoxicity. (A) Cisplatin exposure (100 μM) results in significant loss of outer hair cells (second row). Co-incubation of cisplatin with either insulin or sorafenib results in a small, but significant protection of outer hair cells (3rd, 4th rows). Combined incubation with cisplatin, insulin and sorafenib provided strong protection of cochlear hair cells (5th row). This protection, however, did not persist when organs were cultured for another 24 h in normal growth medium (7th row). Addition of rapamycin, a known inhibitor of the mTOR pathway, did not reverse the protection seen with sorafenib and insulin (6th row). (B) Quantification of outer hair cell numbers demonstrating the protective effect of insulin (Ins), sorafenib (Sora) and the combination of the two after 100 μM cisplatin (Cis) exposure. P -values for analysis of variance (ANOVA) with post hoc test (Tukey): Con to Cis:

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Inhibition of JNK activation with simultaneous stimulation of protein synthesis results in protection from cisplatin ototoxicity. (A) Cisplatin exposure (100 μM) results in significant loss of outer hair cells (second row). Co-incubation of cisplatin with either insulin or sorafenib results in a small, but significant protection of outer hair cells (3rd, 4th rows). Combined incubation with cisplatin, insulin and sorafenib provided strong protection of cochlear hair cells (5th row). This protection, however, did not persist when organs were cultured for another 24 h in normal growth medium (7th row). Addition of rapamycin, a known inhibitor of the mTOR pathway, did not reverse the protection seen with sorafenib and insulin (6th row). (B) Quantification of outer hair cell numbers demonstrating the protective effect of insulin (Ins), sorafenib (Sora) and the combination of the two after 100 μM cisplatin (Cis) exposure. P -values for analysis of variance (ANOVA) with post hoc test (Tukey): Con to Cis:

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Inhibition, Activation Assay, Incubation, Cell Culture

    Bioorthogonal noncanonical amino acid tagging (BONCAT) to study protein synthesis within sensory hair cells of mouse explant cultures. (A) Chemical structure of methionine and its utilized analog, azidohomoalanine (AHA). (B) Schematic of the BONCAT technique using either cell lysates for immunoblot or fixed organs for fluorescence microscopy. (C) AHA-biotin immunoreactivity demonstrates dose-dependent inhibition of protein synthesis on a cell-by-cell basis after treatment with cisplatin. P3–4 organ of Corti explants were cultured in growth medium containing AHA, in the presence of varying cisplatin concentrations. After 4 h, prior to the onset of cisplatin-induced cell death or changes in Myosin 7a (MYO7A) levels, explants were fixed and processed for click-chemistry reaction and imaged using confocal microscopy. Protein synthesis is inhibited in both hair cells and supporting cells. Gentamicin induced inhibition of cellular protein synthesis shown to involve only hair cells (bottom panels). Scale bar 20 μm. (D) Immunoblot showing a decrease in cellular protein synthesis within organ of Corti explant lysates. AHA-biotin was detected with streptavidin (SA)-horseradish peroxidase (HRP). (E) Quantification of AHA uptake relative to immunoreactivity of MYO7A within mouse cochleae and utricles ( n = 4). There is a marked dose-dependent reduction in AHA uptake in both sensory hair cells (HC) and supporting cells (SC). Error bars indicate SEM (standard error of the mean). (F) MYO7A immunoreactivity and nuclear morphology is not affected by short exposure (4 h) to high concentrations of cisplatin, demonstrating the appropriateness of using MYO7A staining to normalize the AHA signal.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity

    doi: 10.3389/fncel.2017.00303

    Figure Lengend Snippet: Bioorthogonal noncanonical amino acid tagging (BONCAT) to study protein synthesis within sensory hair cells of mouse explant cultures. (A) Chemical structure of methionine and its utilized analog, azidohomoalanine (AHA). (B) Schematic of the BONCAT technique using either cell lysates for immunoblot or fixed organs for fluorescence microscopy. (C) AHA-biotin immunoreactivity demonstrates dose-dependent inhibition of protein synthesis on a cell-by-cell basis after treatment with cisplatin. P3–4 organ of Corti explants were cultured in growth medium containing AHA, in the presence of varying cisplatin concentrations. After 4 h, prior to the onset of cisplatin-induced cell death or changes in Myosin 7a (MYO7A) levels, explants were fixed and processed for click-chemistry reaction and imaged using confocal microscopy. Protein synthesis is inhibited in both hair cells and supporting cells. Gentamicin induced inhibition of cellular protein synthesis shown to involve only hair cells (bottom panels). Scale bar 20 μm. (D) Immunoblot showing a decrease in cellular protein synthesis within organ of Corti explant lysates. AHA-biotin was detected with streptavidin (SA)-horseradish peroxidase (HRP). (E) Quantification of AHA uptake relative to immunoreactivity of MYO7A within mouse cochleae and utricles ( n = 4). There is a marked dose-dependent reduction in AHA uptake in both sensory hair cells (HC) and supporting cells (SC). Error bars indicate SEM (standard error of the mean). (F) MYO7A immunoreactivity and nuclear morphology is not affected by short exposure (4 h) to high concentrations of cisplatin, demonstrating the appropriateness of using MYO7A staining to normalize the AHA signal.

    Article Snippet: Cisplatin (TEVA, MD NDC 0703-5748-11, injectable solution, 1 mg/ml) and gentamicin (Sigma, St. Louis, MO, USA) were dissolved in water.

    Techniques: Fluorescence, Microscopy, Inhibition, Cell Culture, Confocal Microscopy, Staining

    Pica induced by cisplatin. Rats had two distinct periods of eating kaolin in the cisplatin groups, and Ginger significantly decreased the weight of kaolin eaten induced by cisplatin during the 72 h observation period. Metoclopramide significantly decreased the weight of kaolin eaten during 24 h following cisplatin injection, but there was no significant decrease in the pica during 24–72 h. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). i.g., intragastrically.

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: Pica induced by cisplatin. Rats had two distinct periods of eating kaolin in the cisplatin groups, and Ginger significantly decreased the weight of kaolin eaten induced by cisplatin during the 72 h observation period. Metoclopramide significantly decreased the weight of kaolin eaten during 24 h following cisplatin injection, but there was no significant decrease in the pica during 24–72 h. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). i.g., intragastrically.

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques: Injection

    TH immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of TH in area postrema and ileum. The figures above (C3, CG3, V3, M3, GL3, GM3, GH3) show the expression in area postrema. The figures below (C4, CG4, V4, M4, GL4, GM4, GH4) show the expression in ileum. B: Mean optical density values of TH. The photographs generated were quantitatively analyzed the optical density of TH with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: TH immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of TH in area postrema and ileum. The figures above (C3, CG3, V3, M3, GL3, GM3, GH3) show the expression in area postrema. The figures below (C4, CG4, V4, M4, GL4, GM4, GH4) show the expression in ileum. B: Mean optical density values of TH. The photographs generated were quantitatively analyzed the optical density of TH with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques: Immunostaining, Expressing, Immunohistochemistry, Generated

    DAT immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of DAT in area postrema and ileum. The figures above (C5, CG5, V5, M5, GL5, GM5, GH5) show the expression in area postrema. The figures below (C6, CG6, V6, M6, GL6, GM6, GH6) show the expression in ileum. B: Mean optical density values of DAT. The photographs generated were quantitatively analyzed the optical density of DAT with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: DAT immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of DAT in area postrema and ileum. The figures above (C5, CG5, V5, M5, GL5, GM5, GH5) show the expression in area postrema. The figures below (C6, CG6, V6, M6, GL6, GM6, GH6) show the expression in ileum. B: Mean optical density values of DAT. The photographs generated were quantitatively analyzed the optical density of DAT with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques: Immunostaining, Expressing, Immunohistochemistry, Generated

    D2R immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of D2R in area postrema and ileum. The figures above (C1, CG1, V1, M1, GL1, GM1, GH1) show the expression in area postrema. The figures below (C2, CG2, V2, M2, GL2, GM2, GH2) show the expression in ileum. B: Mean optical density values of D2R. The photographs generated were quantitatively analyzed the optical density of D2R with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: D2R immunostaining expression in area postrema and ileum. A: Immunohistochemistry expression of D2R in area postrema and ileum. The figures above (C1, CG1, V1, M1, GL1, GM1, GH1) show the expression in area postrema. The figures below (C2, CG2, V2, M2, GL2, GM2, GH2) show the expression in ileum. B: Mean optical density values of D2R. The photographs generated were quantitatively analyzed the optical density of D2R with Image-Pro Plus. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques: Immunostaining, Expressing, Immunohistochemistry, Generated

    Gastric emptying. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 5); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g every day ( n = 5); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 5); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 5); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 5); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 5); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 5). * P

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: Gastric emptying. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 5); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g every day ( n = 5); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 5); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 5); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 5); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 5); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 5). * P

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques:

    Changes of mRNA expression of TH and DAT in area postrema and ileum. Levels of TH and DAT against beta-actin mRNA expression are shown in the above histogram. The left-side graphs show the expression in area postrema. The right-side graphs show the expression in ileum. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Journal: Yonago Acta Medica

    Article Title: Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats

    doi:

    Figure Lengend Snippet: Changes of mRNA expression of TH and DAT in area postrema and ileum. Levels of TH and DAT against beta-actin mRNA expression are shown in the above histogram. The left-side graphs show the expression in area postrema. The right-side graphs show the expression in ileum. C: normal control group, pretreated with sterile saline 3 mL every day ( n = 10); CG: simple gingerol control group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10); V: cisplatin control group, pretreated with sterile saline 3 mL every day ( n = 10); M: cisplatin + metoclopramide group, pretreated with metoclopramide 2.5 mg·kg −1 i.g. every day ( n = 10); GL: cisplatin + low-dose gingerol group, pretreated with gingerol 10 mg·kg −1 i.g. every day ( n = 10); GM: cisplatin + middle-dose gingerol group, pretreated with gingerol 20 mg·kg −1 i.g. every day ( n = 10); GH: cisplatin + high-dose gingerol group, pretreated with gingerol 40 mg·kg −1 i.g. every day ( n = 10). * P

    Article Snippet: Cisplatin (Qilu Pharmaceutical, Jinan, China) was prepared in normal saline at 70 °C followed by gradual cooling to 40 °C and was administered immediately.

    Techniques: Expressing

    IGFBP5 expression vector reversed cisplatin-resistance. ( A ) Relative MTS activity of SLMT-1/CDDP1R, SLMT-1R-IGFBP5 and SLMT-1R-pcMV3 cells. Relative MTS activities were expressed as means ± standard error compared with the MTS activities at zero CDDP concentration and analyzed using one-way ANOVA. SLMT-1R-IGFBP5 was compared to SLMT-1R-pcMV3 with* p

    Journal: Cells

    Article Title: Expression of Insulin-Like Growth Factor Binding Protein-5 (IGFBP5) Reverses Cisplatin-Resistance in Esophageal Carcinoma

    doi: 10.3390/cells7100143

    Figure Lengend Snippet: IGFBP5 expression vector reversed cisplatin-resistance. ( A ) Relative MTS activity of SLMT-1/CDDP1R, SLMT-1R-IGFBP5 and SLMT-1R-pcMV3 cells. Relative MTS activities were expressed as means ± standard error compared with the MTS activities at zero CDDP concentration and analyzed using one-way ANOVA. SLMT-1R-IGFBP5 was compared to SLMT-1R-pcMV3 with* p

    Article Snippet: The cisplatin-resistant cell line, SLMT-1/CDDP1R, was established from the SLMT-1 cells by repeatedly subcultured under an increasing concentration of cisplatin (CDDP) (Sigma-Aldrich, St Louis, MO, USA)starting from 0.1 μg/mL to final concentration of 1.00 μg/mL.

    Techniques: Expressing, Plasmid Preparation, Activity Assay, Concentration Assay

    circELP3 may facilitate cisplatin resistance by targeting cancer stem-like cells. A: Sphere formation by T24 (the upper one) and 5637 cells (the lower one) was photographed microscopically at 40×; B: Spheres were counted under a light microscope; C: Quantitative real-time PCR was used to determine the expression of circELP3 between sphere-forming and non-sphere-forming cells; H, I, J: Quantitative real-time PCR was used to determine the expression of Sox2, Oct-4 and Nanog after treatment with siRNA for circELP3; D: Cell clone formation assays were performed to evaluate the effect of circELP3 on the self-renewal ability of sphere-forming cells and non-sphere-forming cells after treatment with 5 µM cisplatin; F and G show the significant clone numbers in each treatment group. Scale bars: A, 100μM.

    Journal: International Journal of Biological Sciences

    Article Title: Hypoxia-elevated circELP3 contributes to bladder cancer progression and cisplatin resistance

    doi: 10.7150/ijbs.26826

    Figure Lengend Snippet: circELP3 may facilitate cisplatin resistance by targeting cancer stem-like cells. A: Sphere formation by T24 (the upper one) and 5637 cells (the lower one) was photographed microscopically at 40×; B: Spheres were counted under a light microscope; C: Quantitative real-time PCR was used to determine the expression of circELP3 between sphere-forming and non-sphere-forming cells; H, I, J: Quantitative real-time PCR was used to determine the expression of Sox2, Oct-4 and Nanog after treatment with siRNA for circELP3; D: Cell clone formation assays were performed to evaluate the effect of circELP3 on the self-renewal ability of sphere-forming cells and non-sphere-forming cells after treatment with 5 µM cisplatin; F and G show the significant clone numbers in each treatment group. Scale bars: A, 100μM.

    Article Snippet: For the cisplatin treatment, cells were treated with cisplatin (Sigma, USA) at concentrations of 5 µM, 10 µM, 20 µM, 40 µM and 80 µM.

    Techniques: Light Microscopy, Real-time Polymerase Chain Reaction, Expressing

    Hypoxia-elevated circELP3 promotes cisplatin resistance in bladder cancer cells. A, B: CCK8 assay to evaluate the effect of circELP3 on the viability of T24(A) and 5637(B) cell lines after treatment with different concentrations of cisplatin; C, D: Cell clone formation assay to evaluate the effect of circELP3 on the self-renewal ability of T24 and 5637 cell lines treated with 5 μM cisplatin; E, F: Statistically significant clone numbers in each treatment group.

    Journal: International Journal of Biological Sciences

    Article Title: Hypoxia-elevated circELP3 contributes to bladder cancer progression and cisplatin resistance

    doi: 10.7150/ijbs.26826

    Figure Lengend Snippet: Hypoxia-elevated circELP3 promotes cisplatin resistance in bladder cancer cells. A, B: CCK8 assay to evaluate the effect of circELP3 on the viability of T24(A) and 5637(B) cell lines after treatment with different concentrations of cisplatin; C, D: Cell clone formation assay to evaluate the effect of circELP3 on the self-renewal ability of T24 and 5637 cell lines treated with 5 μM cisplatin; E, F: Statistically significant clone numbers in each treatment group.

    Article Snippet: For the cisplatin treatment, cells were treated with cisplatin (Sigma, USA) at concentrations of 5 µM, 10 µM, 20 µM, 40 µM and 80 µM.

    Techniques: CCK-8 Assay, Tube Formation Assay

    NPRL2 enhances the effect of cisplatin that can activate cell cycle checkpoints. H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: NPRL2 enhances the effect of cisplatin that can activate cell cycle checkpoints. H1229 cells treated with empty vector + IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. ( A ) Cdc25A was degraded by the treatment of NPRL2 or IC 20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. ( B ) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC 20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation, Transfection

    Combination treatment of NPRL2 and cisplatin induces strong cell arrest in G2/M phase. The cell cycle in tumor cells treated with NPRL2 and IC 20 value of cisplatin was analyzed by flow cytometry with use of the APO-BRDU KIT. ( A ) The shift in cell cycle parameters of cells treated with NPRL2 is manifested as clear G1 and G2 peaks 72 h after treatment. In contrast, the cell cycle distribution showed an increase in the G2/M population after treatment with empty vector and cisplatin compared with cells treated with NPRL2 alone. The shift in cell cycle distribution was seen in cells treated with NPRL2+ cisplatin: the G2/M phase population strongly increased, whereas the G1 phase population decreased. ( B ) The cell cycle shift was analyzed at 24, 48, and 72 h after treatment. The combination of NPRL2 and cisplatin significantly increased the G2/M population in a time-dependent manner ( P

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Combination treatment of NPRL2 and cisplatin induces strong cell arrest in G2/M phase. The cell cycle in tumor cells treated with NPRL2 and IC 20 value of cisplatin was analyzed by flow cytometry with use of the APO-BRDU KIT. ( A ) The shift in cell cycle parameters of cells treated with NPRL2 is manifested as clear G1 and G2 peaks 72 h after treatment. In contrast, the cell cycle distribution showed an increase in the G2/M population after treatment with empty vector and cisplatin compared with cells treated with NPRL2 alone. The shift in cell cycle distribution was seen in cells treated with NPRL2+ cisplatin: the G2/M phase population strongly increased, whereas the G1 phase population decreased. ( B ) The cell cycle shift was analyzed at 24, 48, and 72 h after treatment. The combination of NPRL2 and cisplatin significantly increased the G2/M population in a time-dependent manner ( P

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Flow Cytometry, Cytometry, Plasmid Preparation

    Prospective role of NPRL2 in combating resistance to cisplatin. NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Prospective role of NPRL2 in combating resistance to cisplatin. NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Activity Assay

    Induction of caspases by exogenous expression of NPRL2 and cisplatin in non-small cell lung cancer cells. ( A ) Induction of apoptosis in H1299 cells treated NPRL2 in the presence or absence of cisplatin ( CDDP ) by flow cytometry analysis with TUNEL staining. The PBS treated and empty vector ( EV ) treated cells were used as mock and negative controls, respectively. Error bars indicate SDs of the mean in three individual experiments (*, P

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Induction of caspases by exogenous expression of NPRL2 and cisplatin in non-small cell lung cancer cells. ( A ) Induction of apoptosis in H1299 cells treated NPRL2 in the presence or absence of cisplatin ( CDDP ) by flow cytometry analysis with TUNEL staining. The PBS treated and empty vector ( EV ) treated cells were used as mock and negative controls, respectively. Error bars indicate SDs of the mean in three individual experiments (*, P

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Expressing, Flow Cytometry, Cytometry, TUNEL Assay, Staining, Plasmid Preparation

    γ-H2AX, induced by cisplatin treatment, is also strongly enhanced by NPRL2. H1229 cells treated with an empty vector and IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment. ( A ) Western blot shows remarkably enhanced expression of γ-H2AX in cells treated with NPRL2 and cisplatin compared with cells treated with empty vector and cisplatin. ( B ) γ-H2AX expression was examined at 48 h after treatment by immunofluorescence staining and flow cytometric analysis. γ-H2AX expression was detected in cells treated with empty vector and cisplatin or NPRL2 but not in cells treated with empty vector only, and this expression was dramatically enhanced in cells treated with NPRL2 and cisplatin. Magnification: ×800. Mean fluorescein isothiocyanate (FITC) intensity was also examined at 24, 48, and 72 h after treatment. At these three time points, γ-H2AX expression in cells treated with NPRL2 and cisplatin was significantly higher than that in cells in all other treatments ( P

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: γ-H2AX, induced by cisplatin treatment, is also strongly enhanced by NPRL2. H1229 cells treated with an empty vector and IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment. ( A ) Western blot shows remarkably enhanced expression of γ-H2AX in cells treated with NPRL2 and cisplatin compared with cells treated with empty vector and cisplatin. ( B ) γ-H2AX expression was examined at 48 h after treatment by immunofluorescence staining and flow cytometric analysis. γ-H2AX expression was detected in cells treated with empty vector and cisplatin or NPRL2 but not in cells treated with empty vector only, and this expression was dramatically enhanced in cells treated with NPRL2 and cisplatin. Magnification: ×800. Mean fluorescein isothiocyanate (FITC) intensity was also examined at 24, 48, and 72 h after treatment. At these three time points, γ-H2AX expression in cells treated with NPRL2 and cisplatin was significantly higher than that in cells in all other treatments ( P

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry

    Activation of ATM signaling pathway by ectopic expression of NPRL2 protein. ( A ), Effects of ectopic expression of NPRL2 on expression and phosphorylation of ATM and NBS1 proteins.H1229 cells treated with an empty vector ( EV ) vector and IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of ataxia telangiectasia mutated (ATM) kinase, phospho-ATM (Ser1981), NBS1, and phospho-NBS1 (Ser343). β-actin was used as a loading control. Increases in phospho-ATM and phospho-NBS1 were observed in cells treated with NPRL2 or NPRL2 and cisplatin in a time-dependent manner, but not in cells treated with the control empty vector and cisplatin. ( B ), Effects of NPRL2-specific siRNA ( Si ) on expression of NPRL2 and ATM proteins in the presence and absence of cisplatin ( CDDP ). The scrambled siRNA ( Sc ) was used as a non-specific control.

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Activation of ATM signaling pathway by ectopic expression of NPRL2 protein. ( A ), Effects of ectopic expression of NPRL2 on expression and phosphorylation of ATM and NBS1 proteins.H1229 cells treated with an empty vector ( EV ) vector and IC 20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of ataxia telangiectasia mutated (ATM) kinase, phospho-ATM (Ser1981), NBS1, and phospho-NBS1 (Ser343). β-actin was used as a loading control. Increases in phospho-ATM and phospho-NBS1 were observed in cells treated with NPRL2 or NPRL2 and cisplatin in a time-dependent manner, but not in cells treated with the control empty vector and cisplatin. ( B ), Effects of NPRL2-specific siRNA ( Si ) on expression of NPRL2 and ATM proteins in the presence and absence of cisplatin ( CDDP ). The scrambled siRNA ( Sc ) was used as a non-specific control.

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Activation Assay, Expressing, Plasmid Preparation

    Lung cancer cell line H1437R was used to analyze apoptosis by flow cytometry after treatment with NPRL2 and IC 20 dose of cisplatin. ( A ) Western blot shows reduction in Chk2 expression using SiRNA specific to Chk2 (SiChk2) and scrambled siRNA (SiScrb) as control in stable clones generated in 1437R lung cancer cell line. ( B ) Measurement of cellular apoptosis by fluorescence-activated cell sorter (FACS) analysis with TUNEL staining. Results show that down-regulated Chk2 expression attenuated NPRL-2-induced apoptosis and its ability to sensitize tumor cells' response to cisplatin.

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Lung cancer cell line H1437R was used to analyze apoptosis by flow cytometry after treatment with NPRL2 and IC 20 dose of cisplatin. ( A ) Western blot shows reduction in Chk2 expression using SiRNA specific to Chk2 (SiChk2) and scrambled siRNA (SiScrb) as control in stable clones generated in 1437R lung cancer cell line. ( B ) Measurement of cellular apoptosis by fluorescence-activated cell sorter (FACS) analysis with TUNEL staining. Results show that down-regulated Chk2 expression attenuated NPRL-2-induced apoptosis and its ability to sensitize tumor cells' response to cisplatin.

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Expressing, Clone Assay, Generated, Fluorescence, FACS, TUNEL Assay, Staining

    Combination of NPRL2 and cisplatin increases the activation of both Chk1 and Chk2. ( A ) H1229 cells treated with empty vector and IC 20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC 20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC 20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC 20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC 20 dose of cisplatin. ( B ) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the Materials and Methods section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. ( C ) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC 20 dose of cisplatin, NPRL2 or NPRL2+ IC 20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P

    Journal: PLoS ONE

    Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    doi: 10.1371/journal.pone.0011994

    Figure Lengend Snippet: Combination of NPRL2 and cisplatin increases the activation of both Chk1 and Chk2. ( A ) H1229 cells treated with empty vector and IC 20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC 20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC 20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC 20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC 20 dose of cisplatin. ( B ) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the Materials and Methods section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. ( C ) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC 20 dose of cisplatin, NPRL2 or NPRL2+ IC 20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P

    Article Snippet: Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ).

    Techniques: Activation Assay, Plasmid Preparation, Expressing, Mouse Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    Cisplatin enhances the sensitivity of melanoma cells to PARP inhibitor olaparib. Five-day SRB growth assay on A375 melanoma cells in 96-well plates showing enhanced sensitivity to olaparib in the presence of 0.5 μM cisplatin. This cisplatin concentration is well below the IC50 value. In order to correct for the toxicity of cisplatin alone, for each curve growth is expressed as the percentage of the non-olaparib-treated control. Values plotted are mean % growth (±SEM) from three independent experiments

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: Cisplatin enhances the sensitivity of melanoma cells to PARP inhibitor olaparib. Five-day SRB growth assay on A375 melanoma cells in 96-well plates showing enhanced sensitivity to olaparib in the presence of 0.5 μM cisplatin. This cisplatin concentration is well below the IC50 value. In order to correct for the toxicity of cisplatin alone, for each curve growth is expressed as the percentage of the non-olaparib-treated control. Values plotted are mean % growth (±SEM) from three independent experiments

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Sulforhodamine B Assay, Growth Assay, Concentration Assay

    Expression of FLAG-tagged RAD51 adversely affects the response of melanoma cells to cisplatin. a No cisplatin-induced reduction in levels of FLAG-RAD51. Western blots from control A375 cells, cells transfected to express FLAG-RAD51 at levels equivalent to endogenous RAD51 and cells of both types treated with 1 μM cisplatin for 24, 48 or 72 h. The blot was probed sequentially for RAD51 (RAD51 37 kDa, FLAG-RAD51 38.2 kDa), the FLAG tag, and β-actin (42 kDa). b Reduced growth in cisplatin-treated cultures expressing FLAG-RAD51. The table shows the mean cell growth (±SEM, n = 4) of wells after 72 h of 1 μM cisplatin treatment for non-transfected A375 cells expressed as a percentage of the untreated control, and for FLAG-RAD51 transfected A375 cells expressed as a percentage of the non-cisplatin-treated FLAG-RAD51 transfection control. The P value for Student’s paired t test, comparing the effect of cisplatin on growth of normal and FLAG-RAD51-expressing cells is also shown. c Increased early apoptosis in cisplatin-treated A375 cultures expressing FLAG-RAD51. Cultures treated as in ( a ) and western blotted for activated caspase-3 and β-actin after 48 h of 1 μM cisplatin treatment. Note the highest levels of cleaved caspase-3 (at 17 and 19 kDa) in the sample transfected with FLAG-RAD51 and treated with cisplatin. d Increased apoptosis in cisplatin-treated A375 cells expressing FLAG-RAD51. Cultures treated as in ( a ) after 72 h of 1 μM cisplatin. A representative flow cytometry profile for each culture condition is shown, with the percentage of cells with subG1 (apoptotic), G1, S and G2/M DNA contents indicated across the top of each profile. Note the highest level of subG1 (apoptotic) material in the cisplatin-treated FLAG-RAD51-expressing cells. The table below shows the mean level of apoptosis (±SEM, n = 4) in wells after 72 h of cisplatin treatment. The P values for Student’s paired t test, comparing the level of apoptosis between cisplatin-treated and untreated control wells, and between cisplatin-treated FLAG-RAD51-expressing wells and untreated FLAG-RAD51-expressing wells are also shown

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: Expression of FLAG-tagged RAD51 adversely affects the response of melanoma cells to cisplatin. a No cisplatin-induced reduction in levels of FLAG-RAD51. Western blots from control A375 cells, cells transfected to express FLAG-RAD51 at levels equivalent to endogenous RAD51 and cells of both types treated with 1 μM cisplatin for 24, 48 or 72 h. The blot was probed sequentially for RAD51 (RAD51 37 kDa, FLAG-RAD51 38.2 kDa), the FLAG tag, and β-actin (42 kDa). b Reduced growth in cisplatin-treated cultures expressing FLAG-RAD51. The table shows the mean cell growth (±SEM, n = 4) of wells after 72 h of 1 μM cisplatin treatment for non-transfected A375 cells expressed as a percentage of the untreated control, and for FLAG-RAD51 transfected A375 cells expressed as a percentage of the non-cisplatin-treated FLAG-RAD51 transfection control. The P value for Student’s paired t test, comparing the effect of cisplatin on growth of normal and FLAG-RAD51-expressing cells is also shown. c Increased early apoptosis in cisplatin-treated A375 cultures expressing FLAG-RAD51. Cultures treated as in ( a ) and western blotted for activated caspase-3 and β-actin after 48 h of 1 μM cisplatin treatment. Note the highest levels of cleaved caspase-3 (at 17 and 19 kDa) in the sample transfected with FLAG-RAD51 and treated with cisplatin. d Increased apoptosis in cisplatin-treated A375 cells expressing FLAG-RAD51. Cultures treated as in ( a ) after 72 h of 1 μM cisplatin. A representative flow cytometry profile for each culture condition is shown, with the percentage of cells with subG1 (apoptotic), G1, S and G2/M DNA contents indicated across the top of each profile. Note the highest level of subG1 (apoptotic) material in the cisplatin-treated FLAG-RAD51-expressing cells. The table below shows the mean level of apoptosis (±SEM, n = 4) in wells after 72 h of cisplatin treatment. The P values for Student’s paired t test, comparing the level of apoptosis between cisplatin-treated and untreated control wells, and between cisplatin-treated FLAG-RAD51-expressing wells and untreated FLAG-RAD51-expressing wells are also shown

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Expressing, Western Blot, Transfection, FLAG-tag, Flow Cytometry, Cytometry

    Increased levels of translesion synthesis DNA Polymerase zeta in melanoma cells improves their response to cisplatin. a and b Increased levels of DNA Polymerase zeta, but not DNA Polymerase eta after cisplatin treatment. Melanoma cell lines (A375, C32, G361), ovarian cancer cell line, PEO4, and immortalised fibroblast line, MRC5v1, were untreated, or treated with 3 μM cisplatin for 24, 48 or 72 h and western blotted for translesion synthesis DNA Polymerases. a DNA Pol ζ. b DNA Pol η. Graphs shows the expression relative to β-actin and normalised to the level in untreated cells. Results are the means from two separate determinations. Representative western blots themselves are shown in Additional file 1 : Figure S6. c Reduced growth in cisplatin-treated cultures following siRNA knockdown of DNA Polymerase zeta. The table shows the mean cell growth (±SEM, n = 4) of wells after 72 h of 1 μM cisplatin treatment for non-transfected A375 cells and cells transfected with DNA Polymerase zeta siRNA or scrambled control siRNA, expressed as a percentage of the untreated control. The P value for Student’s paired t test, comparing the effect of DNA Polymerase zeta siRNA or scrambled control siRNA transfection on cell growth of cisplatin-treated wells is also shown. d Increased apoptosis in cisplatin-treated DNA Pol ζ siRNA-transfected A375 cells. Cultures treated as in ( c ) after 72 h of 1 μM cisplatin. A representative flow cytometry profile for each culture condition is shown, with the percentage of cells with subG1 (apoptotic), G1, S and G2/M DNA contents indicated across the top of each profile. Note the increased level of subG1 (apoptotic) material in the cisplatin-treated DNA Pol ζ siRNA-transfected cells. The table below shows the mean level of apoptosis (±SEM, n = 4) in wells after 72 h of cisplatin treatment. The P values for Student’s paired t test, comparing the level of apoptosis between cisplatin-treated and untreated control wells, and between cisplatin-treated DNA Pol ζ siRNA-transfected wells and cisplatin-treated scrambled control siRNA-transfected wells are also shown

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: Increased levels of translesion synthesis DNA Polymerase zeta in melanoma cells improves their response to cisplatin. a and b Increased levels of DNA Polymerase zeta, but not DNA Polymerase eta after cisplatin treatment. Melanoma cell lines (A375, C32, G361), ovarian cancer cell line, PEO4, and immortalised fibroblast line, MRC5v1, were untreated, or treated with 3 μM cisplatin for 24, 48 or 72 h and western blotted for translesion synthesis DNA Polymerases. a DNA Pol ζ. b DNA Pol η. Graphs shows the expression relative to β-actin and normalised to the level in untreated cells. Results are the means from two separate determinations. Representative western blots themselves are shown in Additional file 1 : Figure S6. c Reduced growth in cisplatin-treated cultures following siRNA knockdown of DNA Polymerase zeta. The table shows the mean cell growth (±SEM, n = 4) of wells after 72 h of 1 μM cisplatin treatment for non-transfected A375 cells and cells transfected with DNA Polymerase zeta siRNA or scrambled control siRNA, expressed as a percentage of the untreated control. The P value for Student’s paired t test, comparing the effect of DNA Polymerase zeta siRNA or scrambled control siRNA transfection on cell growth of cisplatin-treated wells is also shown. d Increased apoptosis in cisplatin-treated DNA Pol ζ siRNA-transfected A375 cells. Cultures treated as in ( c ) after 72 h of 1 μM cisplatin. A representative flow cytometry profile for each culture condition is shown, with the percentage of cells with subG1 (apoptotic), G1, S and G2/M DNA contents indicated across the top of each profile. Note the increased level of subG1 (apoptotic) material in the cisplatin-treated DNA Pol ζ siRNA-transfected cells. The table below shows the mean level of apoptosis (±SEM, n = 4) in wells after 72 h of cisplatin treatment. The P values for Student’s paired t test, comparing the level of apoptosis between cisplatin-treated and untreated control wells, and between cisplatin-treated DNA Pol ζ siRNA-transfected wells and cisplatin-treated scrambled control siRNA-transfected wells are also shown

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Translesion Synthesis, Western Blot, Expressing, Transfection, Flow Cytometry, Cytometry

    Reduced RAD51 levels in melanoma cells after cisplatin treatment. a Levels of Nucleotide Excision Repair proteins increase after cisplatin treatment of A375 melanoma cells, while levels of RAD51 decrease dramatically. Total protein extracts from untreated A375 human melanoma cells and cells treated with 6 μM cisplatin (CDDP) for 48 h were western blotted for the NER proteins, ERCC1 (33 kDa), XPF (104 kDa) and XPA (40 kDa) and the homologous recombination protein, RAD51 (37 kDa). β-actin (42 kDa) served as the loading control. b Cisplatin concentration- and treatment time-dependent decrease in RAD51 levels in A375 melanoma cells. Total protein extracts from untreated A375 human melanoma cells and cells treated with CDDP concentrations ranging from 0.3 to 6 μM for 48 and 72 h were western blotted for RAD51. β-actin served as the loading control. The positions of molecular weight markers (in kDa) adjacent to proteins of interest are shown

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: Reduced RAD51 levels in melanoma cells after cisplatin treatment. a Levels of Nucleotide Excision Repair proteins increase after cisplatin treatment of A375 melanoma cells, while levels of RAD51 decrease dramatically. Total protein extracts from untreated A375 human melanoma cells and cells treated with 6 μM cisplatin (CDDP) for 48 h were western blotted for the NER proteins, ERCC1 (33 kDa), XPF (104 kDa) and XPA (40 kDa) and the homologous recombination protein, RAD51 (37 kDa). β-actin (42 kDa) served as the loading control. b Cisplatin concentration- and treatment time-dependent decrease in RAD51 levels in A375 melanoma cells. Total protein extracts from untreated A375 human melanoma cells and cells treated with CDDP concentrations ranging from 0.3 to 6 μM for 48 and 72 h were western blotted for RAD51. β-actin served as the loading control. The positions of molecular weight markers (in kDa) adjacent to proteins of interest are shown

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Western Blot, Homologous Recombination, Concentration Assay, Molecular Weight

    The reduction in RAD51 levels after cisplatin treatment in melanoma cells is not seen in other cell types. Graphs show the expression of RAD51, determined by western blotting, in five human melanoma cell lines (A375, C32, G361, HBL and WM115), two human ovarian cancer cell lines (PEO1 and PEO4) and the immortalised human fibroblast line, MRC5v1. Cells were untreated, or treated with 0.3, 1 or 3 μM cisplatin for 24, 48 or 72 h. RAD51 expression is plotted relative to β-actin and normalised to the level in untreated cells. Results are the means from two separate determinations. Representative western blots themselves are shown in Additional file 1 : Figure S1

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: The reduction in RAD51 levels after cisplatin treatment in melanoma cells is not seen in other cell types. Graphs show the expression of RAD51, determined by western blotting, in five human melanoma cell lines (A375, C32, G361, HBL and WM115), two human ovarian cancer cell lines (PEO1 and PEO4) and the immortalised human fibroblast line, MRC5v1. Cells were untreated, or treated with 0.3, 1 or 3 μM cisplatin for 24, 48 or 72 h. RAD51 expression is plotted relative to β-actin and normalised to the level in untreated cells. Results are the means from two separate determinations. Representative western blots themselves are shown in Additional file 1 : Figure S1

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Expressing, Western Blot

    Reduced RAD51 mRNA levels in melanoma cells after cisplatin treatment. A375, C32, G361 melanoma cells, PEO4 ovarian cancer cells and MRC5v1 fibroblasts were treated with 3 μM cisplatin for 24, 48 or 72 h. RNA was extracted and RAD51 mRNA levels were determined by quantitative RT-PCR. RAD51 mRNA expression (±SEM, n = 3) is shown relative to β-actin and normalised to the level in untreated control (Cont.) cells

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: Reduced RAD51 mRNA levels in melanoma cells after cisplatin treatment. A375, C32, G361 melanoma cells, PEO4 ovarian cancer cells and MRC5v1 fibroblasts were treated with 3 μM cisplatin for 24, 48 or 72 h. RNA was extracted and RAD51 mRNA levels were determined by quantitative RT-PCR. RAD51 mRNA expression (±SEM, n = 3) is shown relative to β-actin and normalised to the level in untreated control (Cont.) cells

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Quantitative RT-PCR, Expressing

    RAD51 DNA repair foci are not induced by short term cisplatin treatment of melanoma cells. A375 melanoma, MRC5v1 immortalised fibroblasts and PEO4 ovarian cancer cells growing on coverslips were treated for 24 h with 6 μM cisplatin and the number of RAD51 foci in treated and control cultures was determined by RAD51 immunofluorescence of fixed cells. Graph shows the mean number of RAD51 foci per nucleus (±SEM) for treated and control cultures. Between 40 and 60 nuclei were scored for each cell type and treatment. Representative images are shown in Additional file 1 : Figure S2

    Journal: BMC Cancer

    Article Title: Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

    doi: 10.1186/s12885-017-3864-6

    Figure Lengend Snippet: RAD51 DNA repair foci are not induced by short term cisplatin treatment of melanoma cells. A375 melanoma, MRC5v1 immortalised fibroblasts and PEO4 ovarian cancer cells growing on coverslips were treated for 24 h with 6 μM cisplatin and the number of RAD51 foci in treated and control cultures was determined by RAD51 immunofluorescence of fixed cells. Graph shows the mean number of RAD51 foci per nucleus (±SEM) for treated and control cultures. Between 40 and 60 nuclei were scored for each cell type and treatment. Representative images are shown in Additional file 1 : Figure S2

    Article Snippet: Four hours after transfection 1 μM cisplatin (Hospira UK Ltd., Leamington Spa, UK) was added to some transfected and non-transfected wells.

    Techniques: Immunofluorescence

    Aldi-6 inhibits ALDH activity in HNSCC ( A ) ALDH3A1 activity in SCC4 xenograft tumor. Mice with SCC4 xenografts were treated intra-tumorally with Aldi-6 or vehicle control for three consecutive days, and the ALDH3A1 activity was measured using an isoelectric focusing assay (see Methods). Experiment was performed three times. ( B ) Representative FACS analyses of ALDH activity of SCC4 and PCI-13 cells. After a two-day treatment of cisplatin (0.88 μM) and/or Aldi-6 (30 μM), ALDH activity was measured in the surviving cells by Aldefluor assay. Grey line represents the DEAB-treated negative control for each treatment condition. Blue line represents the ALDH activity of each sample. ( C ) Changes in ALDH activity in (B) were quantified as a ratio of the shift of MFI between treated and untreated sample. The ratio in MFI shift was calculated by (MFI of treated sample-(MFI of treated sample+DEAB))/(MFI of untreated sample-(MFI of untreated sample+DEAB)). Results represent the means ± SEMs of 2–3 independent experiments with 10,000 cells each. (* p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Aldi-6 inhibits ALDH activity in HNSCC ( A ) ALDH3A1 activity in SCC4 xenograft tumor. Mice with SCC4 xenografts were treated intra-tumorally with Aldi-6 or vehicle control for three consecutive days, and the ALDH3A1 activity was measured using an isoelectric focusing assay (see Methods). Experiment was performed three times. ( B ) Representative FACS analyses of ALDH activity of SCC4 and PCI-13 cells. After a two-day treatment of cisplatin (0.88 μM) and/or Aldi-6 (30 μM), ALDH activity was measured in the surviving cells by Aldefluor assay. Grey line represents the DEAB-treated negative control for each treatment condition. Blue line represents the ALDH activity of each sample. ( C ) Changes in ALDH activity in (B) were quantified as a ratio of the shift of MFI between treated and untreated sample. The ratio in MFI shift was calculated by (MFI of treated sample-(MFI of treated sample+DEAB))/(MFI of untreated sample-(MFI of untreated sample+DEAB)). Results represent the means ± SEMs of 2–3 independent experiments with 10,000 cells each. (* p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: Activity Assay, Mouse Assay, FACS, Negative Control

    Aldi-6 reduces HNSCC tumor growth rate in vivo ( A ) SCC4 cells (2 × 10 6 ) were subcutaneously injected into the flanks of NOD- scid IL2Rgamma null mice ( n = 3–6 per group). Mice were treated systemically with Aldi-6, using implantable osmotic mini pumps (24 mg/kg/day) for continuous delivery of the compound. Cisplatin was administered by weekly i.p. injection (2 mg/kg) for 3 weeks. Tumor size was measured weekly for three weeks. One-way ANOVA analysis was performed on the final tumor size (* p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Aldi-6 reduces HNSCC tumor growth rate in vivo ( A ) SCC4 cells (2 × 10 6 ) were subcutaneously injected into the flanks of NOD- scid IL2Rgamma null mice ( n = 3–6 per group). Mice were treated systemically with Aldi-6, using implantable osmotic mini pumps (24 mg/kg/day) for continuous delivery of the compound. Cisplatin was administered by weekly i.p. injection (2 mg/kg) for 3 weeks. Tumor size was measured weekly for three weeks. One-way ANOVA analysis was performed on the final tumor size (* p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: In Vivo, Injection, Mouse Assay

    Aldi-6 decreases cell viability of HNSCC cells ( A ) SCC4 and ( B ) PCI-13 cells were treated with Aldi-6 and/or cisplatin for two consecutive days. Then, the cells were analyzed on the fourth day. Cell viability was quantified using MTT assay. Results represent mean ± SEMs of 8–16 replicates. *p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Aldi-6 decreases cell viability of HNSCC cells ( A ) SCC4 and ( B ) PCI-13 cells were treated with Aldi-6 and/or cisplatin for two consecutive days. Then, the cells were analyzed on the fourth day. Cell viability was quantified using MTT assay. Results represent mean ± SEMs of 8–16 replicates. *p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: MTT Assay

    Cisplatin increases ALDH3A1 expression in HNSCC ( A ) Western blot analyses of human primary tumor homogenates from HNSCC patients using GAPDH as a loading control. ( B ) SCC4 and PCI-13 cells were treated with cisplatin (15 μM) for 2 and 4 days and total cell lysates were analyzed by Western blot for ALDH3A1 protein. Densitometric analysis of ALDH bands obtained by Western blot using Image J software shows relative levels after normalization for equal protein loading using β-tubulin as a loading control. Results are expressed as mean±SEM. (* p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Cisplatin increases ALDH3A1 expression in HNSCC ( A ) Western blot analyses of human primary tumor homogenates from HNSCC patients using GAPDH as a loading control. ( B ) SCC4 and PCI-13 cells were treated with cisplatin (15 μM) for 2 and 4 days and total cell lysates were analyzed by Western blot for ALDH3A1 protein. Densitometric analysis of ALDH bands obtained by Western blot using Image J software shows relative levels after normalization for equal protein loading using β-tubulin as a loading control. Results are expressed as mean±SEM. (* p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: Expressing, Western Blot, Software

    ALDH3A1 activation enhances cisplatin-resistance in HNSCC ( A ) Structure of Alda-89, a small molecule ALDH3A1 activator, is shown (4-Allyl-1,2-methylenedioxybenzene, MW 162.1). ( B – C ) SCC4 cells and PCI-13 cells were treated with increasing concentrations of Alda-89 (15–60 μM) and/or cisplatin (15 μM) for four consecutive days. Then, the cells were analyzed on the fourth day. The percentage of live cells is shown compared to that of control cells. Cell viability was quantified using MTT assay, which was performed in 4–8 replicates in two independent experiments. Results represent mean ± SEMs (* p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: ALDH3A1 activation enhances cisplatin-resistance in HNSCC ( A ) Structure of Alda-89, a small molecule ALDH3A1 activator, is shown (4-Allyl-1,2-methylenedioxybenzene, MW 162.1). ( B – C ) SCC4 cells and PCI-13 cells were treated with increasing concentrations of Alda-89 (15–60 μM) and/or cisplatin (15 μM) for four consecutive days. Then, the cells were analyzed on the fourth day. The percentage of live cells is shown compared to that of control cells. Cell viability was quantified using MTT assay, which was performed in 4–8 replicates in two independent experiments. Results represent mean ± SEMs (* p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: Activation Assay, MTT Assay

    Knockdown of ALDH3A1 expression in HNSCC cells increases sensitivity to cisplatin ( A ) Knockdown of ALDH3A1 by lentiviral transduction of shRNA in SCC4 cells was confirmed by Western blot assay. ( B ) Wild type (WT) and ALDH3A1 knockdown cells were treated with cisplatin on days 1 and 2, and cell viability was quantified by MTT on the fourth day. Results were expressed as percent of control (* p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Knockdown of ALDH3A1 expression in HNSCC cells increases sensitivity to cisplatin ( A ) Knockdown of ALDH3A1 by lentiviral transduction of shRNA in SCC4 cells was confirmed by Western blot assay. ( B ) Wild type (WT) and ALDH3A1 knockdown cells were treated with cisplatin on days 1 and 2, and cell viability was quantified by MTT on the fourth day. Results were expressed as percent of control (* p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: Expressing, Transduction, shRNA, Western Blot, MTT Assay

    Cisplatin increases ALDH activity in HNSCC SCC4 and PCI-13 cells were treated for 2 days with 15 μM cisplatin. ALDH activity was determined using an Aldefluor assay in surviving cells and compared with the non-treated cells to evaluate the effect of cisplatin. The fluorescence intensity (ALDH activity) was determined by flow cytometry. Results represent the means and SEMs of two independent experiments. (* and # p

    Journal: Oncotarget

    Article Title: Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

    doi: 10.18632/oncotarget.17017

    Figure Lengend Snippet: Cisplatin increases ALDH activity in HNSCC SCC4 and PCI-13 cells were treated for 2 days with 15 μM cisplatin. ALDH activity was determined using an Aldefluor assay in surviving cells and compared with the non-treated cells to evaluate the effect of cisplatin. The fluorescence intensity (ALDH activity) was determined by flow cytometry. Results represent the means and SEMs of two independent experiments. (* and # p

    Article Snippet: Cisplatin was from Enzo Life Sciences (ALX-400-040-M250, AnnArbor, MI).

    Techniques: Activity Assay, Fluorescence, Flow Cytometry, Cytometry

    Pemetrexed and cisplatin inhibit proliferation, whereas cSBL has a cytotoxic effect in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 0–72 h. The number of cells was estimated using a Muse ™ Count Viability Kit. ( A ) Cell growth rates were calculated as the ratio of the cell number at 72 h to the cell number at 0 h and presented as a fraction of the controls. ( B ) The number of live and dead cells every 24 h is shown. Statistically significant differences in the live cell number at 72 h were observed in the treatment groups compared to the control (***). Statistically significant differences in the dead cell number at 72 h were observed in cSBL-treated cells compared to cisplatin- or pemetrexed-treated cells ( †† ). * p

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Pemetrexed and cisplatin inhibit proliferation, whereas cSBL has a cytotoxic effect in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 0–72 h. The number of cells was estimated using a Muse ™ Count Viability Kit. ( A ) Cell growth rates were calculated as the ratio of the cell number at 72 h to the cell number at 0 h and presented as a fraction of the controls. ( B ) The number of live and dead cells every 24 h is shown. Statistically significant differences in the live cell number at 72 h were observed in the treatment groups compared to the control (***). Statistically significant differences in the dead cell number at 72 h were observed in cSBL-treated cells compared to cisplatin- or pemetrexed-treated cells ( †† ). * p

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques:

    Pharmacological interactions between pemetrexed, cisplatin, and cSBL in H28 cells ( A ) The drug concentration ratios were as follows: pemetrexed + cisplatin (1:2), pemetrexed + cSBL (20:1), cisplatin + cSBL (40:1). Cells were treated with pemetrexed (2 nM–400 μM), cisplatin (4 nM–800 μM), or cSBL (0.1 nM–20 μM) for 72 h. The horizontal axis indicates the concentration of pemetrexed in the pemetrexed + cisplatin or pemetrexed + cSBL combination or the concentration of cisplatin in the cisplatin + cSBL combination. ( B ) CI-Fa curves for H28 cells treated with pemetrexed + cisplatin, pemetrexed + cSBL, or cisplatin + cSBL. CI values

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Pharmacological interactions between pemetrexed, cisplatin, and cSBL in H28 cells ( A ) The drug concentration ratios were as follows: pemetrexed + cisplatin (1:2), pemetrexed + cSBL (20:1), cisplatin + cSBL (40:1). Cells were treated with pemetrexed (2 nM–400 μM), cisplatin (4 nM–800 μM), or cSBL (0.1 nM–20 μM) for 72 h. The horizontal axis indicates the concentration of pemetrexed in the pemetrexed + cisplatin or pemetrexed + cSBL combination or the concentration of cisplatin in the cisplatin + cSBL combination. ( B ) CI-Fa curves for H28 cells treated with pemetrexed + cisplatin, pemetrexed + cSBL, or cisplatin + cSBL. CI values

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques: Concentration Assay

    Pemetrexed, cisplatin, and cSBL, either alone or in combination, induced apoptosis in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. The y-axis indicates the percentage of annexin V-positive cells. The percentage of PI-positive and negative cells is indicated by the different column patterns. The statistical significance of the percentage of annexin V-positive cells compared to the control is shown. ** p

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Pemetrexed, cisplatin, and cSBL, either alone or in combination, induced apoptosis in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. The y-axis indicates the percentage of annexin V-positive cells. The percentage of PI-positive and negative cells is indicated by the different column patterns. The statistical significance of the percentage of annexin V-positive cells compared to the control is shown. ** p

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques:

    Caspase-3 activation is not enhanced by combination treatment Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. ( A ) Cleaved (activated) caspase-3 was detected using western blot analysis. ( B ) Caspase-3 activity was analyzed using a Caspase-Glo ™ 3/7 assay. * p

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Caspase-3 activation is not enhanced by combination treatment Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. ( A ) Cleaved (activated) caspase-3 was detected using western blot analysis. ( B ) Caspase-3 activity was analyzed using a Caspase-Glo ™ 3/7 assay. * p

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques: Activation Assay, Western Blot, Activity Assay, Caspase-Glo Assay

    Pemetrexed, cisplatin, and cSBL, either alone or in combination, alter cell cycle dynamics in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. ( A , B ) Flow cytometry analysis of cell cycle progression in H28 cell lines after 72 h of treatment. ( C ) Western blot analysis of cyclin (A, B1, D1, and E), p21 Waf1/Cip1 , pan-Akt, and phospho-Akt levels.

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Pemetrexed, cisplatin, and cSBL, either alone or in combination, alter cell cycle dynamics in H28 cells Cells were treated with pemetrexed (20 μM), cisplatin (40 μM), or cSBL (1 μM) for 72 h. ( A , B ) Flow cytometry analysis of cell cycle progression in H28 cell lines after 72 h of treatment. ( C ) Western blot analysis of cyclin (A, B1, D1, and E), p21 Waf1/Cip1 , pan-Akt, and phospho-Akt levels.

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques: Flow Cytometry, Cytometry, Western Blot

    Dose-response curves in the mesothelioma cell lines (H28, H2452, MESO-1, MESO-4, and MSTO), and MeT5A mesothelial cells treated with pemetrexed ( A ), cisplatin ( B ), or cSBL ( C ). Cells were treated with pemetrexed (0.1 nM–20 mM), cisplatin (1 nM–1 mM), or cSBL (1 nM–30 μM) for 72 h. The dots and bars represent the mean and SD, respectively. Dose-response curves are depicted as lines or dotted lines. Each data point represents the mean ± SD of at least three independent WST-8 assays. Each sample was plated in triplicate.

    Journal: Oncotarget

    Article Title: Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma

    doi: 10.18632/oncotarget.17198

    Figure Lengend Snippet: Dose-response curves in the mesothelioma cell lines (H28, H2452, MESO-1, MESO-4, and MSTO), and MeT5A mesothelial cells treated with pemetrexed ( A ), cisplatin ( B ), or cSBL ( C ). Cells were treated with pemetrexed (0.1 nM–20 mM), cisplatin (1 nM–1 mM), or cSBL (1 nM–30 μM) for 72 h. The dots and bars represent the mean and SD, respectively. Dose-response curves are depicted as lines or dotted lines. Each data point represents the mean ± SD of at least three independent WST-8 assays. Each sample was plated in triplicate.

    Article Snippet: Cisplatin was purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques:

    ( A ) Steps used in the clonogenic assay; ( B ) Toxicity and effect of cisplatin action in the absence and presence of GNPs. Data are means ± S.D. for n = 9 cell preparations over three independent experimental set-ups.

    Journal: Cancers

    Article Title: Determining the Radiation Enhancement Effects of Gold Nanoparticles in Cells in a Combined Treatment with Cisplatin and Radiation at Therapeutic Megavoltage Energies

    doi: 10.3390/cancers10050150

    Figure Lengend Snippet: ( A ) Steps used in the clonogenic assay; ( B ) Toxicity and effect of cisplatin action in the absence and presence of GNPs. Data are means ± S.D. for n = 9 cell preparations over three independent experimental set-ups.

    Article Snippet: Cisplatin (Tocris Bioscience Bristol, UK) was added to the GNP-RGD construct at approximately 620 molecules/GNP.

    Techniques: Clonogenic Assay

    Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by cisplatin treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by cisplatin treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Mouse Assay

    The viSNE plots illustrate the expression intensity of Gr-1, CD44, IL-17A, B220, CD62L markers within the clouds of main subsets defined in Figure 4 in the splenic samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional to the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not show differential expression are not shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The viSNE plots illustrate the expression intensity of Gr-1, CD44, IL-17A, B220, CD62L markers within the clouds of main subsets defined in Figure 4 in the splenic samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional to the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not show differential expression are not shown.

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Expressing, Mouse Assay

    The trajectories on the radar plots delineate the characteristic marker profile of splenocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of splenic ( A ) CD11b+/Gr-1+ MDSCs, ( B ) CD44+, and in a smaller extent ( C ) IL-17A+ MDSCs is a characteristic of 4T1 breast cancer. Cisplatin restores the percentage of ( D ) B220+ and ( E ) CD62L+ B-cells, ( F ) CD62L+ CD4+ and ( G ) CD8+ T-cells. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text and in Figure S3 . The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The trajectories on the radar plots delineate the characteristic marker profile of splenocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of splenic ( A ) CD11b+/Gr-1+ MDSCs, ( B ) CD44+, and in a smaller extent ( C ) IL-17A+ MDSCs is a characteristic of 4T1 breast cancer. Cisplatin restores the percentage of ( D ) B220+ and ( E ) CD62L+ B-cells, ( F ) CD62L+ CD4+ and ( G ) CD8+ T-cells. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text and in Figure S3 . The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Marker, Mouse Assay, Expressing

    Real-time monitoring of 4T1 cell viability hampered by cisplatin. The 4T1 cells were seeded and the baseline impedance was recorded for 48 h. After that 48 h culturing treatment with 11.111 µM, 33.333 µM, or 100 µM cisplatin reduced viability of 4T1 cells on a time and dose dependent manner. The corresponding dose-response curves with the half maximal inhibitory concentration (IC 50 ) values can be found in Figure S1 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Real-time monitoring of 4T1 cell viability hampered by cisplatin. The 4T1 cells were seeded and the baseline impedance was recorded for 48 h. After that 48 h culturing treatment with 11.111 µM, 33.333 µM, or 100 µM cisplatin reduced viability of 4T1 cells on a time and dose dependent manner. The corresponding dose-response curves with the half maximal inhibitory concentration (IC 50 ) values can be found in Figure S1 .

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Concentration Assay

    The proteolytic activity of the fibroblast activator protein (FAP) increased by the formation of breast cancer, and was significantly decreased by cisplatin treatment. Area under the curve (AUC) values from high pressure liquid chromatography (HPLC) analysis of the peptide digestion product 2 ( Figure S2 ) were plotted. The results are shown as arithmetic mean values of the samples ± standard error of the mean (SEM), statistical significance was set to *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The proteolytic activity of the fibroblast activator protein (FAP) increased by the formation of breast cancer, and was significantly decreased by cisplatin treatment. Area under the curve (AUC) values from high pressure liquid chromatography (HPLC) analysis of the peptide digestion product 2 ( Figure S2 ) were plotted. The results are shown as arithmetic mean values of the samples ± standard error of the mean (SEM), statistical significance was set to *** p

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Activity Assay, High Performance Liquid Chromatography

    Cisplatin treatment reduced 4T1 tumor growth, the number of lung metastatic nodules and the weight of the spleen. The 4T1 cells (1.2 × 10 5 ) were transplanted by the injection into the mammary fat pad of BALB/c mice ( n = 12). Tumor growth was monitored daily ( A ). On the 23rd day mice were euthanized and the weight of the tumors ( B ), the number of metastatic nodules on the lungs ( C ), and the weight of the spleens were measured ( D ). Individual values and arithmetic mean values of the samples ± standard error of the mean (SEM) are plotted, statistical significance was set to *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Cisplatin treatment reduced 4T1 tumor growth, the number of lung metastatic nodules and the weight of the spleen. The 4T1 cells (1.2 × 10 5 ) were transplanted by the injection into the mammary fat pad of BALB/c mice ( n = 12). Tumor growth was monitored daily ( A ). On the 23rd day mice were euthanized and the weight of the tumors ( B ), the number of metastatic nodules on the lungs ( C ), and the weight of the spleens were measured ( D ). Individual values and arithmetic mean values of the samples ± standard error of the mean (SEM) are plotted, statistical significance was set to *** p

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Injection, Mouse Assay

    The viSNE plots illustrate the expression intensity of Gr-1, CD44, and IFN-γ markers within the clouds of main subsets defined in Figure 7 in the blood samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional with the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in the Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not showed different expression are not shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The viSNE plots illustrate the expression intensity of Gr-1, CD44, and IFN-γ markers within the clouds of main subsets defined in Figure 7 in the blood samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional with the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in the Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not showed different expression are not shown.

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Expressing, Mouse Assay

    The trajectories on the radar plots delineate the characteristic marker profile of blood-derived leukocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of peripheral ( A ) CD11b+/Gr-1+ MDSCs and ( B ) CD44+ MDSCs is a characteristic of 4T1 breast cancer. ( C ) Due to cisplatin treatment IFN-γ+ MDSCs were developed at the periphery. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text. The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The trajectories on the radar plots delineate the characteristic marker profile of blood-derived leukocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of peripheral ( A ) CD11b+/Gr-1+ MDSCs and ( B ) CD44+ MDSCs is a characteristic of 4T1 breast cancer. ( C ) Due to cisplatin treatment IFN-γ+ MDSCs were developed at the periphery. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text. The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Marker, Derivative Assay, Mouse Assay, Expressing

    Immunophenotyping of blood showed dramatic expansion of CD11b+ cells in advanced cancer. The viSNE analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets was performed in the blood of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) has significance at p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Immunophenotyping of blood showed dramatic expansion of CD11b+ cells in advanced cancer. The viSNE analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets was performed in the blood of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) has significance at p

    Article Snippet: Cells viability was determined by cisplatin (5 μM 195Pt, Fluidigm) staining for 3 min on ice in 300 μL PBS.

    Techniques: Mouse Assay

    Characterization of infiltrating immune cell populations in responsive and resistant tumors to Ab+Pal combination treatment. ( A ) Preliminary gating for mass cytometry samples. Iridium 191 and cisplatin 195 were used to select live single cells and subsequently CD45+ cells were selected for further analysis. ( B ) Representative plots showing the expression of markers used for cell characterization/gating overlayed on viSNE plots. viSNE plots were generated by Cytobank, based on the t-SNE algorithm. Each point represents a single cell and the color gradient represents marker’s intensity (expression level). ( C , D ) Classification of CD45+ tumor infiltrated immune cell populations on viSNE plots and quantification. ( F ) Representative images and quantification of CD11b and Gr1 immunofluorescence staining for Ctrl, APP and APR tumors. Arrows indicate CD11b and Gr1 double positive cells. Scale bar, 50 μm. P-value by one-way ANOVA with Tukey’s test.

    Journal: bioRxiv

    Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

    doi: 10.1101/671198

    Figure Lengend Snippet: Characterization of infiltrating immune cell populations in responsive and resistant tumors to Ab+Pal combination treatment. ( A ) Preliminary gating for mass cytometry samples. Iridium 191 and cisplatin 195 were used to select live single cells and subsequently CD45+ cells were selected for further analysis. ( B ) Representative plots showing the expression of markers used for cell characterization/gating overlayed on viSNE plots. viSNE plots were generated by Cytobank, based on the t-SNE algorithm. Each point represents a single cell and the color gradient represents marker’s intensity (expression level). ( C , D ) Classification of CD45+ tumor infiltrated immune cell populations on viSNE plots and quantification. ( F ) Representative images and quantification of CD11b and Gr1 immunofluorescence staining for Ctrl, APP and APR tumors. Arrows indicate CD11b and Gr1 double positive cells. Scale bar, 50 μm. P-value by one-way ANOVA with Tukey’s test.

    Article Snippet: Cells were incubated with Cell-ID Cisplatin (Fluidigm, 201064) at 2.5 μM for 2.5 min for viability staining.

    Techniques: Mass Cytometry, Expressing, Generated, Marker, Immunofluorescence, Staining

    Characterization of infiltrating immune cell populations in tumors after Cabo and ICB treatment. ( A ) Preliminary gating for mass cytometry samples after Cabo and ICB treatment. Iridium 191 and cisplatin 195 were used to select live single cells and subsequently CD45+ cells were selected for further analysis. ( B ) Representative plots showing the expression of markers used for cell characterization/-gating overlayed on viSNE plots. viSNE plots were generated by Cytobank, based on the t-SNE algorithm. Each point represents a single cell and the color gradient represents marker’s intensity (expression level). ( C ) Classification of CD45+ tumor infiltrated immune cell populations on viSNE plots (left) and quantification (right).

    Journal: bioRxiv

    Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

    doi: 10.1101/671198

    Figure Lengend Snippet: Characterization of infiltrating immune cell populations in tumors after Cabo and ICB treatment. ( A ) Preliminary gating for mass cytometry samples after Cabo and ICB treatment. Iridium 191 and cisplatin 195 were used to select live single cells and subsequently CD45+ cells were selected for further analysis. ( B ) Representative plots showing the expression of markers used for cell characterization/-gating overlayed on viSNE plots. viSNE plots were generated by Cytobank, based on the t-SNE algorithm. Each point represents a single cell and the color gradient represents marker’s intensity (expression level). ( C ) Classification of CD45+ tumor infiltrated immune cell populations on viSNE plots (left) and quantification (right).

    Article Snippet: Cells were incubated with Cell-ID Cisplatin (Fluidigm, 201064) at 2.5 μM for 2.5 min for viability staining.

    Techniques: Mass Cytometry, Expressing, Generated, Marker

    Comparison of CCL2 and kidney injury molecule-1 (Kim-1) expression in the kidney ( a ) and ( b ) , Total RNA was extracted from isolated proximal tubules ( open circles ) and whole kidneys ( closed circles ) of rats treated with 5 mg/kg cisplatin. This RNA was reverse-transcribed to yield cDNA. Real-time polymerase chain reaction analysis of CCL2 ( a ) and Kim-1 ( b ) was performed using these cDNAs. GAPDH mRNA was used as an internal control. Ratio to day 0 was calculated, and data are expressed as means ± S.E. of 3–5 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Comparison of CCL2 and kidney injury molecule-1 (Kim-1) expression in the kidney ( a ) and ( b ) , Total RNA was extracted from isolated proximal tubules ( open circles ) and whole kidneys ( closed circles ) of rats treated with 5 mg/kg cisplatin. This RNA was reverse-transcribed to yield cDNA. Real-time polymerase chain reaction analysis of CCL2 ( a ) and Kim-1 ( b ) was performed using these cDNAs. GAPDH mRNA was used as an internal control. Ratio to day 0 was calculated, and data are expressed as means ± S.E. of 3–5 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    Measuring urinary and plasma CCL2 and urinary Kim-1 in nephrectomized rats treated with 2 mg/kg cisplatin CCL2 concentrations in bladder urine ( a ) or plasma ( b ) in nephrectomized rats given saline (vehicle, open circles ) or 2 mg/kg cisplatin ( closed circles ) were measured using ELISA kits. Kim-1 urinary levels in nephrectomized rats given saline (vehicle, open circles ) or 2 mg/kg cisplatin ( closed circles ] ( c ). Concentrations of urinary CCL2 and KIM-1 were normalized to urinary creatinine concentration. Plasma creatinine ( open circles ) and blood urea nitrogen levels ( closed circles ) in nephrectomized rats after administration of corn oil (vehicle, open circle ) or 2 mg/kg cisplatin ( closed circle ) are shown ( d ). Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. Data are expressed as means ± S.E. of 8 rats. **P

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Measuring urinary and plasma CCL2 and urinary Kim-1 in nephrectomized rats treated with 2 mg/kg cisplatin CCL2 concentrations in bladder urine ( a ) or plasma ( b ) in nephrectomized rats given saline (vehicle, open circles ) or 2 mg/kg cisplatin ( closed circles ) were measured using ELISA kits. Kim-1 urinary levels in nephrectomized rats given saline (vehicle, open circles ) or 2 mg/kg cisplatin ( closed circles ] ( c ). Concentrations of urinary CCL2 and KIM-1 were normalized to urinary creatinine concentration. Plasma creatinine ( open circles ) and blood urea nitrogen levels ( closed circles ) in nephrectomized rats after administration of corn oil (vehicle, open circle ) or 2 mg/kg cisplatin ( closed circle ) are shown ( d ). Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. Data are expressed as means ± S.E. of 8 rats. **P

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Cytokine and chemokine expression profiles of rats treated with 5 mg/kg cisplatin Interleukin (IL)-1beta ( a ), chemokine (C-C motif) ligand (CCL) 2 ( b ), CCL20 ( c ), chemokine (C-X-C motif) ligand (CXCL) 1 ( d ), and CXCL10 ( e ) mRNA levels were examined. Whole-kidney total RNA was extracted from rats 1, 2, 4, and 7 days after cisplatin treatment and reverse-transcribed to complementary DNA (cDNA). Real-time PCR analysis was performed using these cDNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. Data are expressed as means ± S.E. of 6 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Cytokine and chemokine expression profiles of rats treated with 5 mg/kg cisplatin Interleukin (IL)-1beta ( a ), chemokine (C-C motif) ligand (CCL) 2 ( b ), CCL20 ( c ), chemokine (C-X-C motif) ligand (CXCL) 1 ( d ), and CXCL10 ( e ) mRNA levels were examined. Whole-kidney total RNA was extracted from rats 1, 2, 4, and 7 days after cisplatin treatment and reverse-transcribed to complementary DNA (cDNA). Real-time PCR analysis was performed using these cDNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. Data are expressed as means ± S.E. of 6 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. *P

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Measuring urinary and plasma CCL2 and urinary Kim-1 in rats treated with 5 mg/kg cisplatin CCL2 concentrations in bladder urine ( open circles ) and plasma ( closed circles ) in rats given 5 mg/kg cisplatin were measured using enzyme-linked immunosorbent assay (ELISA) kits ( a ] ( b ). Concentrations of urinary CCL2 and Kim-1 were normalized to urinary creatinine concentration. Plasma creatinine ( open circles ) and blood urea nitrogen ( closed circles ) levels in rats after administration of 5 mg/kg cisplatin are shown ( c ). Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. Data are expressed as means ± S.E. of 5–7 rats. **P

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Measuring urinary and plasma CCL2 and urinary Kim-1 in rats treated with 5 mg/kg cisplatin CCL2 concentrations in bladder urine ( open circles ) and plasma ( closed circles ) in rats given 5 mg/kg cisplatin were measured using enzyme-linked immunosorbent assay (ELISA) kits ( a ] ( b ). Concentrations of urinary CCL2 and Kim-1 were normalized to urinary creatinine concentration. Plasma creatinine ( open circles ) and blood urea nitrogen ( closed circles ) levels in rats after administration of 5 mg/kg cisplatin are shown ( c ). Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way ANOVA. Data are expressed as means ± S.E. of 5–7 rats. **P

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Immunofluorescence analysis of Kim-1 in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. The kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with an antibody specific for Kim-1 ( red ), phalloidin ( green ), and DAPI ( blue ). Scale bar, 100 μm. The solid and dotted frames indicate magnifications of the cortex and medullary regions, respectively. Magnification: left panels, ×40; middle and right panels, ×400.

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Immunofluorescence analysis of Kim-1 in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. The kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with an antibody specific for Kim-1 ( red ), phalloidin ( green ), and DAPI ( blue ). Scale bar, 100 μm. The solid and dotted frames indicate magnifications of the cortex and medullary regions, respectively. Magnification: left panels, ×40; middle and right panels, ×400.

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Immunofluorescence, Staining

    Immunofluorescence analysis of CCL2 in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. Kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with an antibody specific for CCL2 ( red ), phalloidin ( green ), and 4′,6-diamidino-2-phenylindole (DAPI; blue ). Scale bar, 100 μm. The solid and dotted frames indicate magnifications of the cortex and medullary regions, respectively. Magnification: left panels, ×40; middle and right panels, ×400.

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Immunofluorescence analysis of CCL2 in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. Kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with an antibody specific for CCL2 ( red ), phalloidin ( green ), and 4′,6-diamidino-2-phenylindole (DAPI; blue ). Scale bar, 100 μm. The solid and dotted frames indicate magnifications of the cortex and medullary regions, respectively. Magnification: left panels, ×40; middle and right panels, ×400.

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Immunofluorescence, Staining

    Immunofluorescence analysis of IL-1beta (a), CCL20 (b), CXCL1 (c), and CXCL10 (d) in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. The kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with antibodies specific for IL-1beta, CCL20, CXCL1, or CXCL10. Red signals for each section were merged with green signals for phalloidin and with blue signals for DAPI. Scale bar, 100 μm. The solid frame indicates the magnification region. Magnification, ×400.

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Immunofluorescence analysis of IL-1beta (a), CCL20 (b), CXCL1 (c), and CXCL10 (d) in rats treated with cisplatin Rats treated with 5 mg/kg cisplatin were used. The kidneys were perfused, fixed with 4% paraformaldehyde, and embedded. The sections (5 μm) were stained with antibodies specific for IL-1beta, CCL20, CXCL1, or CXCL10. Red signals for each section were merged with green signals for phalloidin and with blue signals for DAPI. Scale bar, 100 μm. The solid frame indicates the magnification region. Magnification, ×400.

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Immunofluorescence, Staining

    Time-dependent changes in renal function and histology of rats treated with 5 mg/kg cisplatin The changes in creatinine clearance (Ccr), blood urea nitrogen (BUN), urinary excretion of albumin, and urinary N -acetyl-β-D-glucosaminidase (NAG) activity 1, 2, 4, and 7 days after cisplatin administration are shown ( a ). Histological analyses of the kidneys are also shown ( b ). *, glomeruli. Data are expressed as means ± standard error (S.E.) of 6 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way analysis of variance (ANOVA). * P

    Journal: Biochemical pharmacology

    Article Title: Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

    doi: 10.1016/j.bcp.2012.12.019

    Figure Lengend Snippet: Time-dependent changes in renal function and histology of rats treated with 5 mg/kg cisplatin The changes in creatinine clearance (Ccr), blood urea nitrogen (BUN), urinary excretion of albumin, and urinary N -acetyl-β-D-glucosaminidase (NAG) activity 1, 2, 4, and 7 days after cisplatin administration are shown ( a ). Histological analyses of the kidneys are also shown ( b ). *, glomeruli. Data are expressed as means ± standard error (S.E.) of 6 rats. Multiple comparisons were performed with Dunnett’s 2-tailed test after 1-way analysis of variance (ANOVA). * P

    Article Snippet: Cisplatin (0.5 mg/mL; Randa® ; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered intraperitoneally; the same volume of saline solution was injected into the sham controls.

    Techniques: Activity Assay

    Figure 1. Effect of GHRH antagonist JMR-132, 5-Fluorouracil (5-FU), irinotecan and cisplatin ( A ) or their combination ( B ) on the proliferation of HCT-116 human colon cancer cells in vitro. Cultured cells were treated for 72 h with indicated concentrations

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Figure 1. Effect of GHRH antagonist JMR-132, 5-Fluorouracil (5-FU), irinotecan and cisplatin ( A ) or their combination ( B ) on the proliferation of HCT-116 human colon cancer cells in vitro. Cultured cells were treated for 72 h with indicated concentrations

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques: In Vitro, Cell Culture

    Effect of GHRH antagonist JMR-132, 5-FU, irinotecan, cisplatin and its combinations on the in vivo growth of HT-29, HCT-116 and HCT-15 xenografted human colon cancer cells

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Effect of GHRH antagonist JMR-132, 5-FU, irinotecan, cisplatin and its combinations on the in vivo growth of HT-29, HCT-116 and HCT-15 xenografted human colon cancer cells

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques: In Vivo

    Figure 5. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Figure 5. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques:

    Figure 4. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Figure 4. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques:

    Figure 2. Effect of treatment with ( B ) GHRH antagonist JMR-132 (5 μM), ( C ) 5-FU (5 μM), ( E ) irinotecan (5 μM) or ( G ) cisplatin (5 μM) or the combinations ( D, F and H ) for 24 h on the cell cycle progression of HCT-116

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Figure 2. Effect of treatment with ( B ) GHRH antagonist JMR-132 (5 μM), ( C ) 5-FU (5 μM), ( E ) irinotecan (5 μM) or ( G ) cisplatin (5 μM) or the combinations ( D, F and H ) for 24 h on the cell cycle progression of HCT-116

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques:

    Figure 3. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Journal: Cell Cycle

    Article Title: GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

    doi: 10.4161/cc.22498

    Figure Lengend Snippet: Figure 3. Effect of GHRH antagonist, JMR-132 (daily s.c. at a dose of 10 μg/20 g bw), 5-FU (30 mg/kg bw i.p. on days 1, 8, 15, 21), irinotecan (50 mg/kg bw i.p. on days 1, 8, 15, 21), cisplatin (5 mg/kg bw i.p. on days 1, 8, 15, 21) or

    Article Snippet: 5-FU and cisplatin were obtained from APP Pharmaceuticals, LLC.

    Techniques:

    PARP1 is a target of miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A ) StarBase online predicted the binding sites of miR-770-5p and PARP1. ( B and C ) Luciferase activity was analyzed in A2780/DDP and SKOV3/DDP cells co-transfected with PARP1 3ʹUTR-WT or PARP1 3ʹUTR-MUT and miR-NC or miR-770-5p. ( D and E ) The mRNA and protein levels of PARP1 were measured in A2780/DDP and SKOV3/DDP cells transfected with miR-NC or miR-770-5p by qRT-PCR and Western blot. ( F ) The expression of PARP1 mRNA was detected in cisplatin-sensitive (n=18) and cisplatin-resistant ovarian cancer tissues (n=19) by qRT-PCR. ( G ) The linear correlation between the expression of PARP1 mRNA and miR-770-5p in ovarian cancer tissues was analyzed. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: PARP1 is a target of miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A ) StarBase online predicted the binding sites of miR-770-5p and PARP1. ( B and C ) Luciferase activity was analyzed in A2780/DDP and SKOV3/DDP cells co-transfected with PARP1 3ʹUTR-WT or PARP1 3ʹUTR-MUT and miR-NC or miR-770-5p. ( D and E ) The mRNA and protein levels of PARP1 were measured in A2780/DDP and SKOV3/DDP cells transfected with miR-NC or miR-770-5p by qRT-PCR and Western blot. ( F ) The expression of PARP1 mRNA was detected in cisplatin-sensitive (n=18) and cisplatin-resistant ovarian cancer tissues (n=19) by qRT-PCR. ( G ) The linear correlation between the expression of PARP1 mRNA and miR-770-5p in ovarian cancer tissues was analyzed. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    NEAT1 expression is enhanced in cisplatin-resistant ovarian cancer tissues and cells. ( A ) The expression of NEAT1 was measured in ovarian cancer tissues and para-tumor tissues (n=37) by qRT-PCR. ( B ) The level of NEAT1 was detected in cisplatin-sensitive (n=18) and cisplatin-resistant tissues (n=19) by qRT-PCR. ( C ) The expression of NEAT1 was examined in cisplatin-sensitive ovarian cancer cells (A2780 and SKOV3) and cisplatin-resistant cells (A2780/DDP and SKOV3/DDP) by qRT-PCR. ( D and E ) The cell viability and IC50 of cisplatin (DDP) were analyzed in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells after treatment of different concentrations of cisplatin for 48 h by MTT. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: NEAT1 expression is enhanced in cisplatin-resistant ovarian cancer tissues and cells. ( A ) The expression of NEAT1 was measured in ovarian cancer tissues and para-tumor tissues (n=37) by qRT-PCR. ( B ) The level of NEAT1 was detected in cisplatin-sensitive (n=18) and cisplatin-resistant tissues (n=19) by qRT-PCR. ( C ) The expression of NEAT1 was examined in cisplatin-sensitive ovarian cancer cells (A2780 and SKOV3) and cisplatin-resistant cells (A2780/DDP and SKOV3/DDP) by qRT-PCR. ( D and E ) The cell viability and IC50 of cisplatin (DDP) were analyzed in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells after treatment of different concentrations of cisplatin for 48 h by MTT. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Expressing, Quantitative RT-PCR, MTT Assay

    Knockdown of miR-770-5p reverses the suppressive effect of NEAT1 silencing on cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A and B ) The expression of miR-770-5p was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by qRT-PCR. ( C and D ) Cell viability and IC50 of cisplatin (DDP) were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p after treatment of cisplatin for 48 h by MTT. ( E and F ) Cell viability was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 0, 24, 48 and 72 h by MTT. ( G and H ) Cell apoptosis was detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 72 h by flow cytometry. ( I and J ) The protein levels of Bcl-2, Bax and Cleaved-casp-3 were examined in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 72 h by Western blot. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: Knockdown of miR-770-5p reverses the suppressive effect of NEAT1 silencing on cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A and B ) The expression of miR-770-5p was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by qRT-PCR. ( C and D ) Cell viability and IC50 of cisplatin (DDP) were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p after treatment of cisplatin for 48 h by MTT. ( E and F ) Cell viability was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 0, 24, 48 and 72 h by MTT. ( G and H ) Cell apoptosis was detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 72 h by flow cytometry. ( I and J ) The protein levels of Bcl-2, Bax and Cleaved-casp-3 were examined in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p at 72 h by Western blot. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Western Blot

    NEAT1 is a decoy of miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A ) The binding sites of NEAT1 and miR-770-5p were predicted by starBase. ( B and C ) Luciferase activity was measured in A2780/DDP and SKOV3/DDP cells co-transfected with WT-NEAT1 or MUT-NEAT1 and miR-NC or miR-770-5p. ( D ) The expression of miR-770-5p was measured in cisplatin-sensitive (n=18) and cisplatin-resistant ovarian cancer tissues (n=19) by qRT-PCR. ( E ) The expression of miR-770-5p was detected in A2780, SKOV3, A2780/DDP and SKOV3/DDP cells by qRT-PCR. ( F ) The linear association between the expression levels of miR-770-5p and NEAT1 in ovarian cancer tissues was assessed. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: NEAT1 is a decoy of miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A ) The binding sites of NEAT1 and miR-770-5p were predicted by starBase. ( B and C ) Luciferase activity was measured in A2780/DDP and SKOV3/DDP cells co-transfected with WT-NEAT1 or MUT-NEAT1 and miR-NC or miR-770-5p. ( D ) The expression of miR-770-5p was measured in cisplatin-sensitive (n=18) and cisplatin-resistant ovarian cancer tissues (n=19) by qRT-PCR. ( E ) The expression of miR-770-5p was detected in A2780, SKOV3, A2780/DDP and SKOV3/DDP cells by qRT-PCR. ( F ) The linear association between the expression levels of miR-770-5p and NEAT1 in ovarian cancer tissues was assessed. The difference was compared with the indicated control group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR

    Silencing of NEAT1 decreases PARP1 expression by regulating miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A and B ) The mRNA level of PARP1 was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by qRT-PCR. ( C and D ) The expression of PARP1 protein was detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by Western blot. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: Silencing of NEAT1 decreases PARP1 expression by regulating miR-770-5p in cisplatin-resistant ovarian cancer cells. ( A and B ) The mRNA level of PARP1 was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by qRT-PCR. ( C and D ) The expression of PARP1 protein was detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and anti-NC or anti-miR-770-5p by Western blot. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot

    Knockdown of NEAT1 inhibits cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A ) The abundance of NEAT1 was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #1, #2 or #3 by qRT-PCR. ( B and C ) Cell viability and IC50 of cisplatin (DDP) were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 after stimulation of different concentrations of cisplatin for 48 h by MTT. ( D and E ) Cell viability was determined in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 at 0, 24, 48 and 72 h by MTT. ( F and G ) The apoptotic rate of A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 was measured at 72 h by flow cytometry. Markers: Annexin V-FITC and PI. ( H and I ) The protein levels of Bcl-2, Bax and Cleaved-casp-3 were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 at 72 h by Western blot. The difference was compared with si-NC group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: Knockdown of NEAT1 inhibits cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A ) The abundance of NEAT1 was measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #1, #2 or #3 by qRT-PCR. ( B and C ) Cell viability and IC50 of cisplatin (DDP) were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 after stimulation of different concentrations of cisplatin for 48 h by MTT. ( D and E ) Cell viability was determined in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 at 0, 24, 48 and 72 h by MTT. ( F and G ) The apoptotic rate of A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 was measured at 72 h by flow cytometry. Markers: Annexin V-FITC and PI. ( H and I ) The protein levels of Bcl-2, Bax and Cleaved-casp-3 were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC or si-NEAT1 #2 at 72 h by Western blot. The difference was compared with si-NC group and analyzed via Student’s t -test or ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Transfection, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Western Blot

    Restoration of PARP1 attenuates the inhibitive effect of NEAT1 knockdown on cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A-D ) The expression levels of PARP1 mRNA and protein were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by qRT-PCR and Western blot. ( E and F ) Cell viability and IC50 of cisplatin (DDP) were measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 after treatment of cisplatin for 48 h by MTT. ( G and H ) Cell viability, ( I and J ) apoptosis and ( K and L ) protein levels of Bcl-2, Bax and Cleaved-casp-3 were examined in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by MTT, flow cytometry and Western blot, respectively. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Journal: Cancer Management and Research

    Article Title: NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis

    doi: 10.2147/CMAR.S257311

    Figure Lengend Snippet: Restoration of PARP1 attenuates the inhibitive effect of NEAT1 knockdown on cisplatin resistance in cisplatin-resistant ovarian cancer cells. ( A-D ) The expression levels of PARP1 mRNA and protein were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by qRT-PCR and Western blot. ( E and F ) Cell viability and IC50 of cisplatin (DDP) were measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 after treatment of cisplatin for 48 h by MTT. ( G and H ) Cell viability, ( I and J ) apoptosis and ( K and L ) protein levels of Bcl-2, Bax and Cleaved-casp-3 were examined in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by MTT, flow cytometry and Western blot, respectively. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. * P

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide (MTT) AssayFor detection of the IC50 of cisplatin, cells were incubated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 and 38.4 μg/mL) of cisplatin for 48 h. Then cells were incubated in fresh RPMI-1640 medium with 0.5 mg/mL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, cell medium was removed and the formed crystal was resolved in dimethyl sulfoxide (Solarbio).

    Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, MTT Assay, Flow Cytometry

    The time profile of platinum remaining in the tumor after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Values are expressed as mean ± STD ( n = 6).

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: The time profile of platinum remaining in the tumor after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Values are expressed as mean ± STD ( n = 6).

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Injection

    In vitro inhibition effect of cisplatin on MBT cells. Cisplatin at increasing concentrations was added 24 hours after cell incubation in wells.

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: In vitro inhibition effect of cisplatin on MBT cells. Cisplatin at increasing concentrations was added 24 hours after cell incubation in wells.

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: In Vitro, Inhibition, Incubation

    Histology of the MBT tumor injected intratumorally with cisplatin/polymer formulation (1% w/w, 50 μ L). (a) Magnification ×10, panoramic view of the slice; (b) magnification ×40, enlargement of the cisplatin/polymer region and the surrounding necrotic area; (c) magnification ×40, enlargement of the border between the end of the necrotic area and start of the intact tumor area; (d) magnification ×40, enlargement of the intact tumor area beyond the effect of cisplatin. The polymeric formulation is assigned with the star (∗), the necrotic tissue with the white circle ( ⚪ ), the infiltration of the inflammation cells with a black circle (•), and the intact tumor cells with a black rhomb (♦).

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: Histology of the MBT tumor injected intratumorally with cisplatin/polymer formulation (1% w/w, 50 μ L). (a) Magnification ×10, panoramic view of the slice; (b) magnification ×40, enlargement of the cisplatin/polymer region and the surrounding necrotic area; (c) magnification ×40, enlargement of the border between the end of the necrotic area and start of the intact tumor area; (d) magnification ×40, enlargement of the intact tumor area beyond the effect of cisplatin. The polymeric formulation is assigned with the star (∗), the necrotic tissue with the white circle ( ⚪ ), the infiltration of the inflammation cells with a black circle (•), and the intact tumor cells with a black rhomb (♦).

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Injection

    Macroscopic view of frozen tumor tissues at cryostat sectioning: (a) tumor treated with cisplatin/polymer formulation and excised 3 days after injection; (b) tumor treated with cisplatin/polymer formulation and excised 7 days after injection; (c) tumor treated with the blank polymer and excised 3 days after injection; (d) untreated tumor. The polymeric formulation is assigned with the star (∗), the necrotic tissue with the white circle ( ⚪ ), the infiltration of the inflammation cells with a black circle (•), and the intact tumor cells with a black rhomb (♦).

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: Macroscopic view of frozen tumor tissues at cryostat sectioning: (a) tumor treated with cisplatin/polymer formulation and excised 3 days after injection; (b) tumor treated with cisplatin/polymer formulation and excised 7 days after injection; (c) tumor treated with the blank polymer and excised 3 days after injection; (d) untreated tumor. The polymeric formulation is assigned with the star (∗), the necrotic tissue with the white circle ( ⚪ ), the infiltration of the inflammation cells with a black circle (•), and the intact tumor cells with a black rhomb (♦).

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Injection

    Effect of cisplatin on MBT tumor growth in s.c. implanted mice ( n = 10). Cisplatin 1% w/w in polymer, 50 μ L (∗: solid line); blank polymer (■, dashed line); cisplatin solution (0.1% w/v in saline, 100 μ L injection, 5 mg/kg) (∆, dashed line) were injected intratumorally. No treatment group is designated as (♦) with solid line, and the group treated IP with cisplatin solution (1% w/v in saline, 100 μ L injection, 5 mg/kg) is designated as (■) with solid line. The tumor volume is expressed as mean ± STD. Statistically significant differences between the groups are signed with a star ( P

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: Effect of cisplatin on MBT tumor growth in s.c. implanted mice ( n = 10). Cisplatin 1% w/w in polymer, 50 μ L (∗: solid line); blank polymer (■, dashed line); cisplatin solution (0.1% w/v in saline, 100 μ L injection, 5 mg/kg) (∆, dashed line) were injected intratumorally. No treatment group is designated as (♦) with solid line, and the group treated IP with cisplatin solution (1% w/v in saline, 100 μ L injection, 5 mg/kg) is designated as (■) with solid line. The tumor volume is expressed as mean ± STD. Statistically significant differences between the groups are signed with a star ( P

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Mouse Assay, Injection

    Cisplatin tumor tissue distribution after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Each curve represents a different time point when the mice were sacrificed and their tumors processed. Values are expressed as mean ( n = 6).

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: Cisplatin tumor tissue distribution after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Each curve represents a different time point when the mice were sacrificed and their tumors processed. Values are expressed as mean ( n = 6).

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Injection, Mouse Assay

    In vitro cumulative release of Pt from P(SA : RA)2 : 8 (triangles, dashed line) and P(SA : RA)3 : 7 (stars, solid line) loaded with 5% w/w cisplatin. Each point represents the mean value ± STD ( n = 3). Release was conducted in 0.1 M phosphate buffer, pH 7.4, at 37°C. Pt concentrations were determined by ICP-MS.

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: In vitro cumulative release of Pt from P(SA : RA)2 : 8 (triangles, dashed line) and P(SA : RA)3 : 7 (stars, solid line) loaded with 5% w/w cisplatin. Each point represents the mean value ± STD ( n = 3). Release was conducted in 0.1 M phosphate buffer, pH 7.4, at 37°C. Pt concentrations were determined by ICP-MS.

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: In Vitro, Mass Spectrometry

    The time profile of platinum maximal concentration ( C max ) in the tumor tissue after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Values are expressed as mean ( n = 6).

    Journal: Chemotherapy Research and Practice

    Article Title: Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

    doi: 10.1155/2011/175054

    Figure Lengend Snippet: The time profile of platinum maximal concentration ( C max ) in the tumor tissue after intratumoral injection of cisplatin/polymer formulation (1% w/w, 50 μ L). Values are expressed as mean ( n = 6).

    Article Snippet: Cisplatin was purchased from AlfaAesar (MA, USA).

    Techniques: Concentration Assay, Injection

    Sox2 inhibits cisplatin-induced apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Sox2 inhibits cisplatin-induced apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P

    Article Snippet: The cisplatin-resistant A549/DDP cell line was purchased from the Bank of Cancer Cell Lines of the Chinese Academy of Medical Science (Beijing, China) and its drug resistance phenotype was maintained in a medium containing 10 nM cisplatin (Cayman Chemical Company, Ann Arbor, MI, USA).

    Techniques: Transfection, Plasmid Preparation, Expressing, Cell Culture, MTT Assay, Transduction, Over Expression, Inhibition, shRNA

    Apoptosis associated proteins determined by an immunoblotting analysis. A549 and A549/DDP cells were transfected with plasmid expressing Sox2 or shSox2, or control pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for additional 24 h prior to being harvested for immunoblotting analysis for indicated proteins. The values labeled on the top of each bands represented the relative expression levels of proteins over their respective pcDNA3.1 control as determined by a densitometric assay. Overexpression of Sox2 demonstrated a trend to reduce the expression of pro-apoptotic proteins (caspase-3, Bax), but increased the expression of anti-apoptotic proteins Bcl-2 in lung cancer cells. Cas 3: caspase-3; Bax, Bcl-2-like protein 4; AIF, apoptosis inducing factor; Bcl-2, B-cell lymphoma 2; Mcl-1, myeloid cell leukemia sequence 1 protein; C, control; Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA.

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Apoptosis associated proteins determined by an immunoblotting analysis. A549 and A549/DDP cells were transfected with plasmid expressing Sox2 or shSox2, or control pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for additional 24 h prior to being harvested for immunoblotting analysis for indicated proteins. The values labeled on the top of each bands represented the relative expression levels of proteins over their respective pcDNA3.1 control as determined by a densitometric assay. Overexpression of Sox2 demonstrated a trend to reduce the expression of pro-apoptotic proteins (caspase-3, Bax), but increased the expression of anti-apoptotic proteins Bcl-2 in lung cancer cells. Cas 3: caspase-3; Bax, Bcl-2-like protein 4; AIF, apoptosis inducing factor; Bcl-2, B-cell lymphoma 2; Mcl-1, myeloid cell leukemia sequence 1 protein; C, control; Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA.

    Article Snippet: The cisplatin-resistant A549/DDP cell line was purchased from the Bank of Cancer Cell Lines of the Chinese Academy of Medical Science (Beijing, China) and its drug resistance phenotype was maintained in a medium containing 10 nM cisplatin (Cayman Chemical Company, Ann Arbor, MI, USA).

    Techniques: Transfection, Plasmid Preparation, Expressing, Cell Culture, Labeling, Over Expression, Sequencing, shRNA

    CSE recovers CD11b levels in cisplatin-treated mice. Cisplatin was administered at 5 mg/kg/day for 4 doses in the absence or presence of CSE (9.6 mL/kg/day) for 7 days. Immunohistochemical analysis was conducted with anti-CD11b to analyze spleen and observed by a microscope at 40x (a–c) and 200x (d–f) magnification; bar, 50 μ m. Shown are representative images ( n = 5‐8 per group).

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE recovers CD11b levels in cisplatin-treated mice. Cisplatin was administered at 5 mg/kg/day for 4 doses in the absence or presence of CSE (9.6 mL/kg/day) for 7 days. Immunohistochemical analysis was conducted with anti-CD11b to analyze spleen and observed by a microscope at 40x (a–c) and 200x (d–f) magnification; bar, 50 μ m. Shown are representative images ( n = 5‐8 per group).

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Mouse Assay, Immunohistochemistry, Microscopy

    CSE reduces apoptosis in cisplatin-treated cells. The HL-60 and THP-1 cells were cultured with 4 μ M and 2 μ M cisplatin, respectively, with or without CSE (25, 50, and 100 μ g/mL) for 72 h. After staining with Annexin V and propidium iodide, the apoptotic cells were analyzed by flow cytometry (a), and the percentages of early apoptotic (top left quadrant) and late apoptotic (top right quadrant) cells were calculated (b). Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ### P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE reduces apoptosis in cisplatin-treated cells. The HL-60 and THP-1 cells were cultured with 4 μ M and 2 μ M cisplatin, respectively, with or without CSE (25, 50, and 100 μ g/mL) for 72 h. After staining with Annexin V and propidium iodide, the apoptotic cells were analyzed by flow cytometry (a), and the percentages of early apoptotic (top left quadrant) and late apoptotic (top right quadrant) cells were calculated (b). Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ### P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Cell Culture, Staining, Flow Cytometry

    CSE diminishes cisplatin-induced caspase-3 and PARP activation. HL-60 cells were treated with CSE (100 μ g/mL) and/or cisplatin (4 μ M) for 48 h. Whole cell lysates were collected and subject to western blot analysis for the indicated proteins. Representative images were shown for cleaved caspase-3 and cleaved PARP. β -Actin was used as an internal control. Quantification of blots was performed by using ImageJ, and the fold changes to untreated controls are presented.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE diminishes cisplatin-induced caspase-3 and PARP activation. HL-60 cells were treated with CSE (100 μ g/mL) and/or cisplatin (4 μ M) for 48 h. Whole cell lysates were collected and subject to western blot analysis for the indicated proteins. Representative images were shown for cleaved caspase-3 and cleaved PARP. β -Actin was used as an internal control. Quantification of blots was performed by using ImageJ, and the fold changes to untreated controls are presented.

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Activation Assay, Western Blot

    Effect of CSE and cisplatin on myeloid cell proliferation. The HL-60 and THP-1 were cultured in CSE (a) or cisplatin (b) at indicated concentrations for 72 h. Cell viability was measured using the Alamar Blue assay. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ∗∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: Effect of CSE and cisplatin on myeloid cell proliferation. The HL-60 and THP-1 were cultured in CSE (a) or cisplatin (b) at indicated concentrations for 72 h. Cell viability was measured using the Alamar Blue assay. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ∗∗∗ P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Cell Culture, Alamar Blue Assay

    CSE restores hematopoietic progenitors in mice after cisplatin administration. One day after the last cisplatin injection, bone marrow (a) and spleen (b) cells were cultured for respective 9 and 13 days, and the colony number of CFU-GM per bone marrow and spleen was counted. Data are represented as mean ± SEM ( n = 4 per group). The significance of the data was analyzed by one-way ANOVA with post hoc Dunnett's test. # P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE restores hematopoietic progenitors in mice after cisplatin administration. One day after the last cisplatin injection, bone marrow (a) and spleen (b) cells were cultured for respective 9 and 13 days, and the colony number of CFU-GM per bone marrow and spleen was counted. Data are represented as mean ± SEM ( n = 4 per group). The significance of the data was analyzed by one-way ANOVA with post hoc Dunnett's test. # P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Mouse Assay, Injection, Cell Culture

    Proposed molecular mechanism for the action of CSE on cisplatin toxicity in human promyelocytic cells. Cisplatin treatment results in mitochondrial damage, release of cytochrome c from mitochondria, activation of caspase-3 and PARP, and apoptosis in HL-60 cells. Upon cisplatin exposure, CSE can preserve mitochondrial mass, reduce cytochrome c release, suppress caspase-3 and PARP activation, and consequently promote cell survival. Ctr1: copper transporter 1; OCT2: organic cation transporter 2; Cyt c: cytochrome c; Casp 3: caspase-3; Casp 9: caspase-9.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: Proposed molecular mechanism for the action of CSE on cisplatin toxicity in human promyelocytic cells. Cisplatin treatment results in mitochondrial damage, release of cytochrome c from mitochondria, activation of caspase-3 and PARP, and apoptosis in HL-60 cells. Upon cisplatin exposure, CSE can preserve mitochondrial mass, reduce cytochrome c release, suppress caspase-3 and PARP activation, and consequently promote cell survival. Ctr1: copper transporter 1; OCT2: organic cation transporter 2; Cyt c: cytochrome c; Casp 3: caspase-3; Casp 9: caspase-9.

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Activation Assay

    Effect of CSE on mice receiving cisplatin. (a) Body weight changes after cisplatin treatment. Mice were administered with CSE (9.6 mL/kg/day) or distilled water (CIS+vehicle) by gavage for 7 days in the i.p. injection of cisplatin (5 mg/kg/day) on days 1, 3, 5, and 7. Data are represented as mean ± SEM ( n = 8 per group). The significance of the data was analyzed by one-way ANOVA with post hoc Dunnett's test. ∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: Effect of CSE on mice receiving cisplatin. (a) Body weight changes after cisplatin treatment. Mice were administered with CSE (9.6 mL/kg/day) or distilled water (CIS+vehicle) by gavage for 7 days in the i.p. injection of cisplatin (5 mg/kg/day) on days 1, 3, 5, and 7. Data are represented as mean ± SEM ( n = 8 per group). The significance of the data was analyzed by one-way ANOVA with post hoc Dunnett's test. ∗∗ P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Mouse Assay, Injection

    CSE reserves mitochondrial mass. HL-60 cells were pretreated with 100 μ M CSE for 30 min followed by incubation with 4 μ M of cisplatin for 24 h. Cells were stained with 500 nM of MitoTracker Green FM for 45 min, and the fluorescence of cell extracts was measured using a fluorescence plate reader. Data are the means ± SEM ( n = 3). ## P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE reserves mitochondrial mass. HL-60 cells were pretreated with 100 μ M CSE for 30 min followed by incubation with 4 μ M of cisplatin for 24 h. Cells were stained with 500 nM of MitoTracker Green FM for 45 min, and the fluorescence of cell extracts was measured using a fluorescence plate reader. Data are the means ± SEM ( n = 3). ## P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Incubation, Staining, Fluorescence

    CSE protects normal myeloid cells from cisplatin-induced toxicity. (a) Mouse bone marrow cells were incubated with CSE (10, 100, and 500 μ g/mL) for 7 days. The colony number of CFU-GM per bone marrow was counted. (b) Mouse PBMCs were cultured in cisplatin at 4 μ M, with or without CSE (25, 50, and 100 μ g/mL) for 7 days. Cell morphology was recorded by a bright-field phase-contrast microscopy. (c) Cell viability was measured using the CyQuant direct cell proliferation assay. (d) Mice were i.p. injected with three doses of cisplatin (5 mg/kg/day) for 3 days and received CSE at indicated concentrations by oral gavage for 7 days. Bone marrow cells were cultured in growth medium for 7 days. The colony number of CFU-GM per bone marrow was counted. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. # P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE protects normal myeloid cells from cisplatin-induced toxicity. (a) Mouse bone marrow cells were incubated with CSE (10, 100, and 500 μ g/mL) for 7 days. The colony number of CFU-GM per bone marrow was counted. (b) Mouse PBMCs were cultured in cisplatin at 4 μ M, with or without CSE (25, 50, and 100 μ g/mL) for 7 days. Cell morphology was recorded by a bright-field phase-contrast microscopy. (c) Cell viability was measured using the CyQuant direct cell proliferation assay. (d) Mice were i.p. injected with three doses of cisplatin (5 mg/kg/day) for 3 days and received CSE at indicated concentrations by oral gavage for 7 days. Bone marrow cells were cultured in growth medium for 7 days. The colony number of CFU-GM per bone marrow was counted. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. # P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Incubation, Cell Culture, Microscopy, CyQUANT Assay, Proliferation Assay, Mouse Assay, Injection

    CSE protects cells from cisplatin-induced cytotoxicity. The HL-60 (a) and THP-1 (b) were cultured in cisplatin at 4 μ M and 2 μ M, respectively, with or without CSE (25, 50, and 100 μ g/mL) for 72 h. Cell viability was measured using the Alamar Blue assay. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ### P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE protects cells from cisplatin-induced cytotoxicity. The HL-60 (a) and THP-1 (b) were cultured in cisplatin at 4 μ M and 2 μ M, respectively, with or without CSE (25, 50, and 100 μ g/mL) for 72 h. Cell viability was measured using the Alamar Blue assay. Data are expressed as mean ± SEM ( n = 3). Differences among groups were analyzed by one-way ANOVA and post hoc Dunnett's test. ### P

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Cell Culture, Alamar Blue Assay

    Study scheme of cisplatin and CSE treatment. Mice were divided into 3 groups: (i) control group: 10 mL/kg/day of 5% Dextrose by i.p. injection on days 1, 3, 5, and 7; distilled water (10 mL/kg/day) by oral gavage on days 1-7. (ii) Cisplatin group: cisplatin (5 mg/kg/day) by i.p. injection on days 1, 3, 5, and 7; distilled water (10 mL/kg/day) by oral gavage on days 1-7. (iii) CSE groups: cisplatin (5 mg/kg/day) by i.p. injection on days 1, 3, 5, and 7; CSE (9.6 mL/kg/day) by oral gavage on days 1-7.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: Study scheme of cisplatin and CSE treatment. Mice were divided into 3 groups: (i) control group: 10 mL/kg/day of 5% Dextrose by i.p. injection on days 1, 3, 5, and 7; distilled water (10 mL/kg/day) by oral gavage on days 1-7. (ii) Cisplatin group: cisplatin (5 mg/kg/day) by i.p. injection on days 1, 3, 5, and 7; distilled water (10 mL/kg/day) by oral gavage on days 1-7. (iii) CSE groups: cisplatin (5 mg/kg/day) by i.p. injection on days 1, 3, 5, and 7; CSE (9.6 mL/kg/day) by oral gavage on days 1-7.

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Mouse Assay, Injection

    CSE alleviates cisplatin-elicited mitochondrial damage. HL-60 cells were treated with CSE (100 μ g/mL) and/or cisplatin (4 μ M) for 48 h. The expression levels of cytochrome c, TOM20, COX IV, and HSP70 were examined in the cytosolic and mitochondrial fractions by western blot analysis. GAPDH was used as an internal control. Quantification of blots was performed by using ImageJ, and the fold changes to untreated control are presented. C: cytosolic fraction; M: mitochondrial fraction.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo

    doi: 10.1155/2020/7353618

    Figure Lengend Snippet: CSE alleviates cisplatin-elicited mitochondrial damage. HL-60 cells were treated with CSE (100 μ g/mL) and/or cisplatin (4 μ M) for 48 h. The expression levels of cytochrome c, TOM20, COX IV, and HSP70 were examined in the cytosolic and mitochondrial fractions by western blot analysis. GAPDH was used as an internal control. Quantification of blots was performed by using ImageJ, and the fold changes to untreated control are presented. C: cytosolic fraction; M: mitochondrial fraction.

    Article Snippet: Cisplatin was purchased from Fresenius Kabi Oncology (Haryana, India).

    Techniques: Expressing, Western Blot

    Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the whole cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Journal: Therapeutic Advances in Medical Oncology

    Article Title: Treatment effects of cumulative cisplatin dose during radiotherapy following induction chemotherapy in nasopharyngeal carcinoma: propensity score analyses

    doi: 10.1177/1758835920937424

    Figure Lengend Snippet: Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the whole cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Article Snippet: – The cumulative cisplatin dose (CCD) during radiotherapy (RT) is an important prognostic factor in NPC.

    Techniques:

    Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the high-risk cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Journal: Therapeutic Advances in Medical Oncology

    Article Title: Treatment effects of cumulative cisplatin dose during radiotherapy following induction chemotherapy in nasopharyngeal carcinoma: propensity score analyses

    doi: 10.1177/1758835920937424

    Figure Lengend Snippet: Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the high-risk cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Article Snippet: – The cumulative cisplatin dose (CCD) during radiotherapy (RT) is an important prognostic factor in NPC.

    Techniques:

    Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the low-risk cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Journal: Therapeutic Advances in Medical Oncology

    Article Title: Treatment effects of cumulative cisplatin dose during radiotherapy following induction chemotherapy in nasopharyngeal carcinoma: propensity score analyses

    doi: 10.1177/1758835920937424

    Figure Lengend Snippet: Kaplan–Meier curves for overall survival ((a), (b)), distant metastasis-free survival ((c), (d)), and locoregional recurrence-free survival ((e), (f)) before and after weighting in the low-risk cohort. CCD, cumulative cisplatin dose (mg/m 2 ); IPTW, inverse probability of treatment weighting.

    Article Snippet: – The cumulative cisplatin dose (CCD) during radiotherapy (RT) is an important prognostic factor in NPC.

    Techniques:

    Pairwise plots assessing the balance of baseline characteristics among CCD groups for the whole ((a)–(c)), low-risk ((d)–(f)), and high-risk ((g)–(i)) cohorts. CCD, cumulative cisplatin dose (mg/m 2 ).

    Journal: Therapeutic Advances in Medical Oncology

    Article Title: Treatment effects of cumulative cisplatin dose during radiotherapy following induction chemotherapy in nasopharyngeal carcinoma: propensity score analyses

    doi: 10.1177/1758835920937424

    Figure Lengend Snippet: Pairwise plots assessing the balance of baseline characteristics among CCD groups for the whole ((a)–(c)), low-risk ((d)–(f)), and high-risk ((g)–(i)) cohorts. CCD, cumulative cisplatin dose (mg/m 2 ).

    Article Snippet: – The cumulative cisplatin dose (CCD) during radiotherapy (RT) is an important prognostic factor in NPC.

    Techniques:

    Forest plots showing results of univariate Cox regression for the weighted whole and subgroup cohorts. CCD, cumulative cisplatin dose (mg/m 2 ); CI, confidence interval; DMFS, distant metastasis-free survival; HR, hazard ratio; LRFS, locoregional recurrence-free survival; OS, overall survival.

    Journal: Therapeutic Advances in Medical Oncology

    Article Title: Treatment effects of cumulative cisplatin dose during radiotherapy following induction chemotherapy in nasopharyngeal carcinoma: propensity score analyses

    doi: 10.1177/1758835920937424

    Figure Lengend Snippet: Forest plots showing results of univariate Cox regression for the weighted whole and subgroup cohorts. CCD, cumulative cisplatin dose (mg/m 2 ); CI, confidence interval; DMFS, distant metastasis-free survival; HR, hazard ratio; LRFS, locoregional recurrence-free survival; OS, overall survival.

    Article Snippet: – The cumulative cisplatin dose (CCD) during radiotherapy (RT) is an important prognostic factor in NPC.

    Techniques:

    Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by cisplatin treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Single cell mass cytometric immunophenotyping showed the accumulation of CD11b+ myeloid cells in 4T1 tumor bearing animals at the expense of lymphoid subsets which was reverted by cisplatin treatment. The visualization of stochastic neighbor embedding (viSNE) analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets were performed in the spleen of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) and its decrease by cisplatin treatment ( F ) vs. ( E ) has statistical significance at p

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Mouse Assay

    The viSNE plots illustrate the expression intensity of Gr-1, CD44, IL-17A, B220, CD62L markers within the clouds of main subsets defined in Figure 4 in the splenic samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional to the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not show differential expression are not shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The viSNE plots illustrate the expression intensity of Gr-1, CD44, IL-17A, B220, CD62L markers within the clouds of main subsets defined in Figure 4 in the splenic samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional to the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not show differential expression are not shown.

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Expressing, Mouse Assay

    The trajectories on the radar plots delineate the characteristic marker profile of splenocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of splenic ( A ) CD11b+/Gr-1+ MDSCs, ( B ) CD44+, and in a smaller extent ( C ) IL-17A+ MDSCs is a characteristic of 4T1 breast cancer. Cisplatin restores the percentage of ( D ) B220+ and ( E ) CD62L+ B-cells, ( F ) CD62L+ CD4+ and ( G ) CD8+ T-cells. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text and in Figure S3 . The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The trajectories on the radar plots delineate the characteristic marker profile of splenocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of splenic ( A ) CD11b+/Gr-1+ MDSCs, ( B ) CD44+, and in a smaller extent ( C ) IL-17A+ MDSCs is a characteristic of 4T1 breast cancer. Cisplatin restores the percentage of ( D ) B220+ and ( E ) CD62L+ B-cells, ( F ) CD62L+ CD4+ and ( G ) CD8+ T-cells. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text and in Figure S3 . The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Marker, Mouse Assay, Expressing

    Real-time monitoring of 4T1 cell viability hampered by cisplatin. The 4T1 cells were seeded and the baseline impedance was recorded for 48 h. After that 48 h culturing treatment with 11.111 µM, 33.333 µM, or 100 µM cisplatin reduced viability of 4T1 cells on a time and dose dependent manner. The corresponding dose-response curves with the half maximal inhibitory concentration (IC 50 ) values can be found in Figure S1 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Real-time monitoring of 4T1 cell viability hampered by cisplatin. The 4T1 cells were seeded and the baseline impedance was recorded for 48 h. After that 48 h culturing treatment with 11.111 µM, 33.333 µM, or 100 µM cisplatin reduced viability of 4T1 cells on a time and dose dependent manner. The corresponding dose-response curves with the half maximal inhibitory concentration (IC 50 ) values can be found in Figure S1 .

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Concentration Assay

    The proteolytic activity of the fibroblast activator protein (FAP) increased by the formation of breast cancer, and was significantly decreased by cisplatin treatment. Area under the curve (AUC) values from high pressure liquid chromatography (HPLC) analysis of the peptide digestion product 2 ( Figure S2 ) were plotted. The results are shown as arithmetic mean values of the samples ± standard error of the mean (SEM), statistical significance was set to *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The proteolytic activity of the fibroblast activator protein (FAP) increased by the formation of breast cancer, and was significantly decreased by cisplatin treatment. Area under the curve (AUC) values from high pressure liquid chromatography (HPLC) analysis of the peptide digestion product 2 ( Figure S2 ) were plotted. The results are shown as arithmetic mean values of the samples ± standard error of the mean (SEM), statistical significance was set to *** p

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Activity Assay, High Performance Liquid Chromatography

    Cisplatin treatment reduced 4T1 tumor growth, the number of lung metastatic nodules and the weight of the spleen. The 4T1 cells (1.2 × 10 5 ) were transplanted by the injection into the mammary fat pad of BALB/c mice ( n = 12). Tumor growth was monitored daily ( A ). On the 23rd day mice were euthanized and the weight of the tumors ( B ), the number of metastatic nodules on the lungs ( C ), and the weight of the spleens were measured ( D ). Individual values and arithmetic mean values of the samples ± standard error of the mean (SEM) are plotted, statistical significance was set to *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Cisplatin treatment reduced 4T1 tumor growth, the number of lung metastatic nodules and the weight of the spleen. The 4T1 cells (1.2 × 10 5 ) were transplanted by the injection into the mammary fat pad of BALB/c mice ( n = 12). Tumor growth was monitored daily ( A ). On the 23rd day mice were euthanized and the weight of the tumors ( B ), the number of metastatic nodules on the lungs ( C ), and the weight of the spleens were measured ( D ). Individual values and arithmetic mean values of the samples ± standard error of the mean (SEM) are plotted, statistical significance was set to *** p

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Injection, Mouse Assay

    The viSNE plots illustrate the expression intensity of Gr-1, CD44, and IFN-γ markers within the clouds of main subsets defined in Figure 7 in the blood samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional with the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in the Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not showed different expression are not shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The viSNE plots illustrate the expression intensity of Gr-1, CD44, and IFN-γ markers within the clouds of main subsets defined in Figure 7 in the blood samples of naive, 4T1 tumor bearing, and cisplatin treated 4T1 tumorous mice. The coloration is proportional with the expression intensity (blue = low, red = high). The list of the antibodies can be found in Table 1 in the Section 4.5 . Representative viSNE plots are shown from the pooled samples of 6 mice per group. The markers of the panel which were not detected or did not showed different expression are not shown.

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Expressing, Mouse Assay

    The trajectories on the radar plots delineate the characteristic marker profile of blood-derived leukocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of peripheral ( A ) CD11b+/Gr-1+ MDSCs and ( B ) CD44+ MDSCs is a characteristic of 4T1 breast cancer. ( C ) Due to cisplatin treatment IFN-γ+ MDSCs were developed at the periphery. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text. The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: The trajectories on the radar plots delineate the characteristic marker profile of blood-derived leukocytes in naive, 4T1 tumor bearing, and cisplatin treated tumorous mice. The accumulation of peripheral ( A ) CD11b+/Gr-1+ MDSCs and ( B ) CD44+ MDSCs is a characteristic of 4T1 breast cancer. ( C ) Due to cisplatin treatment IFN-γ+ MDSCs were developed at the periphery. The percentage of the given populations is demonstrated on the radar plots within the CD45+ living singlets determined by manual gating in Cytobank. The gating hierarchy is explained in the text. The markers of the panel which were not detected or did not show differential expression are not shown. Representative radar plots are shown from the pooled samples of 6 mice per group as described in Section 4.5 .

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Marker, Derivative Assay, Mouse Assay, Expressing

    Immunophenotyping of blood showed dramatic expansion of CD11b+ cells in advanced cancer. The viSNE analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets was performed in the blood of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) has significance at p

    Journal: International Journal of Molecular Sciences

    Article Title: Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

    doi: 10.3390/ijms21010170

    Figure Lengend Snippet: Immunophenotyping of blood showed dramatic expansion of CD11b+ cells in advanced cancer. The viSNE analysis was run on the ( A ) naive, ( B ) 4T1 tumor bearing, and ( C ) cisplatin treated 4T1 tumorous animals within the CD45+ living singlets. Quantitative analysis of the main lymphoid subsets: CD4+ T-cells = lilac, CD8+ T-cells = blue, CD19+ B-cells = orange and the myeloid CD11b+ = green, CD11c + = red subsets was performed in the blood of ( D ) naive, ( E ) 4T1 tumor bearing, and ( F ) cisplatin treated 4T1 tumorous mice within the CD45+ living singlets. Representative viSNE plots and column bars are shown from the pooled samples of 6 mice per group. Data are shown as arithmetic means on the column bars ± SEM. Pairwise comparison of the emergence of CD11b+ population in ( E ) vs. ( D ) has significance at p

    Article Snippet: Treatment by cisplatin (Ebewe Pharma, Unterach am Attersee, Austria) was started after 10 days of inoculation and followed in every 5th day in 5 mg/kg dose administered intraperitoneally twice on the day of the treatment.

    Techniques: Mouse Assay

    TRAIL treatment stimulates phosphorylation of H2AX. The right Y-axes and gray lines show the proportion of MEF cells ( a , c , e ) or LN18 cells ( b , d , f ) bearing phosphorylated H2AX after 1 or 5 h exposure to cross-linked TRAIL ( a , b ), soluble TRAIL ( c , d ) or cisplatin ( e , f ). The left Y-axes and black lines represent the percentage of viable cells after same drug exposures. Error bars indicate s.e.m. from three independent experiments.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: TRAIL treatment stimulates phosphorylation of H2AX. The right Y-axes and gray lines show the proportion of MEF cells ( a , c , e ) or LN18 cells ( b , d , f ) bearing phosphorylated H2AX after 1 or 5 h exposure to cross-linked TRAIL ( a , b ), soluble TRAIL ( c , d ) or cisplatin ( e , f ). The left Y-axes and black lines represent the percentage of viable cells after same drug exposures. Error bars indicate s.e.m. from three independent experiments.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques:

    CrmA expression prevents the formation of 6-TG-resistant mouse embryo fibroblast (MEF) colonies following TRAIL treatment. MEF cells were stably transfected with empty vector (pEF), a wild-type FLAG-tagged crmA expression plasmid or a loss-of-function FLAG-crmA expression plasmid. Genomic DNA from pEF clones was amplified using PCR ( a ). Products of reactions containing either no template or 100 ng of plasmid are denoted ‘pos cont' and ‘neg cont' respectively. CrmA expression was analyzed using anti-FLAG immunoblotting, relative to a GAPDH loading control ( b ). ( c ) The phenotypes of crmA wild-type and mutant clones were tested by transiently transfecting the clones with empty vector (pEF), crmA and/or caspase-8 expression plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for morphological characteristics of apoptosis. The proportions of each transiently transfected plasmid are indicated under the graph, as are the stable clones into which this DNA was introduced. Clonogenic survival of each clone was monitored following 1 h exposure to cross-linked TRAIL ( d ) or cisplatin ( f ). Parental MEF cells and the indicated stable cell lines were treated with cross-linked TRAIL( e ) or cisplatin ( g ) for 1 h then subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: CrmA expression prevents the formation of 6-TG-resistant mouse embryo fibroblast (MEF) colonies following TRAIL treatment. MEF cells were stably transfected with empty vector (pEF), a wild-type FLAG-tagged crmA expression plasmid or a loss-of-function FLAG-crmA expression plasmid. Genomic DNA from pEF clones was amplified using PCR ( a ). Products of reactions containing either no template or 100 ng of plasmid are denoted ‘pos cont' and ‘neg cont' respectively. CrmA expression was analyzed using anti-FLAG immunoblotting, relative to a GAPDH loading control ( b ). ( c ) The phenotypes of crmA wild-type and mutant clones were tested by transiently transfecting the clones with empty vector (pEF), crmA and/or caspase-8 expression plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for morphological characteristics of apoptosis. The proportions of each transiently transfected plasmid are indicated under the graph, as are the stable clones into which this DNA was introduced. Clonogenic survival of each clone was monitored following 1 h exposure to cross-linked TRAIL ( d ) or cisplatin ( f ). Parental MEF cells and the indicated stable cell lines were treated with cross-linked TRAIL( e ) or cisplatin ( g ) for 1 h then subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Amplification, Polymerase Chain Reaction, Mutagenesis, Staining

    TRAIL or FasL treatment of mouse embryo fibroblast (MEF) cells provokes generation of 6-TG-resistant colonies. MEF cells were incubated with various doses of ethane methyl sulphonate ( a ), cisplatin ( b ), cross-linked TRAIL ( c ), soluble TRAIL ( d ) or Fas ligand ( e ), for 1hr or 24 h, prior to propidium iodide uptake ( a – d ) or clonogenicity assays ( a – e ). Cells were exposed to selected doses of the drugs for 1 h for HPRT mutagenicity assays. The left Y-axis and black and gray lines depict survival as measured in acute cell death and clonogenicity assays. The right Y-axis and white columns show the number of 6-TG-resistant colonies in mutational assays. Doses, which failed to generate any 6-TG-resistant colonies are labeled ‘no col'. Error bars indicate standard errors of the means from either three ( a , b , e ), four ( c ) or five ( d ) independent experiments.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: TRAIL or FasL treatment of mouse embryo fibroblast (MEF) cells provokes generation of 6-TG-resistant colonies. MEF cells were incubated with various doses of ethane methyl sulphonate ( a ), cisplatin ( b ), cross-linked TRAIL ( c ), soluble TRAIL ( d ) or Fas ligand ( e ), for 1hr or 24 h, prior to propidium iodide uptake ( a – d ) or clonogenicity assays ( a – e ). Cells were exposed to selected doses of the drugs for 1 h for HPRT mutagenicity assays. The left Y-axis and black and gray lines depict survival as measured in acute cell death and clonogenicity assays. The right Y-axis and white columns show the number of 6-TG-resistant colonies in mutational assays. Doses, which failed to generate any 6-TG-resistant colonies are labeled ‘no col'. Error bars indicate standard errors of the means from either three ( a , b , e ), four ( c ) or five ( d ) independent experiments.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Incubation, Labeling

    Caspase-activated CAD is required for TRAIL to provoke mutations in surviving cells. ( a ) Fluorescent microscopy was used to investigate CAD localization and SR-DEVD-FMK binding, as a measure of caspase-3/7 activation, in untreated MEF cells or cells incubated for 24 h with cisplatin (5.4 μg/ml) or cross-linked TRAIL (300 ng/ml). The scale bar is 100 μm. ( b ) The proportions of cells bearing nuclear CAD or SR-DEVD-FMK fluorescence after each treatment were counted. At least 250 cells were scored for each treatment per experiment for untreated and TRAIL-treated samples; at least 100 cisplatin-treated cells were scored per experiment. Error bars indicate s.e.m. from four, three or seven replicates of untreated, cisplatin-treated or TRAIL-treated cells respectively. P -values were calculated using a Student's t -test. ( c ) After exposure to 300 ng/ml cross-linked TRAIL for 24 h, SR-DEVD-FMK positive and negative cells were flow cytometrically sorted and their clonogenicity assayed following treatment with 300 ng/ml cross-linked TRAIL for 1 h. SR-DEVD-FMK positive and negative sorted cells were also subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted. Error bars show the s.e.m. from six or nine independent experiments for clonogenicity and mutagenicity experiments respectively. MEF cells were transfected with the indicated concentrations of small interfering RNAs targeting CAD or GFP then ( d ) subjected to immunoblotting or ( e ) treated with 300 ng/ml cross-linked TRAIL and assayed for H2AX phosphorylation. The left Y-axis and black lines represent the percentage of viable cells. The right Y-axis and gray lines show the proportion of cells bearing phosphorylated H2AX. Error bars indicate s.e.m. from three independent experiments.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: Caspase-activated CAD is required for TRAIL to provoke mutations in surviving cells. ( a ) Fluorescent microscopy was used to investigate CAD localization and SR-DEVD-FMK binding, as a measure of caspase-3/7 activation, in untreated MEF cells or cells incubated for 24 h with cisplatin (5.4 μg/ml) or cross-linked TRAIL (300 ng/ml). The scale bar is 100 μm. ( b ) The proportions of cells bearing nuclear CAD or SR-DEVD-FMK fluorescence after each treatment were counted. At least 250 cells were scored for each treatment per experiment for untreated and TRAIL-treated samples; at least 100 cisplatin-treated cells were scored per experiment. Error bars indicate s.e.m. from four, three or seven replicates of untreated, cisplatin-treated or TRAIL-treated cells respectively. P -values were calculated using a Student's t -test. ( c ) After exposure to 300 ng/ml cross-linked TRAIL for 24 h, SR-DEVD-FMK positive and negative cells were flow cytometrically sorted and their clonogenicity assayed following treatment with 300 ng/ml cross-linked TRAIL for 1 h. SR-DEVD-FMK positive and negative sorted cells were also subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted. Error bars show the s.e.m. from six or nine independent experiments for clonogenicity and mutagenicity experiments respectively. MEF cells were transfected with the indicated concentrations of small interfering RNAs targeting CAD or GFP then ( d ) subjected to immunoblotting or ( e ) treated with 300 ng/ml cross-linked TRAIL and assayed for H2AX phosphorylation. The left Y-axis and black lines represent the percentage of viable cells. The right Y-axis and gray lines show the proportion of cells bearing phosphorylated H2AX. Error bars indicate s.e.m. from three independent experiments.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Microscopy, Binding Assay, Activation Assay, Incubation, Fluorescence, Flow Cytometry, Clone Assay, Transfection

    Mutation frequencies of TRAIL and cisplatin. Mean mutation frequencies of LN18 ( a ) and mouse embryo fibroblast ( b ) cells are expressed as the percentage of clonogenically competent cells that generated 6-TG-resistant colonies. The X-axes show the doses analyzed (in log scale). Only doses that enabled the clonogenic survival of more than five cells per experiment were included in this analysis.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: Mutation frequencies of TRAIL and cisplatin. Mean mutation frequencies of LN18 ( a ) and mouse embryo fibroblast ( b ) cells are expressed as the percentage of clonogenically competent cells that generated 6-TG-resistant colonies. The X-axes show the doses analyzed (in log scale). Only doses that enabled the clonogenic survival of more than five cells per experiment were included in this analysis.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Mutagenesis, Generated

    TRAIL or FasL treatment of LN18 cells provokes generation of 6-TG-resistant colonies. LN18 cells were incubated with various doses of ethane methyl sulfonate ( a ), cisplatin ( b ), cross-linked TRAIL ( c ), soluble TRAIL ( d ) or Fas ligand ( e ), either for 24 h or for 1 h followed by 23 h incubation in normal media. Propidium iodide uptake ( a – d ) or clonogenicity assays ( a – e ) were then performed, as outlined in the Materials and methods. Cells were exposed to selected doses of the drugs for 1 h for HPRT mutagenicity assays. The left Y-axis and black and gray lines depict survival as measured in acute cell death and clonogenicity assays. The right Y-axis and white columns show the number of 6-TG-resistant colonies in mutational assays. Error bars indicate standard errors of the means from either three ( a , b , e ), four ( c ) or five ( d ) independent experiments.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: TRAIL or FasL treatment of LN18 cells provokes generation of 6-TG-resistant colonies. LN18 cells were incubated with various doses of ethane methyl sulfonate ( a ), cisplatin ( b ), cross-linked TRAIL ( c ), soluble TRAIL ( d ) or Fas ligand ( e ), either for 24 h or for 1 h followed by 23 h incubation in normal media. Propidium iodide uptake ( a – d ) or clonogenicity assays ( a – e ) were then performed, as outlined in the Materials and methods. Cells were exposed to selected doses of the drugs for 1 h for HPRT mutagenicity assays. The left Y-axis and black and gray lines depict survival as measured in acute cell death and clonogenicity assays. The right Y-axis and white columns show the number of 6-TG-resistant colonies in mutational assays. Error bars indicate standard errors of the means from either three ( a , b , e ), four ( c ) or five ( d ) independent experiments.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Incubation

    CrmA expression prevents TRAIL-induced DNA damage in LN18 cells. ( a ) LN18 cells were transiently transfected with the indicated plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for apoptosis based on morphological criteria. The percentage of the transiently transfected plasmids are indicated under the graph. ( b ) LN18 cells were transiently transfected with an empty vector (pEF) or plasmids encoding wild-type or mutant crmA, then incubated with the indicated doses of cross-linked TRAIL, soluble TRAIL or cisplatin for 5 h. The proportions of cells bearing phosphorylated H2AX were quantitated by flow cytometry. Error bars indicate standard errors of the means from three independent experiments.

    Journal: Oncogene

    Article Title: TRAIL treatment provokes mutations in surviving cells

    doi: 10.1038/onc.2010.242

    Figure Lengend Snippet: CrmA expression prevents TRAIL-induced DNA damage in LN18 cells. ( a ) LN18 cells were transiently transfected with the indicated plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for apoptosis based on morphological criteria. The percentage of the transiently transfected plasmids are indicated under the graph. ( b ) LN18 cells were transiently transfected with an empty vector (pEF) or plasmids encoding wild-type or mutant crmA, then incubated with the indicated doses of cross-linked TRAIL, soluble TRAIL or cisplatin for 5 h. The proportions of cells bearing phosphorylated H2AX were quantitated by flow cytometry. Error bars indicate standard errors of the means from three independent experiments.

    Article Snippet: Drugs used in this study were cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia), EMS (Sigma Aldrich, St Louis, MO, USA), 6-TG (Sigma Aldrich), Superkiller (cross-linked) TRAIL (Alexis Biochemicals, Lausen, Switzerland), recombinant human (soluble) TRAIL (Chemicon, Temecula, CA, USA) and SuperFas Ligand soluble (Alexis Biochemicals).

    Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Mutagenesis, Incubation, Flow Cytometry, Cytometry