Journal: eLife

Article Title: Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates p53 function in suppressing metastasis

doi: 10.7554/eLife.10727

Figure Lengend Snippet: ( A ) GLS2-Flag preferentially interacted with the DN Myc-Rac1-T17N but not the CA Myc-Rac1-G12V in Huh-1 cells. Cells were transduced with GLS2-Flag vectors together with Rac1-T17N or Rac1-G12V vectors for co-IP assays. ( B ) GLS2-Flag preferentially bound to Rac1-GDP but not Rac1-GTP in cell lysates. Cell lysates from Huh-1 cells transduced with vectors expressing Myc-Rac1 and GLS2-Flag were pretreated with GDP or GTPγS to convert Rac1 into Rac1-GDP or Rac1-GTP form before co-IP assays. ( C ) Ectopic expression of GLS2 inhibited Rac1 activities represented by decreased levels of Rac1-GTP in HCC cells measured by the GST-p21-binding domain of PAK1 pull-down assays. Left panels: Represented results of Rac1 activity analysis in Huh-1 and HepG2 cells. Right panels: relative Rac1-GTP/total Rac1/Actin levels in Huh-1, HepG2, Hep3B and Huh-7 cells. Data present mean ± SD (n=3). * p <0.01; Student’s t -test. ( D ) Knockdown of GLS2 by shRNA vectors increased Rac1 activities in HCC cells. Left panels: Represented results of Rac1 activity analysis in Huh-1 and HepG2 cells. Right panels: relative Rac1-GTP/total Rac1 /Actin levels in Huh-1, HepG2, Hep3B and Huh-7 cells. Data present mean ± SD (n=3). * p <0.01; # p <0.05; Student’s t -test. ( E ) Ectopic expression of GLS2-Flag decreased the levels of p-PAK1 at Ser199/204 in Huh-1 and HepG2 cells. ( F ) Knockdown of GLS2 by shRNA vectors increased the levels of p-PAK1 at Ser199/204 in Huh-1 and HepG2 cells. ( G ) The C-terminus of GLS2, GLS2-C139, interacted with DN Myc-Rac1-T17N but not CA Myc-Rac1-G12V in Huh-1 cells detected by co-IP assays. ( H ) The C-terminus of GLS2, GLS2-C139, inhibited the Rac1 activity in Huh-1 and HepG2 cells. Left panels: Represented results of Rac1 activity analysis in Huh-1 cells transduced with different GLS2-Flag vectors. Right panels: relative Rac1-GTP/total Rac1/Actin levels in Huh-1 and HepG2. Data present mean ± SD (n=3). * p <0.01; # p <0.05; Student’s t -test. GDP, guanosine 5′-diphosphate; GLS, glutaminase; GTP, guanosine 5'-triphosphate; HCC, hepatocellular carcinoma; IP, immunoprecipitation; shRNA, short hairpin RNA; WT, wild type. DOI: http://dx.doi.org/10.7554/eLife.10727.004

Article Snippet: HepG2 (p53-WT) and Hep3B (p53-null) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transduction, Co-Immunoprecipitation Assay, Expressing, Binding Assay, Activity Assay, Knockdown, shRNA, Immunoprecipitation

Journal: eLife

Article Title: Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates p53 function in suppressing metastasis

doi: 10.7554/eLife.10727

Figure Lengend Snippet: ( A ) Ectopic expression of GLS2-Flag inhibited Rac1 activities represented by decreased Rac1-GTP levels in Hep3B and Huh-7 cells. ( B ) Knockdown of GLS2 by 2 different shRNA vectors in different HCC cells detected by Taqman real-time Polymerase chain reaction assays. The messenger RNA expression of GLS2 was normalized with Actin. Data are presented as mean ± SD (n=3). ** p <0.001; Student’s t -test. ( C ) Knockdown of endogenous GLS2 by shRNA enhanced Rac1 activities represented by increased Rac1-GTP levels in Hep3B and Huh-7 cells. GLS2 knockdown was presented in . ( D ) Ectopic expression of GLS1 did not clearly affect the Rac1 activity in Huh-1 or HepG2 cells. ( E ) Knockdown of endogenous GLS1 by shRNA vectors did not clearly affect the Rac1 activity in Huh-1 or HepG2 cells. In D and E: data present mean ± SD (n=3). GLS, glutaminase; GTP, guanosine 5'-triphosphate; HCC, hepatocellular carcinoma; shRNA, short hairpin RNA DOI: http://dx.doi.org/10.7554/eLife.10727.005

Article Snippet: HepG2 (p53-WT) and Hep3B (p53-null) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Knockdown, shRNA, Real-time Polymerase Chain Reaction, RNA Expression, Activity Assay

Journal: International journal of oncology

Article Title: The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

doi: 10.3892/ijo.2015.3087

Figure Lengend Snippet: Figure 1. Effect of panobinostat (LBH589) on gene expression of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and fms-like tyrosine kinase-1 (FLT-1) in vitro in HepG2 and Hep3B cells. (A) Quantitative real-time RT-PCR analysis after 24, 48 and 72 h treatment with 0.01 µM panobinostat (A) and 0.1 µM panobinostat (B) in HepG2 cells, with 0.01 µM panobinostat (C) and 0.1 µM panobinostat (D) in Hep3B cells. Gene expression was normalized to β-actin and expressed relative to untreated controls (set at 1.0). Asterisks indicate statistically significant differences in gene expression vs. the untreated controls (p<0.01). The symbol (x) indicates that very few viable cells remained after 72 h of treatment in these cells.

Article Snippet: The human hepatoma cell lines HepG2 (p53wt) and Hep3B (p53null) were obtained from the German collection of microorganisms and cell cultures (DSMZ, braunschweig, Germany) and maintained under standard conditions as described previously (41).

Techniques: Gene Expression, In Vitro, Quantitative RT-PCR

Journal: International journal of oncology

Article Title: The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

doi: 10.3892/ijo.2015.3087

Figure Lengend Snippet: Figure 2. Western blot analysis of expression of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase-1 (FLT-1), kinase insert domain containing receptor (KDR), MAPK, p-MAPK in vitro and densitometric quantification. HepG2 and Hep3B cells were incubated with 0.1 and 0.01 µM panobinostat for 12, 24 and 48 h. Western blot results show representative examples for expression of CTGF, VEGF, FLT-1, KDR, MAPK and p-MAPK as well as β-actin, which served as loading control (A). Protein expression analyzed using western blot analysis was quantified by densitometry (B). Shown are expression values for CTGF, MAPK, p-MAPK, KDR and VEGF after treatment of HepG2 and Hep3B cell lines with 0.01 and 0.1 µM of panobinostat for 12-48 h.

Article Snippet: The human hepatoma cell lines HepG2 (p53wt) and Hep3B (p53null) were obtained from the German collection of microorganisms and cell cultures (DSMZ, braunschweig, Germany) and maintained under standard conditions as described previously (41).

Techniques: Western Blot, Expressing, In Vitro, Incubation, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) p53; (b) ATM in HepG2 and Hep3B cell lines. Single asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisks indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: HepG2 (p53 ++ , carrying wild-type p53) and Hep3B (p53 −− carrying p53 null) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India.

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) cytoplasmic PARP-1; (b) nuclear PARP-1 in HepG2 and Hep3B cell lines. Single asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisk indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: HepG2 (p53 ++ , carrying wild-type p53) and Hep3B (p53 −− carrying p53 null) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India.

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) cytoplasmic AIF; (b) nuclear AIF in HepG2 and Hep3B cell lines. Single asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisks indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: HepG2 (p53 ++ , carrying wild-type p53) and Hep3B (p53 −− carrying p53 null) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India.

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) cytochrome c ; (b) Apaf-1; (c) caspase-9; (d) caspase-3 in HepG2 and Hep3B cell lines. Single asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisks indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: HepG2 (p53 ++ , carrying wild-type p53) and Hep3B (p53 −− carrying p53 null) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India.

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) ICAD; (b) cytoplasmic CAD; (c) nuclear CAD in HepG2 and Hep3B cell lines. Asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisks indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: HepG2 (p53 ++ , carrying wild-type p53) and Hep3B (p53 −− carrying p53 null) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India.

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: Dose–response curves of BJ extract in liver cancer cell lines. Notes: ( A ) BJ inhibited growth of Hep3B cells. Liver cancer cells Huh7, Hep3B, and HepG2 were cultured in 96-well plate for 28 hours, then treated with various concentrations (0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, and 1.0 mg/mL) of aqueous BJ extract or water for 12 hours, and stained with MTT. The cell viabilities were converted from the absorbance of drug-treated cells against that of water control. The relative viabilities were obtained by conversion into percentages compared with water treatment control as 100%. The data are the averages of quadruplicate. The results were represented as mean ± SD of three independent experiments. * P <0.05 and ** P <0.01. ( B ) BJ induced sub-G 1 populations. Hep3B, Huh7, and HepG2 cells cultured in 1% serum-supplemented RPMI-1640 were incubated with various concentrations of BJ (0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL and 1.0 mg/mL) and water control for 12 hours before being labeled with PI followed by flow cytometry analysis. ( C ) Two-dimensional flow analysis. Hep3B cells were treated with BJ (0.1 mg/mL, 0.2 mg/ml, 0.5 mg/mL, and 1.0 mg/mL) or water for 12 hours and the trypsinized cells were analyzed by Annexin V/PI-stained flow cytometry. ( D ) Quantitative analysis of Annexin V-positive cell populations. Hep3B cells were treated with various concentrations (0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, and 1.0 mg/mL) of BJ as indicated for 12 hours and the trypsinized cells were analyzed by flow cytometry. The early (dark) and late (light) apoptotic population distributions were expressed as mean values from three independent experiments. Abbreviations: BJ, Brucea javanica ; FITC, fluorescein isothiocyanate; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide; PI, propidium iodide; RPMI, Roswell Park Memorial Institute; SD, standard deviation.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Culture, Staining, Control, Incubation, Labeling, Flow Cytometry, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: Release of cytochrome c from mitochondria. Notes: Hep3B cells treated with various concentrations of BJ (0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, and 1.0 mg/mL) and water control for 6 hours were fixed and incubated with antibody against cytochrome c followed by secondary antibody conjugated with TRITC (red). The slides were counterstained with Mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The arrows indicate the colocalization of red color cytochrome c and green color mitochondria, while blue color indicates nucleus (scale bar =10 µm and applies to all images). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; TRITC, tetramethylrhodamine isothiocyanate.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Control, Incubation, Confocal Microscopy

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: Effects of BJ on apoptotic features in Hep3B cells. Notes: Liver cancer cells Huh7, Hep3B, and HepG2 cultured in 1% serum-supplemented DMEM or RPMI-1640 and incubated with various concentrations of BJ (0.1 mg/mL, 0.5 mg/mL, and 1.0 mg/mL) plus vehicle control H 2 O for 12 hours were collected. The protein lysates were conducted for Western blot analysis. The blots were incubated with various primary antibodies, including EGFR, Akt, phosphorylated Akt S473 , caspase-9, caspase-3, Bcl-2, and PARP followed by horseradish peroxidase-conjugated secondary antibodies with GAPDH as loading control. The blots were visualized by ECL staining system. The values shown underneath each blot indicate relative intensities compared with the results of ddH 2 O treatment for each cell line. Abbreviations: BJ, Brucea javanica ; DMEM, Dulbecco’s Modified Eagle’s Medium; ECL, enhanced chemiluminescence; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly(adenosine diphosphate-ribose) polymerase; RPMI, Roswell Park Memorial Institute.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Culture, Incubation, Control, Western Blot, Staining, Modification

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: BJ suppressed the growth of Hep3B spheroids. Notes: ( A ) Effects of BJ on the growth of multicellular spheroids. Multicellular spheroids were prepared. The representative image of Hep3B spheroid in suspension treated at each condition with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ for 12 hours, 24 hours, and 48 hours, respectively, was taken under an inverted microscope (scale bar =150 µm and applies to all images). ( B ) Representative colony images of soft agar assay. The spheroids derived from Huh7, Hep3B, and HepG2 cells that were treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ, respectively, for 24 hours, were seeded and grown in soft agar for 28 days and stained with crystal violet. Each representative image of colony grown in soft agar at different concentrations and water control was taken from an inverted microscope (scale bar =150 µm and applies to all images). ( C ) Soft agar colony forming assay. Soft agar plates of Huh7, Hep3B, and HepG2 spheroids as treated with various concentrations of BJ extract were incubated at 37°C for 28 days before being stained with 0.002% crystal violet. Colonies with >50 cells were counted as positive. ( D ) Statistical analysis. The numbers of colonies of soft agar formation of liver cancer spheroids from Huh7, Hep3B, and HepG2 cells treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ were counted and plotted. * P <0.05 and ** P <0.01 indicates significant difference between treatment groups and water control as determined from three independent experiments. Abbreviation: BJ, Brucea javanica .

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Suspension, Inverted Microscopy, Soft Agar Assay, Derivative Assay, Staining, Control, Incubation

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: BJ induced apoptotic characteristics in Hep3B spheroids. Notes: ( A ) Reduction of BrdU incorporation. Hep3B spheroids were treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ, respectively, for 24 hours followed by evaluation with BrdU incorporation assay. The merged images of green BrdU and blue nucleus marker DAPI were taken by a fluorescence microscope (scale bar =100 µm and applies to all images). ( B ) BJ reduced BrdU incorporation. The numbers of fluorescence positive spheres with incorporated BrdU were counted in each field of different concentrations. The numbers as counted were the average of at least three different fields. The data are expressed as mean ± SD of three individual experiments (* P <0.05). ( C ) Dose-dependent increase of TUNEL staining. The spheroids were treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ, respectively, for 24 hours followed by TUNEL staining evaluation. The merged images of green TUNEL and blue nucleus marker DAPI were taken by a fluorescence microscope (scale bar =100 µm and applies to all images). ( D ) BJ increased TUNEL positive nuclei. The numbers of fluorescent TUNEL positive spheres in each field of BJ concentration of 2.0 and 6.0 mg/mL were counted. The numbers counted were the average of at least three different fields. The data are expressed as mean ± SD of three individual experiments (* P <0.05). Abbreviations: BJ, Brucea javanica ; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: BrdU Incorporation Assay, Marker, Fluorescence, Microscopy, TUNEL Assay, Staining, Concentration Assay, End Labeling

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: BJ reduced stemness marker of Hep3B spheres. Notes: ( A ) Protein lysates from Hep3B spheroids treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ extract for 12 hours were resolved by SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were analyzed for expressions of different stemness markers. They included Nanog, CD133, EpCAM, and ALDH1A1 with GAPDH as loading control. The values shown underneath each blot signify relative intensities compared with the results for vehicle control water treatment. ( B ) Merged images of immunofluorescent detection with CD133 antibody (green) of Hep3B spheroids after BJ treatment at 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ for 24 hours after counterstaining with DAPI (blue) (scale bar =100 µm and applies to all images). ( C ) Merged images of immunofluorescent detection with Nanog antibody (green) of Hep3B spheroids after BJ treatment with counterstaining of DAPI (blue) (scale bar =100 µm and applies to all images). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Marker, SDS Page, Control, Polyacrylamide Gel Electrophoresis

Journal: Drug Design, Development and Therapy

Article Title: The aqueous extract of Chinese medicinal herb Brucea javanica suppresses the growth of human liver cancer and the derived stem-like cells by apoptosis

doi: 10.2147/DDDT.S107909

Figure Lengend Snippet: BJ increased apoptotic characteristics of Hep3B spheres. Notes: ( A ) Protein lysates from Hep3B spheroids treated with 2.0 mg/mL, 4.0 mg/mL, and 6.0 mg/mL of BJ extract for 12 hours were resolved and transferred. The blots were analyzed for expressions of PARP, Akt, and caspase-3 with GAPDH as loading control. The values shown underneath each plot signify relative intensities compared with the results of vehicle control water treatment. ( B ) Merged images of immunofluorescent detection with EGFR antibody (green) of Hep3B spheroids after BJ treatment for 24 hours and DAPI counterstaining (blue) (scale bar =100 µm and applies to all images). Abbreviations: BJ, Brucea javanica ; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly(adenosine diphosphate-ribose) polymerase.

Article Snippet: Human hepatocellular carcinoma cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Control