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  • br secondary antibodies for hours at room temperature Specif

    2020-08-28

    
    secondary 113617-63-3 for 2 hours at room temperature. Specific pro-teins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL) and analyzed by Chemidoc MP Im-aging System (Bio-Rad, Hercules, CA). a-Tubulin was used as an internal control to confirm equal gel loading.
    Gene Expression Analyses
    All analyses of tumor samples were carried out at the ISO 15189ecertified Pangaea Oncology laboratory located in the Quiron Dexeus University Hospital (Barcelona, Spain). RNA was isolated from
    Cancer Stem Cell Biomarkers
    Figure 2 Effect of EGFR Blockade EGFR TKIs on ALDH-Activated Cell Subpopulation and CSC Protein Marker Expression. (A) PC9 and H1975 Cells Were Treated With EGFR TKIs (Gefitinib, 40 nM; Osimertinib 0.17 mM; Afatinib, 3.1 nM) for 7 Days. Identification of ALDH Positivity Was Made Using Aldefluor assay. Cells Were Separated by Flow Cytometry. (B) PC9 and H1975 Cells Were Treated With EGFR TKIs as in Figure 3A for Indicated Time Intervals; Western Blot Analysis for CSC Markers Was Performed. a-Tubulin Was Used as Housekeeping Protein. Independent Experiments Were Conducted at Least Twice
    A PC9 DEAB
    Control
    Gefi nib
    ALDH+
    ALDH+
    ALDH+
    PC-9 DEAB
    Control
    Osimer nib
    ALDH+
    ALDH+
    ALDH+
    DEAB
    Control
    Osimer nib
    ALDH+
    ALDH+
    ALDH+
    B
    Gefi nib
    Afa nib
    Osimer nib
    Osimer nib
    NOTCH3 cleaved
    Bmi1
    HES1
    ALDH1A3
    α-Tubulin
    Abbreviations: ALDH ¼ aldehyde dehydrogenase; CSC ¼ cancer stem cell; EGFR ¼ epidermal growth factor receptor; TKI ¼ tyrosine kinase inhibitor.
    the tumor tissue specimens in accordance with a proprietary procedure (European patent EP1945764-B1), as previously described.11,13 The primer and probe sets were designed using Primer Express 3.0 Software (Applied Biosystems; Thermo Fisher Scientific) according to their Ref Seq (http://www.ncbi.nlm.nih.gov/LocusLink). Gene-specific primers are as follows: b-actin, forward: 50-TGAGCGCGGCTACAGCTT-30 and reverse: 50-TCCTTAATGTCACGCACGATTT-30; HES1, for-ward: 50-GGACATTCTGGAAATGACAGTGAA-30 and reverse: 50-CAGCACACTTGGGTCTGTGC-30; ALDH1A1, forward: 50-TGCAACTGAGGAGGAGCTCTG-30 and reverse: 50-CTTCAC 
    TGCCTTGTCAACATCC-30. Gene expression of ALDH1A3 and Bmi-1 were analyzed with Hs00167476_m1 and Hs00995536_m1 (Applied Biosystems), respectively. Quantification of gene expression was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Quantification of gene expression was calculated according to the comparative cycle threshold (Ct) method. Final results were determined as follows: 2 (DCtsample-DCtcalibrator), where DCt values of the calibrator and sample are determined by subtracting the Ct value of the target gene from the value of the endogenous gene (b-actin). Com-mercial RNA controls were used as calibrators (liver and lung; Stratagene,
    Jordi Codony-Servat et al
    Figure 3 Effect of EGFR TKI Alone and Combined With STAT3 and Src Inhibitors on ALDH Subpopulation and CSC Protein Marker Expression. (A) Measurement of ALDHD Subpopulation of PC9 and H1975 Cell Lines Treated for 7 days With Indicated Drugs (Gefitinib, 40 nM; Osimertinib, 0.17 mM; Saracatinib, 10 mM; and TPCA-1, 4 nM) Using Aldefluor Assay and Separation by Flow Cytometry. (B) Protein Expression of CSC Markers Was Analyzed by Western Blot Test in PC9 and H1975 Cell Lines Treated for 24 Hours With Gefitinib (40 nM), Osimertinib (0.17 mM), Saracatinib (10 mM), and TPCA-1 (4 nM) in Combination or as Single Agent, as Indicated. (C) Endogenous Protein Levels in 5 OR Cell Lines Compared to Parental Cell Line PC9. a-Tubulin Was Used as Housekeeping Protein. Independent Experiments Were Conducted at Least Twice
    Abbreviations: ALDH ¼ aldehyde dehydrogenase; CSC ¼ cancer stem cell; EGFR ¼ epidermal growth factor receptor; OR ¼ osimertinib resistant; STAT3 ¼ signal transducer and activator of transcription 3; TKI ¼ tyrosine kinase inhibitor.
    La Jolla, CA). In all quantitative experiments, a sample was considered not evaluable when the standard deviation of the Ct values was > 0.30 in 2 independent analyses. As a result, the number of evaluable samples varied among the genes examined. 
    (CI). Each analysis was performed with the use of a 2-sided 5% significance level and a 95% CI. Association between biomarkers was assessed by Pearson correlation analysis. Statistical analyses were performed by SAS 9.4 software (SAS Institute, Cary, NC).