Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: The relative percentage migration compared to MDA-MB-231 ( A ), A549 ( B ), and DU145 ( C ) cells at 0 h exposure when cells were exposed to PPV (10–150 µM). The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The blue bar represents the percentage of the cell migration of the cells after 18 h of exposure, the orange bar represents the percentage of cell migration of cells after 24 h of exposure, and the grey bar represents the percentage of cell migration of cells after 48 h of exposure. The statistical significance of cell migration after 18 h of exposure is indicated with an asterisk (**), the statistical significance of cell migration after 24 h of exposure is indicated with two asterisks (***), and the statistical significance of cell migration after 48 h of exposure indicated with three asterisks (*). The positive control for the migration assay-included cells exposed to 2-ethyl-17-hydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6-cyclopenta[a]phenanthren-3-yl sulphamate (ESE-ol) (0.4 µM) for 48 h. Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Migration, Standard Deviation, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF B ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF B ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 72 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF R1 ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a P value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF R2 ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of FAK ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of there independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Quantitation and characterization of tumor-cell derived exosomes. NanoSight and ImageStream analyses of A549-derived exosomes determined size, concentration, and characterization. Human lung cancer cells A549, H358, and non-tumor cells HEK293 were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant by using differential centrifugation ( A ) Mean size and concentration of A549 cell-derived exosome determined by NanoSight analysis, mean size was recorded as 182.6 nm, 154.22 nm, and 142.7 nm for A549, H358 and HEK293 cells respectively. ( B ) NanoSight data analysis showing the small size of H358 and HEK293 exosomes compared to A549 cell-derived exosomes. ( C ) NanoSight data analysis showing significantly reduced concentration of exosomes produced from 160 mL of culture media collected of p53 null human lung cancer cells H358 compared to A549 and HEK293 cell exosomes. ( D ) Representative Figure of ImageStream analysis to characterize A549 derived exosomes by expression signals of CD63, CD9, TSG-101, CD81, and EpCAM. ( E ) Percentage of total expression of conventional exosomes markers expressed over A549, H358 and HEK293 cell-exosomes respectively * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Quantitation Assay, Derivative Assay, Concentration Assay, Cell Culture, Isolation, Centrifugation, Produced, Expressing

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: ImageStream and flow cytometry analyses quantify time-dependent internalization of tumor cell-derived exosomes by THP-1 cells. THP-1 cells were seeded and differentiated into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained A549-derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. Image Stream analysis showing Bright field, CD64 + , PKH-26 + , and composite images. ( A ) Time-dependent internalization of exosomes by CD64 + populations assessed by internalization of PKH-26 stained A549-derived exosomes. CD64 + population, without internalization indicates M0 phenotype. ( B ) MATLAB analysis of percentage of PKH-26 + and CD64 + PKH-26 + signals to show time-dependent internalization of exosomes. Fluorescent signals were collected from 300 cells for each time point. ( C ) Representative flow cytometry of exosome internalization analysis at 24 h and 72 h. Group comparisons of 24 h exosome-, 24 h exosome+, and 72h exosome+ were made. After co-culture, cells were prepared for flow cytometry and stained with CD64, CD11b, and PKH-26. The experiment was repeated twice, with three replicates per sample in each experiment. Exosome- sample was used as control ( D ) Percentage of internalization by THP-1 macrophages as CD11b + CD64 + PKH-26 + showing a significant increase of uptake in 72 h compared to 24 h * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Staining, Co-Culture Assay

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: A549 cell-derived exosomes differentiate non-committed M0 macrophages to M2 phenotype. A549 human lung cancer cells were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant using differential centrifugation. Exosomes were stained with PKH-26 for overnight. THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained, A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. ( A ) Representative flow cytometry plot showing in-vitro induction of M2 phenotype (CD11b + CD64 + CD163 + CD206 + ) with A549-derived exosomes. ( B ) Left to right panel-Total percentage of CD163 + macrophages, showing significant increase in CD163 + macrophages that have internalized PKH + exosomes. Total percentage of CD206 + macrophages, showing significant induction in CD206 + macrophages that have internalized PKH + exosomes. Total percentage of M2 macrophages as CD11b + CD64 + CD163 + CD206 + showing significantly induced M2 phenotypes with exosome-positive sample. ( C ) THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20 ng/mL). M0 macrophages were then co-cultured with A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 h M2 gene signature, CHI3L1 (Ym1), IL-10, RETNLB (Fizz1), and Arg1 were upregulated on 24 h of A549-derived exosomes treatment, assessed by qRT-PCR in the cells. ( D ) ImageStream analyses showing CD64 + , PKH-26 + , CD206 + CD163 + macrophages before and after internalization of PKH+ exosomes. Left panel shows composite images from several M0 macrophages without exosome internalization. The right panel shows composite images of M0 macrophages with exosome internalization that have polarized to become M2 (CD206 + CD163 + ) ( E ) Time-dependent increase of M2 polarization induced by A549-derived exosomes showing by percentage of CD163 + , CD64 + CD163 + and PKH-26 + CD163 + analyzed by using MATLAB analysis with the signals collected from 300 cells in each time points. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Derivative Assay, Cell Culture, Isolation, Centrifugation, Staining, Transformation Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Alterations in cellular bioenergetics of macrophages co-cultured with Tumor-derived exosomes by extracellular flux analysis. ( A ) Cellular bioenergetic profiles and ( B ) the cellular bioenergetic parameters (Basal OCR, ATP-linked OCR, Proton Leak, Maximal OCR, Reserve Capacity and Non-Mitochondrial OCR) of M0, M1 and M2 macrophages as determined by sequential injection of oligomycin (Oligo), FCCP, antimycin A (AntiA). Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M1 macrophages. ( C ) Comparison of the cellular bioenergetic profiles of untreated M0 macrophages and those co-cultured with normal cell (HEK293) or tumor cell (A549)–derived exosomes, and ( D ) quantitation of the bioenergetic parameters of C. Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes. ( E ) Inhibition of nonmitochondrial OCR in nonpermeabilized macrophages using DPI. Mean values from 4–8 replicates with ± sem. # p < 0.005 compared to M0 macrophages; * p < 0.01 compared to M1 macrophages. ( F ) PMP-sensitive mitochondrial OCR in macrophages. Mean values from 3–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Cell Culture, Derivative Assay, Injection, Comparison, Quantitation Assay, Co-Culture Assay, Inhibition

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: Hyperoxia-mediated p53 phosphorylation is dependent on the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR) in A549 cells. A: A549 cells were transfected with either nontargeting small interfering RNA (siRNA) (NT) or with ATR siRNA as described in experimental procedures. After 48 h, cells were harvested using lysis buffer, and equal amounts of protein were resolved by 10% SDS-PAGE. ATR, ATM, and β-actin expression were analyzed using Western blot analysis. B: A549 cells were seeded and RNA interference of ATR was performed as described for A. After 48 h, cells were exposed to either 21% oxygen or 95% oxygen for 24 h. Cells were harvested, total p53 was immunoprecipitated, and samples were resolved by 10% SDS-PAGE and blotted to polyvinylidene difluoride (PVDF) membranes for detection of phosphorylation (p-) of p53 on Ser6, −15, −37, and −392 by Western analysis. C: densitometry of Western analysis presented in B. ***Significantly lower than that of NT-transfected cells exposed to 95% oxygen.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Small Interfering RNA, Lysis, SDS Page, Expressing, Western Blot, Immunoprecipitation

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: ATR-interacting protein (ATRIP) downregulation by RNA interference decreases hyperoxia-mediated phosphorylation of p53 on Ser6, −15, −37, and −392. A: A549 cells were transfected with either NT or ATRIP siRNA as described in experimental procedures. After 48 h, cells were harvested using lysis buffer, and equal amounts of protein were resolved by 10% SDS-PAGE. ATRIP and β-actin expression were analyzed using Western blot analysis. B: A549 cells were seeded and RNA interference of ATR was performed as described for Fig. 1A. After 48 h, cells were exposed to either 21% oxygen or 95% oxygen for 24 h. Cells were harvested, total p53 was immunoprecipitated, and samples were resolved by 10% SDS-PAGE and blotted to PVDF membranes for detection of phosphorylation of p53 on Ser6, −15, −37, and −392 by Western analysis. C: densitometry of Western analysis presented in B.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Transfection, Lysis, SDS Page, Expressing, Western Blot, Immunoprecipitation

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: Hyperoxia-mediated p53 Ser6 phosphorylation is dependent on ATM, but phosphorylations of p53 on Ser15, −37, and −392 are independent of ATM in A549 cells. A: A549 cells were transfected with either NT or ATM siRNA. After 48 h, cells were harvested using lysis buffer, and equal amounts of protein were resolved by 10% SDS-PAGE. ATM, ATR, and β-actin expression were analyzed using Western blot analysis. B: A549 cells were seeded and RNA interference of ATM was performed as described for Fig. 1A. After 48 h, cells were exposed to either 21% oxygen or 95% oxygen for 24 h. Cells were harvested in lysis buffer, total p53 was immunoprecipitated, and the samples were resolved by 10% SDS-PAGE and blotted to PVDF membrane for detection of phosphorylation of p53 on Ser6, −15, −37, and −392 as described in experimental procedures. C: densitometry of B.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Transfection, Lysis, SDS Page, Expressing, Western Blot, Immunoprecipitation, Membrane

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: Casein kinase inhibitor does not inhibit phosphorylation of p53 (Ser392) in hyperoxia. A: A549 cells either untreated or treated with 10 μM 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) for 2 h. Following incubation, cells were either exposed to room air or hyperoxia for 24 h. Total p53 protein was immunoprecipitated and Western analysis of phospho-p53 (Ser392) was performed as described in experimental procedures. B: densitometry of p53 (Ser392) band normalized to total p53.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: A: time course of phosphorylation of p53 Ser6, −15, 37, and −392 in hyperoxia. A549 cells were exposed to normoxia (21% oxygen) or 95% oxygen for 4, 8, 12, or 16 h, followed by immunoprecipitation and detection of phospho-p53 as described in experimental procedures. B: relative densitometry of p53 phosphorylations presented in A.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Immunoprecipitation

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: Phosphorylation of histone H2AX on Ser139 in hyperoxia is dependent on both ATR and ATM. A: A549 cells were exposed to normoxia (21% oxygen) or hyperoxia for 0, 4, 8, 12, or 16 h. Cell lysates were prepared and histone H2AX (Ser139) was detected by Western analysis as described in experimental procedures B: A549 cells were either treated with NT, ATM, or ATR siRNA as described in experimental procedures. After 48 h, transfected cells were exposed either to normoxia or hyperoxia for 24 h. Phosphorylation of histone H2AX on Ser139 was detected by Western analysis.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Phospho-proteomics, Western Blot, Transfection

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: Checkpoint kinase 1 (Chk1; Ser345) is phosphorylated in hyperoxia in an ATR-dependent manner but independent of ATRIP. A549 cells were either treated with NT, ATM, or ATR siRNA as described in experimental procedures. After 48 h, transfected cells were exposed either to normoxia or hyperoxia for 24 h. Phosphorylation of Chk1 on Ser345 was detected by Western analysis. A: effect of ATR siRNA. B: densitometry of A. C: effect of ATRIP siRNA. D: densitometry of C. E: effect of ATM siRNA. F: densitometry of E.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Transfection, Phospho-proteomics, Western Blot

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

doi: 10.1152/ajplung.00004.2008

Figure Lengend Snippet: A: ATRIP associates with ATR in response to hyperoxia. Human embryonic kidney HEK293T cells were transfected with pcDNA3-hemagglutinin (HA)-ATRIP overexpression vector, and, after 48 h, cells were exposed to normoxia or hyperoxia for 24 h. ATRIP was immunoprecipitated using anti-HA antibody. ATR Western analysis was performed on ATRIP immunoprecipitates. Top, ATR; bottom, HA. B: hyperoxia activates ATR followed by activation of ATM. A549 cells were either exposed to normoxia or hyperoxia for 8, 16, or 24 h, and activation of ATR or ATM activation was determined by ATR or ATM kinase assay as described in experimental procedures. Top, phospho-Chk1; bottom, ATR as detected in each kinase reaction. Con, control; 1–96AA, amino acids 1–96. C: ATR is required for ATM activation in hyperoxia. A549 cells were transfected with ATR siRNA followed by exposure to hyperoxia (16 h). Following incubation, ATM kinase assay was performed using glutathione S-transferase (GST)-Chk2 as substrate as described in experimental procedures.

Article Snippet: A549 cell lysate was immunoprecipitated with p53-agarose conjugate antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating at 4°C in a rotating mixer for 2 h. The mixture was then centrifuged at 1,000 g for 30 s, the supernatant was carefully aspirated, and the pellet containing the agarose beads was washed three times with lysis buffer.

Techniques: Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Western Blot, Activation Assay, Kinase Assay, Control, Incubation

Journal: mAbs

Article Title: Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

doi: 10.1080/19420862.2017.1319023

Figure Lengend Snippet: Expression levels (receptors per cell) of EGFR, HER2, and HER3 on various cancer cell lines and binding of IgG-43 as analyzed by flow cytometry.

Article Snippet: A549 cells were obtained from CLS Cell Lines Services (Eppelheim, Germany).

Techniques: Expressing, Binding Assay, Flow Cytometry

Journal: mAbs

Article Title: Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

doi: 10.1080/19420862.2017.1319023

Figure Lengend Snippet: Inhibition of ligand-dependent and ligand-independent proliferation. (a) Inhibition of proliferation of MCF-7, NCI-N87, and A549 cells incubated for one week under low (0.2%) serum concentrations in the presence of 10 ng/ml heregulin and either IgG 3–43 (3–43) or rituximab (Control) as a negative control antibody. (b) Inhibition of proliferation of FaDu cells without the addition of heregulin under the same conditions as in (a). (c) Colony formation assay. SKBR3 or BT474 were grown in 12-well plates (1,000 cells per well) and incubated with IgG 3–43 (50 nM) or trastuzumab (Tras.) for 12 d. Medium and antibodies were exchanged after 7 d. Cells incubated without antibody were included as a control (con). Shown are crystal violet-stained wells and the quantification of 2 independent experiments performed with triplicate wells.

Article Snippet: A549 cells were obtained from CLS Cell Lines Services (Eppelheim, Germany).

Techniques: Inhibition, Incubation, Negative Control, Colony Assay, Staining