Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: TKI-sensitive (K562/KBM7) and resistant (K562R/KBM7R) cell lines were harvested and stained for ROS using DCF as described. A representative histogram is shown in A. , and staining quantified in B. . Bars are indicative of mean and SEM. * indicates p < 0.05. C. K562 and K562R cells were immobilized using Cell-Tak, and then oxygen consumption rates (OCR) measured over time with indicated treatments by Seahorse Bioanalyzer. All injections were 1 μM. D. Intracellular ROS levels were measured by flow cytometry using DCF staining as described after treatment with 30 μM DPI, 1 μM Rotenone, or 20 μM Antimycin A for 4 hours. Mean fluorescence intensity was normalized to control for each experiment. Bars indicate mean and SEM. * indicates p < 0.05 Unstained cells were utilized as a negative staining control. E. K562 (black bar) and K562R (grey bar) cells were plated at a density of 5×10 5 cells and grown or treated with 30 μM diphenyleneiodonium (white spotted bar) for 4 hours. Cells were then lysed by freeze/thaw and lysates subjected to NOX activity assay as described. Bars indicate mean and SEM. * indicates p < 0.05. F. 72 hours post transfection with control (black bar) or p47phox (white bar) siRNA, NOX activity levels were measured in K562R cells as described. Bars indicate mean and SEM G. 72 hours post transfection with control (black bar) or p47phox (white bar) siRNA, superoxide levels were measured in K562R cells using HE staining as described. Mean fluorescence intensity was normalized to control for each experiment. Bars indicate mean and SEM. * indicates p < 0.05 Unstained cells were utilized as a negative staining control. H. Microarray data were mined comparing TKI- resistant patients (IR, gray bar, n = 15) to blast crisis (BC, black bar, n = 28). Log (ratio) values were converted to ratios then normalized to blast crisis. I. TKI-sensitive (K562/KBM7) and -resistant (K562R/KBM7R) cell lines were harvested and cDNA made. qRTPCR was performed using p47phox directed primers. Bars indicate mean and SEM. * indicates p < 0.05. J. TKI-sensitive (K562/KBM7) and -resistant (K562R/KBM7R) cell lines were harvested and lysates subjected to SDS-PAGE followed by western blotting using p47phox and Actin antibodies. All data are representative of at least three individual experiments.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Staining, Flow Cytometry, Fluorescence, Control, Negative Staining, Activity Assay, Transfection, Microarray, SDS Page, Western Blot

Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: A. 96 hours post transfection with control or p47phox siRNA, K562R cells were lysed and subjected to SDS-PAGE followed by immunoblotting for p47phox, Fyn, and Actin. B. K562, KBM7, K562R, and KBM7R cells were lysed and subjected to SDS-PAGE followed by immunoblotting for Fyn and Actin as a loading control. C. K562 and K562R cells were lysed then subjected to immunoprecipitation using antibodies directed against Fyn as described. Samples were washed in lysis buffer then boiled in loading dye prior to SDS-PAGE. Proteins were then immunoblotted using antibodies directed against pSrc Y416 and total Fyn. Densitometry was performed using ImageJ. D. Microarray data were mined comparing Fyn mRNA expression between TKI-resistant patients (IR, gray bar, n = 15) to blast crisis (BC, black bar, n = 28). Log(ratio) values were converted to ratios then normalized to blast crisis. E. Viable cell number was counted 24 hours after nucleofection using siRNA directed against Fyn (white bar) or control siRNA (black bar) in K562R cells. Bars indicate mean viable cell yield and SEM. * indicates p < 0.05 Inset: Western blot of lysates after nucleofection with control or Fyn siRNA utilizing antibodies directed against Fyn or Actin. All data are representative of at least three individual experiments.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Transfection, Control, SDS Page, Western Blot, Immunoprecipitation, Lysis, Microarray, Expressing

Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: A. K562R cells were treated with control or Egr-1 directed siRNA for 24 hours then harvested. Lysates were subjected to SDS-PAGE followed by immunoblotting for Egr-1, Fyn, and Actin. B. Tissue microarrays containing samples from chronic phase (CP; n = 10), accelerated phase (AP; n = 6) and blast crisis (BC; n = 10) patients were probed with antibodies directed against Egr-1 then scored by a pathologist. The percent positive patients were calculated compared to negative and positive staining controls as described. C. Lysates from K562/K562R and KBM7/KBM7R cells were subjected to SDS-PAGE followed by immunoblotting with antibodies directed against Egr-1 and Actin. D. Viable cell number was counted 24 hours after nucleofection using siRNA directed against Egr-1 (white bar) or control siRNA (black bar) in K562R cells. Bars indicate mean viable cell yield and SEM. Inset: Western blot of lysates post-nucleofection with control or Egr-1 siRNA utilizing antibodies directed against Egr-1 and Actin. E. 96 hours post-transfection with control or p47phox siRNA, K562R cells were lysed the subjected to SDS-PAGE followed by immunoblotting for Egr-1 and Actin. F. 96 hours post-transfection with control or p47phox siRNA, K562R cells were lysed then subjected to qRTPCR using primers against Egr-1. Bars indicate mean and SEM. * indicates p < 0.05. All data are representative of at least three individual experiments.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control, SDS Page, Western Blot, Staining, Transfection

Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: A. K562R cells were nucleofected with siRNA directed against Fyn (white bars) or control siRNA (black bars). At 24 hours, cells were re-plated then treated with DMSO or 6 nM dasatinib for an additional 48 hours. Bars indicate mean percent growth and SEM. * indicates p < 0.05. B. Twenty four hours post nucleofection with control (black bar) or Egr-1-directed siRNA (white bar), K562R cells were re-plated and treated with 500 nM imatinib or 6 nM dasatinib for an additional 48 hours. Cells were then counted by ViCell. Bars indicate mean percent growth and SEM. * indicates p < 0.05. C. K562R cells were plated then pre-treated for 30 min with 24 mM NAC followed by 48 hour incubation with 500 nM Imatinib or 6 nM Dasatinib. Viable cell number was then counted by ViCell using trypan blue staining. Percent growth was calculated compared to untreated control. Bars indicate mean and SEM. * indicates p < 0.05. D. Seventy-two hours post transfection with control (black bar) or p47phox-directed siRNA (white bar), K562R and KBM7R cells were re-plated and treated with 500 nM imatinib or 6 nM dasatinib for an additional 48 hours. Cells were then counted by ViCell. Bars indicate mean and SEM. * indicates p < 0.05. E. K562R cells were treated with 250 μM NSC23766 alone or in combination with imatinib (doses as shown, left panel) or dasatinib (doses as shown, right panel). Cells were then stained with propidium iodide and analyzed by flow cytometry on the FL-3 channel. The percent sub-diploid was then measured. Bars indicate mean and SEM. * indicates p < 0.05. All data are representative of at least three individual experiments.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control, Incubation, Staining, Transfection, Flow Cytometry

Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: A. Data were mined from Sun and colleagues using Oncomine. The number 1 indicates normal brain tissues and the number 2 indicates GBMs. B. The U87 cell line model system expressing vector, wtEGFR, EGFRΔIII, or R108K-EGFR (R108K) were lysed then subjected to SDS-PAGE followed by immunoblotting for p47phox, Egr-1, Fyn, and Actin. C. The panel of U87 cell lines were lysed then subjected to qRTPCR using primers directed against NOX4 or p47phox. Bars indicate mean and SEM. * indicates p < 0.01. D. U87-vector, wtEGFR, EGFRΔIII or R108K cell lines were stained with HE then analyzed by flow cytometry on the FL-3 channel. Representative histograms are displayed. E. EGFRΔIII or R108K expressing U87 cell lines were treated with control, DPI (5 μM; 4 h), apocynin (100 μM; 24 h) or rotenone (1 μM; 4 h). Following treatment, intracellular ROS levels were measured by HE staining then analyzed by flow cytometry. F. NOX activity was measured in vector, wtEGFR, and EGFRΔIII-expressing U87 cells as described. Bars indicate mean and SEM. * indicates p < 0.05. U87-EGFRΔIII cells were transfected with control (black bar) or p47phox (white bar) siRNA. Forty-eight hours post-transfection, G. NOX activity and H. ROS levels were measured as described in materials and methods. Bars indicate mean and SEM. * indicates p < 0.05. I. U87-EGFRΔIII cells were transfected with either control or p47phox siRNA then harvested at 48h and subjected to SDS-PAGE followed by immunoblotting for p47phox, Egr-1, Fyn, and Actin. All data are representative of at least three individual experiments.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Plasmid Preparation, SDS Page, Western Blot, Staining, Flow Cytometry, Control, Activity Assay, Transfection

Journal: Oncotarget

Article Title: A NOX2/Egr-1/Fyn pathway delineates new targets for TKI-resistant malignancies

doi:

Figure Lengend Snippet: A. CD34 + and CD34 − PBMCs were isolated as described and together with Jurkat cells (as a positive control) were stained with DCF. Representative histograms are shown. B. CD34 + and CD34 − PBMCs were isolated from a patient as described. Lysates were subjected to SDS-PAGE followed by immunoblotting for Fyn, Egr-1, or Actin. C. Microarray data were mined comparing Fyn mRNA expression between LSCs from patients with chronic phase CML (CP, black bar) and blast crisis CML (BC, grey bar). D. Patient derived normal progenitor (NPC) or glioblastoma stem cell (GSC) lines were stained with HE then analyzed by flow cytometry on the FL-3 channel. Representative histograms are displayed. E. Glioblastoma stem cell (GSC) lines were lysed then subjected to SDS-PAGE followed by immunoblotting for Fyn and Actin. Densitometry was performed using ImageJ. F. GSC line 7-2 was transfected with control or Fyn siRNA and sphere-forming capacity was measured as described in materials and methods. Images are shown at 20x magnification. Bars indicate mean and SEM. * indicates p < 0.05.

Article Snippet: Egr-1 siRNA was purchased from ThermoScientific (Pittsburgh, PA) and p47phox and Fyn siRNA from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Positive Control, Staining, SDS Page, Western Blot, Microarray, Expressing, Derivative Assay, Flow Cytometry, Transfection, Control