polyclonal rabbit anti-human s15 antibody  (Absolute Biotech Inc)

Journal: Cancers

Article Title: Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

doi: 10.3390/cancers10040126

Figure Lengend Snippet: APR-246 synergistically enhanced the cytotoxic effect of CDDP in a p53-mutant background under both normoxic and hypoxic conditions. ( A1 – A3 ) To determine possible synergism, cells were simultaneously treated for 72 h with a CDDP (cisplatin) dose range of 0, 0.5, 1, 2, 5, 10 μM (A549 wt ) or 0, 0.5, 1, 2, 5, 10, 20 μM (NCI-H2228 Q331 * and NCI-H1975 R273H ) and fixed concentrations of APR-246, based on the drug sensitivity of each cell line . Cell confluence was determined using the SRB-assay and IC 50 values were calculated using WinNonlin. IC 50 values for CDDP monotherapy or combined with APR-246 are presented as mean ± SD of at least 3 independent experiments. ( B1 – B3 ) The corresponding combination index was calculated using the ‘Additive Model’ for each concentration presented, in correlation with the affected fraction (FA) of the cells. CI = 1.0 ± 0.2 indicates an additive effect, <0.8 indicates moderate synergism, <0.5 strong synergism, and <0.2 very strong synergism (** p < 0.05 compared to CDDP monotherapy). The corresponding values are presented in . Nx: normoxia; Hx: hypoxia.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p53 (Cell Signalling Technology, Danvers, MA, USA, no. 9282; 1:1000); rabbit polyclonal anti-phospho-p53 (S15, R&D systems, #AF1043, 1:1000); mouse monoclonal anti-HIF-1α (Becton Dickinson Pharmingen, clone 54/HIF-1α; 1:1000, 96 kDa: unprocessed; 116 kDa: fully post-translationally modified); Apoptosis Western Blot Cocktail (procaspase 3, cleaved caspase 3, β-actin; Abcam no. ab136812); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA; clone AC-15; 1:2500); anti-mouse and anti-rabbit HRP-labelled secondary antibodies (Cell Signalling; no. 7076S and no. 7074S, respectively; 1:2500).

Techniques: Mutagenesis, Sulforhodamine B Assay, Concentration Assay

Journal: Cancers

Article Title: Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

doi: 10.3390/cancers10040126

Figure Lengend Snippet: Balance between HIF-1α and p53 protein levels and transcriptional activity. ( A ) A549 wt , NCI-H1975 R273H , and NCI-H2228 Q331 * cell lines were treated with either vehicle or CDDP under normoxic and hypoxic conditions. Protein levels of HIF-1α 116 kDa (processed), HIF-1α 96 kDa (unprocessed), full p53 wt/R273H , and truncated p53 Q331 * were determined by Western blotting. β-actin was used as internal control. ( B – D ) NCI-H2228 cells were treated for 24 h with with CDDP, APR-246, or combined therapy under normoxic and hypoxic conditions. ( B ) Protein levels of HIF-1α 116 kDa (processed), HIF-1α 96 kDa (unprocessed), p53 Q331 *, and p53-serine 15 were determined by Western blotting. β-actin was used as internal control. ( C , D ) Relative mRNA expression levels of p53 ( C ) and HIF-1α ( D ) transcriptional targets. Expression levels are presented relative to the vehicle-treated sample under normoxia as mean ± SEM of 3 independent experiments (★ p < 0.05 normoxia vs. hypoxia; * p < 0.05 compared to vehicle-treated sample under normoxia; ** p < 0.05 compared to vehicle-treated sample under hypoxia.). ARD: Adjusted Relative Density; Nx: normoxia; Hx: hypoxia.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p53 (Cell Signalling Technology, Danvers, MA, USA, no. 9282; 1:1000); rabbit polyclonal anti-phospho-p53 (S15, R&D systems, #AF1043, 1:1000); mouse monoclonal anti-HIF-1α (Becton Dickinson Pharmingen, clone 54/HIF-1α; 1:1000, 96 kDa: unprocessed; 116 kDa: fully post-translationally modified); Apoptosis Western Blot Cocktail (procaspase 3, cleaved caspase 3, β-actin; Abcam no. ab136812); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA; clone AC-15; 1:2500); anti-mouse and anti-rabbit HRP-labelled secondary antibodies (Cell Signalling; no. 7076S and no. 7074S, respectively; 1:2500).

Techniques: Activity Assay, Western Blot, Control, Expressing

Journal: Cancers

Article Title: Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

doi: 10.3390/cancers10040126

Figure Lengend Snippet: Role of p53 in CDDP resistance. ( A ) P53 protein levels determined by Western blotting after 48 h treatment with 5 µM CDDP of the NCI-H2228 Q331 * parental cell line, nontemplate control (NTC), and three TP53 shRNA monoclonal subclones under normoxia. β-actin was used as an internal standard. ( B ) Cell proliferation presented as mean ± SD of 3 replicates Green Object Confluence using the IncuCyte system. Cells were treated with either vehicle or 5 µM CDDP under normoxia or 1% O 2 . ( C ) Protein levels of HIF-1α 116 kDa (processed), HIF-1α 96 kDa (unprocessed), and p53 were determined by Western blotting after treatment with vehicle, CDDP, APR-246, or combination therapy for 24 h in NTC and shRNA.1 ( TP53 shRNA) cell lines under normoxic and hypoxic conditions. β-actin was used as internal control. ( D ) mRNA levels of p53 (NOXA, PUMA, p21) and HIF-1α (CA9, BNIP3, VEGF) transcription targets. NTC and shRNA.1 cells were treated with 5 µM CDDP for 24 h under normoxia or 1% O 2 and expression levels are presented relative to the vehicle-treated sample under normoxia. (* p < 0.05 compared to vehicle-treated sample at the 72 h time-point). ARD: Adjusted Relative Density; Nx: normoxia; Hx: hypoxia.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p53 (Cell Signalling Technology, Danvers, MA, USA, no. 9282; 1:1000); rabbit polyclonal anti-phospho-p53 (S15, R&D systems, #AF1043, 1:1000); mouse monoclonal anti-HIF-1α (Becton Dickinson Pharmingen, clone 54/HIF-1α; 1:1000, 96 kDa: unprocessed; 116 kDa: fully post-translationally modified); Apoptosis Western Blot Cocktail (procaspase 3, cleaved caspase 3, β-actin; Abcam no. ab136812); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA; clone AC-15; 1:2500); anti-mouse and anti-rabbit HRP-labelled secondary antibodies (Cell Signalling; no. 7076S and no. 7074S, respectively; 1:2500).

Techniques: Western Blot, Control, shRNA, Expressing

Journal: Cancers

Article Title: Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

doi: 10.3390/cancers10040126

Figure Lengend Snippet: Role of p53 Q331 * and ROS in the synergistic combinatory effect of CDDP and APR-246 under hypoxia. ( A ) NCI-H2228 Q331 * parental cell line, non-template control (NTC), and three TP53 shRNA monoclonal subclones were treated with APR-246 or CDDP/APR-246 under 1% O 2 . Caspase-3/7 activity was measured using the IncuCyte Caspase 3/7 Red reagent under hypoxia and presented as mean ± SD of the number of Red Object Counts/mm 2 of 3 replicates. ( B ) NCI-H2228 Q331 * cells were treated with vehicle, CDDP, APR-246, or CDDP/APR-246 for 24 h and total glutathione (GSH) content was measured under normoxia and hypoxia. Data is presented as mean µM GSH/well ± SD of 3 independent experiments. ( C ) NCI-H2228 Q331 * cells were treated with vehicle, CDDP, APR-246, or CDDP/APR-246 for 72 h in the absence or presence of 5 mM NAC under 1% O 2 . Intracellular mean intensity (GCU) of the CellROX dye was measured using the IncuCyte system and presented as mean fold change to untreated sample ± SEM of 4 independent experiments ( D ) NCI-H2228 cells were treated with vehicle, CDDP, APR-246, or CDDP/APR-246 for 72 h in the absence or presence of 5 mM NAC under 1% O 2 . The percentage of viable cells (AnnV−/PI−) was measured by flow cytometry and presented as mean percentage ± SD of 3 independent experiments. (★ p < 0.05 Nx vs. Hx; * p < 0.05 compared to vehicle-treated sample; ** p < 0.05 compared to vehicle-, CDDP-, and APR-246-treated samples) Nx: normoxia; Hx: hypoxia.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p53 (Cell Signalling Technology, Danvers, MA, USA, no. 9282; 1:1000); rabbit polyclonal anti-phospho-p53 (S15, R&D systems, #AF1043, 1:1000); mouse monoclonal anti-HIF-1α (Becton Dickinson Pharmingen, clone 54/HIF-1α; 1:1000, 96 kDa: unprocessed; 116 kDa: fully post-translationally modified); Apoptosis Western Blot Cocktail (procaspase 3, cleaved caspase 3, β-actin; Abcam no. ab136812); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA; clone AC-15; 1:2500); anti-mouse and anti-rabbit HRP-labelled secondary antibodies (Cell Signalling; no. 7076S and no. 7074S, respectively; 1:2500).

Techniques: Control, shRNA, Activity Assay, Flow Cytometry

Journal: Cancers

Article Title: Hypoxia-Induced Cisplatin Resistance in Non-Small Cell Lung Cancer Cells Is Mediated by HIF-1α and Mutant p53 and Can Be Overcome by Induction of Oxidative Stress

doi: 10.3390/cancers10040126

Figure Lengend Snippet: Underlying mechanism of the synergistic interaction between CDDP and APR-246 under hypoxic conditions. Cisplatin induces upregulation of mutant, truncated p53, which in turn degrades HIF-1α protein. HIF-1α reduction leads to a shift in metabolism, resulting in induction of reactive oxygen species (ROS). APR-246 further increases oxidative stress through inhibition of glutathione (GSH) leading to a synergistic induction of intracellular ROS, which in turn induces massive cell death.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p53 (Cell Signalling Technology, Danvers, MA, USA, no. 9282; 1:1000); rabbit polyclonal anti-phospho-p53 (S15, R&D systems, #AF1043, 1:1000); mouse monoclonal anti-HIF-1α (Becton Dickinson Pharmingen, clone 54/HIF-1α; 1:1000, 96 kDa: unprocessed; 116 kDa: fully post-translationally modified); Apoptosis Western Blot Cocktail (procaspase 3, cleaved caspase 3, β-actin; Abcam no. ab136812); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA; clone AC-15; 1:2500); anti-mouse and anti-rabbit HRP-labelled secondary antibodies (Cell Signalling; no. 7076S and no. 7074S, respectively; 1:2500).

Techniques: Mutagenesis, Inhibition