h1975  (ATCC)


Bioz Verified Symbol ATCC is a verified supplier
Bioz Manufacturer Symbol ATCC manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    ATCC h1975
    The effect of hinokitiol on cell cycle distribution. <t>H1975</t> cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p
    H1975, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h1975/product/ATCC
    Average 97 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    h1975 - by Bioz Stars, 2022-09
    97/100 stars

    Images

    1) Product Images from "Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells"

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104203

    The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p
    Figure Legend Snippet: The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p

    Techniques Used: Flow Cytometry, Cytometry, Staining, BrdU Incorporation Assay, Western Blot, Expressing, Microscopy

    The effects of hinokitiol on apoptosis and autophagy. ( A ) Apoptosis was assessed using an annexin-V/PI binding assay in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment. Western blot analysis of PARP in H1975 cells and lung stromal fibroblasts ( B ), LC3, p62 and ATG5 expression in ( C ) H1975 cells and ( F ) lung stromal fibroblasts. The treatment of 100 nM rapamycin for 48 h was used as a positive control for LC3 expression. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. ( D ) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in H1975 cells treated with 5 µM hinokitiol for 8 h. ( E ) H1975 cells were pretreated with 2.5 mM of 3-MA for 1 h, followed by exposure to 5 µM hinokitiol for 48 h. Cell proliferation was analyzed through a trypan blue staining assay. The results are representative of three different experiments and are expressed as the mean ± SD. ** indicates a significant difference at the level of p
    Figure Legend Snippet: The effects of hinokitiol on apoptosis and autophagy. ( A ) Apoptosis was assessed using an annexin-V/PI binding assay in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment. Western blot analysis of PARP in H1975 cells and lung stromal fibroblasts ( B ), LC3, p62 and ATG5 expression in ( C ) H1975 cells and ( F ) lung stromal fibroblasts. The treatment of 100 nM rapamycin for 48 h was used as a positive control for LC3 expression. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. ( D ) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in H1975 cells treated with 5 µM hinokitiol for 8 h. ( E ) H1975 cells were pretreated with 2.5 mM of 3-MA for 1 h, followed by exposure to 5 µM hinokitiol for 48 h. Cell proliferation was analyzed through a trypan blue staining assay. The results are representative of three different experiments and are expressed as the mean ± SD. ** indicates a significant difference at the level of p

    Techniques Used: Binding Assay, Western Blot, Expressing, Positive Control, Flow Cytometry, Cytometry, Staining

    The effects of hinokitiol on cell proliferation. (A) The chemical structure of hinokitiol. (B) The effect of a 72-h hinokitiol treatment on H1975 and PC9-IR cell proliferation, as assayed through trypan blue staining. (C) The effect of hinokitiol on the colony formation ability of H1975 cells. (D) The effect of hinokitiol on the colony formation ability of PC9-IR cells. In (B), (C), and (D), the results are representative of three different experiments and are expressed as the mean ± SD and as % of control. *, **, and *** indicate a significant difference at the level of p
    Figure Legend Snippet: The effects of hinokitiol on cell proliferation. (A) The chemical structure of hinokitiol. (B) The effect of a 72-h hinokitiol treatment on H1975 and PC9-IR cell proliferation, as assayed through trypan blue staining. (C) The effect of hinokitiol on the colony formation ability of H1975 cells. (D) The effect of hinokitiol on the colony formation ability of PC9-IR cells. In (B), (C), and (D), the results are representative of three different experiments and are expressed as the mean ± SD and as % of control. *, **, and *** indicate a significant difference at the level of p

    Techniques Used: Staining

    Hinokitiol induced cellular senescence in H1975 cells and lung stromal fibroblasts. (A) The senescent cells were quantified at 200× magnification under a standard light microscope. (B) Hinokitiol induced cellular senescence was attenuated by autophagy inhibitors in H1975 cells. (C) Hinokitiol induced cellular senescence was attenuated by transfection of siRNA against ATG5 in H1975 cells. Corresponding protein expression was detected by western blot. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. In (A) , (B) and (C) , each value is the mean ± SD of 3-5 fields of three different experiments. * and ** indicate a significant difference at the level of p
    Figure Legend Snippet: Hinokitiol induced cellular senescence in H1975 cells and lung stromal fibroblasts. (A) The senescent cells were quantified at 200× magnification under a standard light microscope. (B) Hinokitiol induced cellular senescence was attenuated by autophagy inhibitors in H1975 cells. (C) Hinokitiol induced cellular senescence was attenuated by transfection of siRNA against ATG5 in H1975 cells. Corresponding protein expression was detected by western blot. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. In (A) , (B) and (C) , each value is the mean ± SD of 3-5 fields of three different experiments. * and ** indicate a significant difference at the level of p

    Techniques Used: Light Microscopy, Transfection, Expressing, Western Blot

    In vivo antitumor activity of hinokitiol. ( A ) The growth curves of subcutaneous xenografts of H1975 are shown. ( B ) The excised tumors were weighed and imaged. All results are given as the mean ± SD; n = 5 - 7 for each group. *indicates a significant difference at the level of p
    Figure Legend Snippet: In vivo antitumor activity of hinokitiol. ( A ) The growth curves of subcutaneous xenografts of H1975 are shown. ( B ) The excised tumors were weighed and imaged. All results are given as the mean ± SD; n = 5 - 7 for each group. *indicates a significant difference at the level of p

    Techniques Used: In Vivo, Activity Assay

    The effects of hinokitiol on the expression of DNA damage regulatory proteins. (A) The effect of hinokitiol (5 µM) or cisplatin (25 µM) on the level of γ-H2AX phosphorylation and total p53 expression in H1975 cells, as assayed using western blots. (B) Assessment of hinokitiol-induced DNA damage in H1975 cells through an immunofluorescence γ-H2AX focus assay. (C) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation and total p53 expression in lung stromal fibroblasts, as assayed using western blots. (D) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation in H1299 cells. (E) The effect of hinokitiol (25 µM) or cisplatin (CDDP, 25 µM) on the phosphorylation and total level of ATM, SMC3, and p53 in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control.
    Figure Legend Snippet: The effects of hinokitiol on the expression of DNA damage regulatory proteins. (A) The effect of hinokitiol (5 µM) or cisplatin (25 µM) on the level of γ-H2AX phosphorylation and total p53 expression in H1975 cells, as assayed using western blots. (B) Assessment of hinokitiol-induced DNA damage in H1975 cells through an immunofluorescence γ-H2AX focus assay. (C) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation and total p53 expression in lung stromal fibroblasts, as assayed using western blots. (D) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation in H1299 cells. (E) The effect of hinokitiol (25 µM) or cisplatin (CDDP, 25 µM) on the phosphorylation and total level of ATM, SMC3, and p53 in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control.

    Techniques Used: Expressing, Western Blot, Immunofluorescence

    The effects of hinokitiol on gene expression. (A) Microarray profiling of H1975 cells and PC9-IR cells treated with 5 µM hinokitiol for 48 h. (B) Q-PCR array validation of the expression of genes related to DNA damage and autophagy in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment for 24 h. The results are representative of those obtained in three different experiments and are expressed as the fold change compared with control. * and ** indicate a significant difference at the level of p
    Figure Legend Snippet: The effects of hinokitiol on gene expression. (A) Microarray profiling of H1975 cells and PC9-IR cells treated with 5 µM hinokitiol for 48 h. (B) Q-PCR array validation of the expression of genes related to DNA damage and autophagy in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment for 24 h. The results are representative of those obtained in three different experiments and are expressed as the fold change compared with control. * and ** indicate a significant difference at the level of p

    Techniques Used: Expressing, Microarray, Polymerase Chain Reaction

    2) Product Images from "The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor"

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S117443

    EGFR shRNA reduced BJ sensitivities by suppressing apoptosis. ( A ) Viability determination. H1975 cells transfected with EGFR shRNA or NS control for 24 hours were treated with 2 mg/mL of BJ extract for 12 hours and the collected cells counted by trypan blue exclusion assay. Symbol (-) meant no transfection. The results were expressed as mean values from three independent experiments. * P
    Figure Legend Snippet: EGFR shRNA reduced BJ sensitivities by suppressing apoptosis. ( A ) Viability determination. H1975 cells transfected with EGFR shRNA or NS control for 24 hours were treated with 2 mg/mL of BJ extract for 12 hours and the collected cells counted by trypan blue exclusion assay. Symbol (-) meant no transfection. The results were expressed as mean values from three independent experiments. * P

    Techniques Used: shRNA, Transfection, Trypan Blue Exclusion Assay

    The aqueous BJ extract increased apoptotic distributions of H1975 cells by ( A ) dose-dependent appearance of sub-G 1 cells. Human NSCLC cells H1975 cultured in 1% serum-supplemented DMEM were incubated with various concentrations of BJ extract (1, 2, and 5 mg/mL) or water for 12 hours before being labeled with PI followed by flow cytometry analysis. ( B ) Cell cycle distribution. The percentages of cell cycle distribution in H1975 cells following treatment were analyzed. The results of BJ treatment were compared with those of water control. * P
    Figure Legend Snippet: The aqueous BJ extract increased apoptotic distributions of H1975 cells by ( A ) dose-dependent appearance of sub-G 1 cells. Human NSCLC cells H1975 cultured in 1% serum-supplemented DMEM were incubated with various concentrations of BJ extract (1, 2, and 5 mg/mL) or water for 12 hours before being labeled with PI followed by flow cytometry analysis. ( B ) Cell cycle distribution. The percentages of cell cycle distribution in H1975 cells following treatment were analyzed. The results of BJ treatment were compared with those of water control. * P

    Techniques Used: Cell Culture, Incubation, Labeling, Flow Cytometry, Cytometry

    Dose-response growth curves of BJ extract in NSCLC H1975 cells. The increased concentration of aqueous BJ extracts after 12 hours of treatments reduced viable H1975 cells. The trypsinized cells were counted for the numbers of viable cells using trypan blue exclusion assay. The percentages of viabilities were obtained by comparing cell numbers at each concentration with water control that was assigned as 100%. The data are the average of quadruplicate for each experiment. The results are presented as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: Dose-response growth curves of BJ extract in NSCLC H1975 cells. The increased concentration of aqueous BJ extracts after 12 hours of treatments reduced viable H1975 cells. The trypsinized cells were counted for the numbers of viable cells using trypan blue exclusion assay. The percentages of viabilities were obtained by comparing cell numbers at each concentration with water control that was assigned as 100%. The data are the average of quadruplicate for each experiment. The results are presented as mean ± SD of three independent experiments. * P

    Techniques Used: Concentration Assay, Trypan Blue Exclusion Assay

    Oral administration of BJ reduced xenograft tumor growth. ( A ) BJ extract suppressed the growth of xenograft tumors in animal models. Nude BALB/c mice with established xenograft tumors of H1975 cells were administered orally with 1 and 2 g BJ/kg daily for 7 consecutive days (arrows). The graph represented tumor growth variation ( y -axis) from the start of feeding ( x -axis). Mice fed with aqueous BJ extract showed reduced tumor growth compared with those with water. * P
    Figure Legend Snippet: Oral administration of BJ reduced xenograft tumor growth. ( A ) BJ extract suppressed the growth of xenograft tumors in animal models. Nude BALB/c mice with established xenograft tumors of H1975 cells were administered orally with 1 and 2 g BJ/kg daily for 7 consecutive days (arrows). The graph represented tumor growth variation ( y -axis) from the start of feeding ( x -axis). Mice fed with aqueous BJ extract showed reduced tumor growth compared with those with water. * P

    Techniques Used: Mouse Assay

    Histological and fluorescence examination of the reduced tumors. ( A ) Examination by HE staining. The paraffin-embedded H1975 tumors treated with BJ (2 g/kg) and water control were dissected, stained by HE and analyzed by confocal microscopy (scale bar =100 μm). The rectangular inset of the image were zoomed-in to the right of the panel for each treatment (scale bar =25 μm). The white arrow signifies apoptotic body location. ( B ) Release of mitochondrial cytochrome c in tumors treated with BJ. The dissected specimens of H1975 tumors as treated with BJ (1 and 2 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria signified the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 μm). ( C ) Images of immunofluorescent EGFR H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with EGFR antibody (green) followed by FITC-conjugated secondary antibody treatment before being counterstained with DAPI (blue) (scale bar =50 μm). ( D ) Images of immunofluorescent pEGFR Y1068 H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with pEGFR Y1068 antibody ( green ) followed by FITC-conjugated secondary antibody incubation before being counterstained with DAPI (blue) (scale bar =50 μm). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; FITC, fluorescein; HE, hematoxylin and eosin; TRITC, tetramethylrhodamine.
    Figure Legend Snippet: Histological and fluorescence examination of the reduced tumors. ( A ) Examination by HE staining. The paraffin-embedded H1975 tumors treated with BJ (2 g/kg) and water control were dissected, stained by HE and analyzed by confocal microscopy (scale bar =100 μm). The rectangular inset of the image were zoomed-in to the right of the panel for each treatment (scale bar =25 μm). The white arrow signifies apoptotic body location. ( B ) Release of mitochondrial cytochrome c in tumors treated with BJ. The dissected specimens of H1975 tumors as treated with BJ (1 and 2 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria signified the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 μm). ( C ) Images of immunofluorescent EGFR H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with EGFR antibody (green) followed by FITC-conjugated secondary antibody treatment before being counterstained with DAPI (blue) (scale bar =50 μm). ( D ) Images of immunofluorescent pEGFR Y1068 H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with pEGFR Y1068 antibody ( green ) followed by FITC-conjugated secondary antibody incubation before being counterstained with DAPI (blue) (scale bar =50 μm). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; FITC, fluorescein; HE, hematoxylin and eosin; TRITC, tetramethylrhodamine.

    Techniques Used: Fluorescence, Staining, Confocal Microscopy, Incubation, Mouse Assay

    BJ induced apoptosis and reduced EGFR and pEGFR Y1068 levels in H1975 cells. ( A ) Western blot analysis. The protein lysates from H1975 cells as treated with 1, 2, and 5 mg/mL of BJ extract for 12 hours were collected and used for Western blot analysis. The blots were incubated with various primary antibodies, including EGFR, phosphorylated EGFR Y1068 , Akt, phosphorylated Akt S473 , caspase-3, and PARP as specified, which were followed by HRP-conjugated secondary antibodies. GAPDH was used as loading control. The blots were visualized by ECL detection system. ( B ) Densitometric determination of EGFR and phosphorylated EGFR Y1068 amelioration. The densitometric ratios of EGFR and phosphorylated EGFR Y1068 in H1975 cells from Western blot analysis were obtained by first normalizing individual band intensity at each concentration to that of the loading control and compared with those of water treatment. The results were expressed as mean values of three independent experiments (* P
    Figure Legend Snippet: BJ induced apoptosis and reduced EGFR and pEGFR Y1068 levels in H1975 cells. ( A ) Western blot analysis. The protein lysates from H1975 cells as treated with 1, 2, and 5 mg/mL of BJ extract for 12 hours were collected and used for Western blot analysis. The blots were incubated with various primary antibodies, including EGFR, phosphorylated EGFR Y1068 , Akt, phosphorylated Akt S473 , caspase-3, and PARP as specified, which were followed by HRP-conjugated secondary antibodies. GAPDH was used as loading control. The blots were visualized by ECL detection system. ( B ) Densitometric determination of EGFR and phosphorylated EGFR Y1068 amelioration. The densitometric ratios of EGFR and phosphorylated EGFR Y1068 in H1975 cells from Western blot analysis were obtained by first normalizing individual band intensity at each concentration to that of the loading control and compared with those of water treatment. The results were expressed as mean values of three independent experiments (* P

    Techniques Used: Western Blot, Incubation, Concentration Assay

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • h1975  (ATCC)
    97
    ATCC h1975
    The effect of hinokitiol on cell cycle distribution. <t>H1975</t> cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p
    H1975, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h1975/product/ATCC
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h1975 - by Bioz Stars, 2022-09
    97/100 stars
      Buy from Supplier

    Image Search Results


    The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Flow Cytometry, Cytometry, Staining, BrdU Incorporation Assay, Western Blot, Expressing, Microscopy

    The effects of hinokitiol on apoptosis and autophagy. ( A ) Apoptosis was assessed using an annexin-V/PI binding assay in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment. Western blot analysis of PARP in H1975 cells and lung stromal fibroblasts ( B ), LC3, p62 and ATG5 expression in ( C ) H1975 cells and ( F ) lung stromal fibroblasts. The treatment of 100 nM rapamycin for 48 h was used as a positive control for LC3 expression. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. ( D ) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in H1975 cells treated with 5 µM hinokitiol for 8 h. ( E ) H1975 cells were pretreated with 2.5 mM of 3-MA for 1 h, followed by exposure to 5 µM hinokitiol for 48 h. Cell proliferation was analyzed through a trypan blue staining assay. The results are representative of three different experiments and are expressed as the mean ± SD. ** indicates a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: The effects of hinokitiol on apoptosis and autophagy. ( A ) Apoptosis was assessed using an annexin-V/PI binding assay in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment. Western blot analysis of PARP in H1975 cells and lung stromal fibroblasts ( B ), LC3, p62 and ATG5 expression in ( C ) H1975 cells and ( F ) lung stromal fibroblasts. The treatment of 100 nM rapamycin for 48 h was used as a positive control for LC3 expression. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. ( D ) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in H1975 cells treated with 5 µM hinokitiol for 8 h. ( E ) H1975 cells were pretreated with 2.5 mM of 3-MA for 1 h, followed by exposure to 5 µM hinokitiol for 48 h. Cell proliferation was analyzed through a trypan blue staining assay. The results are representative of three different experiments and are expressed as the mean ± SD. ** indicates a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Binding Assay, Western Blot, Expressing, Positive Control, Flow Cytometry, Cytometry, Staining

    The effects of hinokitiol on cell proliferation. (A) The chemical structure of hinokitiol. (B) The effect of a 72-h hinokitiol treatment on H1975 and PC9-IR cell proliferation, as assayed through trypan blue staining. (C) The effect of hinokitiol on the colony formation ability of H1975 cells. (D) The effect of hinokitiol on the colony formation ability of PC9-IR cells. In (B), (C), and (D), the results are representative of three different experiments and are expressed as the mean ± SD and as % of control. *, **, and *** indicate a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: The effects of hinokitiol on cell proliferation. (A) The chemical structure of hinokitiol. (B) The effect of a 72-h hinokitiol treatment on H1975 and PC9-IR cell proliferation, as assayed through trypan blue staining. (C) The effect of hinokitiol on the colony formation ability of H1975 cells. (D) The effect of hinokitiol on the colony formation ability of PC9-IR cells. In (B), (C), and (D), the results are representative of three different experiments and are expressed as the mean ± SD and as % of control. *, **, and *** indicate a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Staining

    Hinokitiol induced cellular senescence in H1975 cells and lung stromal fibroblasts. (A) The senescent cells were quantified at 200× magnification under a standard light microscope. (B) Hinokitiol induced cellular senescence was attenuated by autophagy inhibitors in H1975 cells. (C) Hinokitiol induced cellular senescence was attenuated by transfection of siRNA against ATG5 in H1975 cells. Corresponding protein expression was detected by western blot. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. In (A) , (B) and (C) , each value is the mean ± SD of 3-5 fields of three different experiments. * and ** indicate a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: Hinokitiol induced cellular senescence in H1975 cells and lung stromal fibroblasts. (A) The senescent cells were quantified at 200× magnification under a standard light microscope. (B) Hinokitiol induced cellular senescence was attenuated by autophagy inhibitors in H1975 cells. (C) Hinokitiol induced cellular senescence was attenuated by transfection of siRNA against ATG5 in H1975 cells. Corresponding protein expression was detected by western blot. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. In (A) , (B) and (C) , each value is the mean ± SD of 3-5 fields of three different experiments. * and ** indicate a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Light Microscopy, Transfection, Expressing, Western Blot

    In vivo antitumor activity of hinokitiol. ( A ) The growth curves of subcutaneous xenografts of H1975 are shown. ( B ) The excised tumors were weighed and imaged. All results are given as the mean ± SD; n = 5 - 7 for each group. *indicates a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: In vivo antitumor activity of hinokitiol. ( A ) The growth curves of subcutaneous xenografts of H1975 are shown. ( B ) The excised tumors were weighed and imaged. All results are given as the mean ± SD; n = 5 - 7 for each group. *indicates a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: In Vivo, Activity Assay

    The effects of hinokitiol on the expression of DNA damage regulatory proteins. (A) The effect of hinokitiol (5 µM) or cisplatin (25 µM) on the level of γ-H2AX phosphorylation and total p53 expression in H1975 cells, as assayed using western blots. (B) Assessment of hinokitiol-induced DNA damage in H1975 cells through an immunofluorescence γ-H2AX focus assay. (C) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation and total p53 expression in lung stromal fibroblasts, as assayed using western blots. (D) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation in H1299 cells. (E) The effect of hinokitiol (25 µM) or cisplatin (CDDP, 25 µM) on the phosphorylation and total level of ATM, SMC3, and p53 in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control.

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: The effects of hinokitiol on the expression of DNA damage regulatory proteins. (A) The effect of hinokitiol (5 µM) or cisplatin (25 µM) on the level of γ-H2AX phosphorylation and total p53 expression in H1975 cells, as assayed using western blots. (B) Assessment of hinokitiol-induced DNA damage in H1975 cells through an immunofluorescence γ-H2AX focus assay. (C) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation and total p53 expression in lung stromal fibroblasts, as assayed using western blots. (D) The effect of hinokitiol (5 µM) on the level of γ-H2AX phosphorylation in H1299 cells. (E) The effect of hinokitiol (25 µM) or cisplatin (CDDP, 25 µM) on the phosphorylation and total level of ATM, SMC3, and p53 in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control.

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Expressing, Western Blot, Immunofluorescence

    The effects of hinokitiol on gene expression. (A) Microarray profiling of H1975 cells and PC9-IR cells treated with 5 µM hinokitiol for 48 h. (B) Q-PCR array validation of the expression of genes related to DNA damage and autophagy in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment for 24 h. The results are representative of those obtained in three different experiments and are expressed as the fold change compared with control. * and ** indicate a significant difference at the level of p

    Journal: PLoS ONE

    Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

    doi: 10.1371/journal.pone.0104203

    Figure Lengend Snippet: The effects of hinokitiol on gene expression. (A) Microarray profiling of H1975 cells and PC9-IR cells treated with 5 µM hinokitiol for 48 h. (B) Q-PCR array validation of the expression of genes related to DNA damage and autophagy in H1975 cells and lung stromal fibroblasts after 5 µM hinokitiol treatment for 24 h. The results are representative of those obtained in three different experiments and are expressed as the fold change compared with control. * and ** indicate a significant difference at the level of p

    Article Snippet: Cell lines and culture conditions The human lung adenocarcinoma cell lines, A549 (EGFR wild type), H1975 (EGFR L858R/T790M, gefitinib-resistant), H1299 (EGFR wild type, p53 null), and H3255 (EGFR L858R) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Expressing, Microarray, Polymerase Chain Reaction

    EGFR shRNA reduced BJ sensitivities by suppressing apoptosis. ( A ) Viability determination. H1975 cells transfected with EGFR shRNA or NS control for 24 hours were treated with 2 mg/mL of BJ extract for 12 hours and the collected cells counted by trypan blue exclusion assay. Symbol (-) meant no transfection. The results were expressed as mean values from three independent experiments. * P

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: EGFR shRNA reduced BJ sensitivities by suppressing apoptosis. ( A ) Viability determination. H1975 cells transfected with EGFR shRNA or NS control for 24 hours were treated with 2 mg/mL of BJ extract for 12 hours and the collected cells counted by trypan blue exclusion assay. Symbol (-) meant no transfection. The results were expressed as mean values from three independent experiments. * P

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: shRNA, Transfection, Trypan Blue Exclusion Assay

    The aqueous BJ extract increased apoptotic distributions of H1975 cells by ( A ) dose-dependent appearance of sub-G 1 cells. Human NSCLC cells H1975 cultured in 1% serum-supplemented DMEM were incubated with various concentrations of BJ extract (1, 2, and 5 mg/mL) or water for 12 hours before being labeled with PI followed by flow cytometry analysis. ( B ) Cell cycle distribution. The percentages of cell cycle distribution in H1975 cells following treatment were analyzed. The results of BJ treatment were compared with those of water control. * P

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: The aqueous BJ extract increased apoptotic distributions of H1975 cells by ( A ) dose-dependent appearance of sub-G 1 cells. Human NSCLC cells H1975 cultured in 1% serum-supplemented DMEM were incubated with various concentrations of BJ extract (1, 2, and 5 mg/mL) or water for 12 hours before being labeled with PI followed by flow cytometry analysis. ( B ) Cell cycle distribution. The percentages of cell cycle distribution in H1975 cells following treatment were analyzed. The results of BJ treatment were compared with those of water control. * P

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: Cell Culture, Incubation, Labeling, Flow Cytometry, Cytometry

    Dose-response growth curves of BJ extract in NSCLC H1975 cells. The increased concentration of aqueous BJ extracts after 12 hours of treatments reduced viable H1975 cells. The trypsinized cells were counted for the numbers of viable cells using trypan blue exclusion assay. The percentages of viabilities were obtained by comparing cell numbers at each concentration with water control that was assigned as 100%. The data are the average of quadruplicate for each experiment. The results are presented as mean ± SD of three independent experiments. * P

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: Dose-response growth curves of BJ extract in NSCLC H1975 cells. The increased concentration of aqueous BJ extracts after 12 hours of treatments reduced viable H1975 cells. The trypsinized cells were counted for the numbers of viable cells using trypan blue exclusion assay. The percentages of viabilities were obtained by comparing cell numbers at each concentration with water control that was assigned as 100%. The data are the average of quadruplicate for each experiment. The results are presented as mean ± SD of three independent experiments. * P

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: Concentration Assay, Trypan Blue Exclusion Assay

    Oral administration of BJ reduced xenograft tumor growth. ( A ) BJ extract suppressed the growth of xenograft tumors in animal models. Nude BALB/c mice with established xenograft tumors of H1975 cells were administered orally with 1 and 2 g BJ/kg daily for 7 consecutive days (arrows). The graph represented tumor growth variation ( y -axis) from the start of feeding ( x -axis). Mice fed with aqueous BJ extract showed reduced tumor growth compared with those with water. * P

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: Oral administration of BJ reduced xenograft tumor growth. ( A ) BJ extract suppressed the growth of xenograft tumors in animal models. Nude BALB/c mice with established xenograft tumors of H1975 cells were administered orally with 1 and 2 g BJ/kg daily for 7 consecutive days (arrows). The graph represented tumor growth variation ( y -axis) from the start of feeding ( x -axis). Mice fed with aqueous BJ extract showed reduced tumor growth compared with those with water. * P

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: Mouse Assay

    Histological and fluorescence examination of the reduced tumors. ( A ) Examination by HE staining. The paraffin-embedded H1975 tumors treated with BJ (2 g/kg) and water control were dissected, stained by HE and analyzed by confocal microscopy (scale bar =100 μm). The rectangular inset of the image were zoomed-in to the right of the panel for each treatment (scale bar =25 μm). The white arrow signifies apoptotic body location. ( B ) Release of mitochondrial cytochrome c in tumors treated with BJ. The dissected specimens of H1975 tumors as treated with BJ (1 and 2 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria signified the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 μm). ( C ) Images of immunofluorescent EGFR H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with EGFR antibody (green) followed by FITC-conjugated secondary antibody treatment before being counterstained with DAPI (blue) (scale bar =50 μm). ( D ) Images of immunofluorescent pEGFR Y1068 H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with pEGFR Y1068 antibody ( green ) followed by FITC-conjugated secondary antibody incubation before being counterstained with DAPI (blue) (scale bar =50 μm). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; FITC, fluorescein; HE, hematoxylin and eosin; TRITC, tetramethylrhodamine.

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: Histological and fluorescence examination of the reduced tumors. ( A ) Examination by HE staining. The paraffin-embedded H1975 tumors treated with BJ (2 g/kg) and water control were dissected, stained by HE and analyzed by confocal microscopy (scale bar =100 μm). The rectangular inset of the image were zoomed-in to the right of the panel for each treatment (scale bar =25 μm). The white arrow signifies apoptotic body location. ( B ) Release of mitochondrial cytochrome c in tumors treated with BJ. The dissected specimens of H1975 tumors as treated with BJ (1 and 2 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria signified the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 μm). ( C ) Images of immunofluorescent EGFR H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with EGFR antibody (green) followed by FITC-conjugated secondary antibody treatment before being counterstained with DAPI (blue) (scale bar =50 μm). ( D ) Images of immunofluorescent pEGFR Y1068 H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with pEGFR Y1068 antibody ( green ) followed by FITC-conjugated secondary antibody incubation before being counterstained with DAPI (blue) (scale bar =50 μm). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; FITC, fluorescein; HE, hematoxylin and eosin; TRITC, tetramethylrhodamine.

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: Fluorescence, Staining, Confocal Microscopy, Incubation, Mouse Assay

    BJ induced apoptosis and reduced EGFR and pEGFR Y1068 levels in H1975 cells. ( A ) Western blot analysis. The protein lysates from H1975 cells as treated with 1, 2, and 5 mg/mL of BJ extract for 12 hours were collected and used for Western blot analysis. The blots were incubated with various primary antibodies, including EGFR, phosphorylated EGFR Y1068 , Akt, phosphorylated Akt S473 , caspase-3, and PARP as specified, which were followed by HRP-conjugated secondary antibodies. GAPDH was used as loading control. The blots were visualized by ECL detection system. ( B ) Densitometric determination of EGFR and phosphorylated EGFR Y1068 amelioration. The densitometric ratios of EGFR and phosphorylated EGFR Y1068 in H1975 cells from Western blot analysis were obtained by first normalizing individual band intensity at each concentration to that of the loading control and compared with those of water treatment. The results were expressed as mean values of three independent experiments (* P

    Journal: Drug Design, Development and Therapy

    Article Title: The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor

    doi: 10.2147/DDDT.S117443

    Figure Lengend Snippet: BJ induced apoptosis and reduced EGFR and pEGFR Y1068 levels in H1975 cells. ( A ) Western blot analysis. The protein lysates from H1975 cells as treated with 1, 2, and 5 mg/mL of BJ extract for 12 hours were collected and used for Western blot analysis. The blots were incubated with various primary antibodies, including EGFR, phosphorylated EGFR Y1068 , Akt, phosphorylated Akt S473 , caspase-3, and PARP as specified, which were followed by HRP-conjugated secondary antibodies. GAPDH was used as loading control. The blots were visualized by ECL detection system. ( B ) Densitometric determination of EGFR and phosphorylated EGFR Y1068 amelioration. The densitometric ratios of EGFR and phosphorylated EGFR Y1068 in H1975 cells from Western blot analysis were obtained by first normalizing individual band intensity at each concentration to that of the loading control and compared with those of water treatment. The results were expressed as mean values of three independent experiments (* P

    Article Snippet: Cell culture Human NSCLC cells, including H1975 (two mutations in EGFR, L858R/T790M, erlotinib-insensitive), H3255 (one mutation in EGFR, L858R, erlotinib-sensitive), A549, H1299, and H460, were acquired from American Type Culture Collection (Manassas, VA, USA) and cultured in 75 cm2 tissue culture flasks.

    Techniques: Western Blot, Incubation, Concentration Assay