Journal: International Journal of Nanomedicine

Article Title: Differences in the Cell Type-Specific Toxicity of Diamond Nanoparticles to Endothelial Cells Depending on the Exposure of the Cells to Nanoparticles

doi: 10.2147/IJN.S411424

Figure Lengend Snippet: Diamond nanoparticles are cytotoxic to HUVEC but not to HS-5 and HMEC. HUVEC, HS-5, and HMEC 48-h proliferation ( A ) and 24-h membrane perforation ( B ) after treatment with NDs at concentrations of 5, 10, 20, 50, and 100 μg/mL. Statistical significance is indicated with asterisks: *P < 0.033, **P < 0.002, ***P < 0.001 (multifactor ANOVA; P < 0.05; n = 3 with 4 individual replicates for proliferation and 6 individual replicates for membrane perforation). All values are expressed as mean ± standard deviation. ( C ) HUVEC metabolic activity after treatment with NDs at concentrations of 5, 10, 20, 50, and 100 mg/l for 24 h. Statistical significance is indicated with different superscripts: P<0.001 (a, b, c, d, e) (ANOVA; P < 0.05; n = 3 with 6 individual replicates). ( D ) Live/Dead assay confocal microscopy images of nontreated HUVEC cells (“C”) and treated cells with 50 mg/l for 24 h (“ND”). The nuclei of dead cells are labelled with a green stain (NucGreen Dead 488), whereas all nuclei are stained with a red stain (NucRed Live 647). Images taken using Nomarski interference contrast (“NOM”) showed light-reflecting nanoparticle agglomerates, suggesting strong endocytosis of NDs.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human mammary epithelial cells (HMEC) were obtained from Thermo Fisher Scientific, whereas HS-5 cells were obtained from ATCC (LGC Standards, Łomianki, Poland).

Techniques: Membrane, Standard Deviation, Activity Assay, Live Dead Assay, Confocal Microscopy, Staining

Journal: International Journal of Nanomedicine

Article Title: Differences in the Cell Type-Specific Toxicity of Diamond Nanoparticles to Endothelial Cells Depending on the Exposure of the Cells to Nanoparticles

doi: 10.2147/IJN.S411424

Figure Lengend Snippet: Toxicity of diamond nanoparticles to HUVEC depends on cell density and surface conjugation of nanoparticles. ( A ) HUVEC 48-h proliferation was analysed for untreated cells (“C”) or cells treated with NDs (“ND”, “-RGD -FBS”) and NDs conjugated with a peptide containing an RGD motive (Gly-Arg-Gly-Asp-Ser; “ND”, “+RGD”) and at concentrations of 5 and 50 mg/l in standard HUVEC culture medium and in medium with the addition of 10% FBS (“+FBS”). Statistical significance is indicated with different superscripts: P<0.001 (a, b, c, d, e) (ANOVA; P < 0.05; n = 3 with 4 individual replicates). ( B ) Dependence of ND toxicity on HUVEC cell density was assessed in a 96-well plate at densities of 5 x 10 3 , 1 x 10 4 , 1.5 x 10 4 , and 2×10 4 . After 24-h incubation, cells were treated with NDs at a concentration of 0.4 ng/cell (the final ND concentration was 20, 40, 60, and 80 mg/L in 100 µL medium, respectively), and the metabolic activity was assessed. The relative toxicity of HUVEC was expressed as the ratio of the metabolic activity value of the ND-treated cells to the metabolic activity of the untreated cells. Statistical significance is indicated with different superscripts: P<0.001 (a, b, c, d) (ANOVA; P < 0.05; n = 3 with 6 individual replicates). All values are expressed as mean ± standard deviation. ( C ) HUVEC time-lapse images of a 12-h cell culture with NDs at a final concentration of 20 mg/l. The full video of time-lapse images is available as Supplementary Video . Abbreviations: RV, relative value; FUs, fluorescent units; C, control; NDs, diamond nanoparticles; RGD, Gly-Arg-Gly-Asp-Ser peptide; FBS, fetal bovine serum. ( D ) HUVEC spheroid morphology after 24-h treatment with NDs at a concentration of 10 and 100 mg/l. HUVEC spheroid membrane perforation ( E ) and spheroid size ( F ) for a after 24-h incubation with NDs at a concentration of 5, 10, 20, 50, and 100 mg/l. Statistical significance is indicated with different superscripts:: P<0.001 (a, b, c) (ANOVA; P < 0.05; n = 3 with 6 individual replicates).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human mammary epithelial cells (HMEC) were obtained from Thermo Fisher Scientific, whereas HS-5 cells were obtained from ATCC (LGC Standards, Łomianki, Poland).

Techniques: Conjugation Assay, Incubation, Concentration Assay, Activity Assay, Standard Deviation, Cell Culture, Control, Membrane

Journal: International Journal of Nanomedicine

Article Title: Differences in the Cell Type-Specific Toxicity of Diamond Nanoparticles to Endothelial Cells Depending on the Exposure of the Cells to Nanoparticles

doi: 10.2147/IJN.S411424

Figure Lengend Snippet: Diamond nanoparticles led to an NADPH-dependent induction of ROS in HUVEC. ( A ) Intercellular level of NADPH analysed in lysates of control HUVEC cells (“C”) and HUVEC cells incubated for 4 h with NDs at a concentration of 20 and 50 mg/l (“ND”). Statistical significance is indicated with different superscripts: P<0.001 (a, b, c) (ANOVA; P < 0.05; n = 2 with 4 individual replicates). ( B ) Merged images of mitochondrial superoxide staining (red channel; MitoSox Red, Thermo Fisher Scientific) and transmitted light with Nomarski interference contrast of control HUVEC cells (“C”) and HUVEC cells incubated for 4 h with NDs at a concentration of 50 mg/l (“ND 50”). ( C ) Confocal images of ROS and NO in control HUVEC cells (“C”) and HUVEC cells incubated for 4 h with NDs at a concentration of 20 and 50 mg/l (“ND 20”, “ND 50”) detected using the general oxidative stress indicator CM-H2CFDA (green channel; Thermo Fisher Scientific) and the NO synthesis level indicator DAF-FM (green channel; Thermo Fisher Scientific). Cell nuclei were stained with NucRed Live 647 (red channel; Thermo Fisher Scientific). Graphs showing ROS ( D ) and NO ( E ) levels expressed as a sum of the pixel values per cell. Statistical significance is indicated with different superscripts: P<0.001 (a, b) (ANOVA; P < 0.05; n = 2 with 7 individual replicates, and each well was imaged in 4 fields of view). All values are expressed as mean ± standard deviation.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human mammary epithelial cells (HMEC) were obtained from Thermo Fisher Scientific, whereas HS-5 cells were obtained from ATCC (LGC Standards, Łomianki, Poland).

Techniques: Control, Incubation, Concentration Assay, Staining, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Differences in the Cell Type-Specific Toxicity of Diamond Nanoparticles to Endothelial Cells Depending on the Exposure of the Cells to Nanoparticles

doi: 10.2147/IJN.S411424

Figure Lengend Snippet: NDs increase the synthesis level of stress-related proteins. ( A ) Antibody array analysis of cytokine synthesis in control HUVEC cells (“C”) incubated with NDs at a concentration of 20 mg/l for 24 h (“ND”). One assay consists of two membranes, which are labelled “I” and “II”, and the proteins are analysed in duplicate. The localization of the positive controls: I_A1,2; I_B1,2; I_H7,8; II_A1,2; II_B1,2; II_H7,8. The localization of the selected proteins in the order of their fold (log(2)) change shown in the graph ( B ): IL-6, I_D3,4; EGF, I_F1,2; VEGF, I_G5,6; TIMP2, I_E5,6; IL-8, I_E3,4; endostatin II_H1,2; IGF-1, I_C3,4; CCL-5, I_B5,6; PECAM-1, II_F5,6; VEGF-D, I_H5,6; uPAR, II_A7,8; VEGFR3, II_C7,8; PLG, II_G1,2; MCP-1, I_G3,4; CXCL11, II_A5,6; ANGPT1, II_E1,2; TIMP1, I_D5,6; GRO A, I_A1,2; MMP-1, II_D5,6; VEGFR2, II_B7,8; TIE-2, II_G5,6; bFGF, I_H1,2; MMP-9, II_E5,6; ANGPT2 II_F1,2. The full array map is available in Tables S1 and S2 . ( C ) Images of control HUVEC tube formation (“C”) and tube formation during treatment with NDs at a final concentration of 20 mg/l (“ND”). Graphs show the mean number of junctions of HUVEC tubes in the field of view ( D ), mean tube length in the field of view ( E ), and mean number of meshes in the field of view ( F ). Data were obtained by analysing the images using ImageJ software and the Angiogenesis Analyzer macro. Statistical significance is indicated with asterisks: *P = 0.003, P = 0.002 and P < 0.001, respectively ( t -test; P < 0.05; n = 2 with 4 individual replicates). All values are expressed as mean ± standard deviation.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human mammary epithelial cells (HMEC) were obtained from Thermo Fisher Scientific, whereas HS-5 cells were obtained from ATCC (LGC Standards, Łomianki, Poland).

Techniques: Ab Array, Control, Incubation, Concentration Assay, Software, Standard Deviation

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 1. The lncGCLC expression in MCLR-exposed cells and population samples. (A) Changes of lncGCLC expression in WRL68 cells after exposure to 0 or 10 µg/L of MCLR for 0, 10, 15, and 25 passages. * p < 0.05 compared with passage-matched control cells. (B) Comparison of lncGCLC expression in WRL68, HepG2, and SMMC7721 cells. ** p < 0.01 compared with WRL68 cells. (C) Com- parison of lncGCLC expression in tumor tissue and adjacent normal tissue from HCC patients with MC exposure (n = 30), ** p < 0.01. (D) LncGCLC expression in HCC patients with high MC exposure (serum MCs ≥0.14 µg/L, n = 17) was lower than that in those with low MC exposure (serum MCs < 0.14 µg/L, n = 13), ** p < 0.01. Data are presented as the mean ± SD of three independent experiments.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Comparison

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 2. Knockdown of lncGCLC promoted the proliferation of MCLR-treated WRL68 cells. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) The expression of lncGCLC was detected using qRT-PCR. (B) Cell proliferation activity was detected by CCK-8. (C) Cell apoptosis was analyzed using flow cytometry. (D) Apoptosis rate in each group. (E) The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow cytometry. (F) Cell cycle distribution quantification. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Transfection, Expressing, Quantitative RT-PCR, Activity Assay, CCK-8 Assay, Cytometry, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 3. Knockdown of lncGCLC promoted the migration and invasion of MCLR-treated WRL68 cells. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) Representative images of a cell migration assay (100×), scale bar = 200 µm. (B) Quantification of cell migration. (C) Representative images of a cell invasion assay (Original magnification ×100, scale bar = 200 µm). (D) Quantification of cell invasion. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Migration, Plasmid Preparation, Transfection, Cell Migration Assay, Invasion Assay, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 4. Knockdown of lncGCLC promoted the growth of MCLR-induced malignantly transformed WRL68 cells in nude mice. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) Representative images of a soft agar assay (Original magnifica- tion ×200, scale bar = 100 µm). (B) Quantification of colony formation in soft agar. (C) Tumorigenicity test of BALB/c nude mice was performed. Solid tumors were removed after the sacrifice at 22 days. (D) Representative image showing subcutaneous tumor size. (E) The weights of solid tumors. The values given are mean ± SD (n = 3 for both male and female nude mice per group). (F) Tumor volume was monitored every 3 days after injection of WRL68 cells. (G) Pathological changes of the tumor tissue in nude mice in each group (HE stains, original magnification ×400, scale bar = 50 µm). ND—not detected. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Transformation Assay, Plasmid Preparation, Transfection, Soft Agar Assay, Injection, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 5. Verification of the interrelationships among lncGCLC, miR-122-5p, and GCLC. (A) The mRNA expression level of neighboring genes which are located nearly 1.5 Mb downstream of lncGCLC in lncGCLC knockdown cells. * p < 0.05 and ** p < 0.01 compared with vector control cells. (B) The expression level of lncGCLC in the nuclear and cytoplasmic fractions of WRL68 cells. (C) The correlation between the lncGCLC expression and the GCLC expression in HCC tissues (n = 30). Correlation coefficient (r) and P value were calculated by Pearson correlation analysis. (D) Comparison of GCLC expression in tumor tissue and adjacent normal tissue from HCC patients with MC exposure (n = 30), ** p < 0.01. (E) GCLC expression in HCC patients with high MC exposure (serum MCs ≥0.14 µg/L, n =17) was lower than in those with low MC exposure (serum MCs < 0.14 µg/L, n = 13), ** p < 0.01. (F) After exposure to 10 µg/L of MCLR for 0, 10, 15, and 25 passages, expression of lncGCLC, miR-122-5p and GCLC mRNA was detected in WRL68 cells. * p < 0.05 and ** p < 0.01 compared with passage-matched control cells. (G,H) Expression of GCLC protein in P0, P10, P15 and P25 MCLR-induced malignantly transformed WRL68 cells. ** p < 0.01 compared with passage-matched control cells. (I) Changes of miR-122-5p in both vector- and sh-lncGCLC-transfected WRL68 cells treated with 0 or 10 µg/L of MCLR for 25 passages. (J–L) Changes of GCLC mRNA (J) and protein (K,L) expression in both vector- and sh-lncGCLC-transfected WRL68 cells treated with or without 10 µg/L of MCLR for 25 passages. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Knockdown, Plasmid Preparation, Control, Comparison, Transformation Assay, Transfection

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 6. Knockdown of lncGCLC reduced GSH levels and induced oxidative DNA damage in MCLR-treated WRL68 cells. Alterations in GCL activity (A), GSH (B), and 8-OHdG content (C) in vector- or sh-lncGCLC-transfected cells exposed to 0 or 10 µg/L of MCLR for 25 passages. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Activity Assay, Plasmid Preparation, Transfection, Control

Journal: Molecular pharmaceutics

Article Title: Targeting Triple Negative Breast Cancer with a Nucleus-Directed p53 Tetramerization Domain Peptide

doi: 10.1021/acs.molpharmaceut.0c00978

Figure Lengend Snippet: Cy5p53Tet penetrates into MDA-MB-468 TNBC cells expressing mtp53 R273H. (A) Schematic of the structure of Cy5p53Tet. (B) Live cell imaging staining of MCF7 and MDA-MB-468 cells after 2 h of incubation with 500 nM Cy5p53Tet (red). Hoechst staining (blue) was used to stain the nuclei. Two independent experiments with biological replicates were performed. (C) p53 protein levels in MCF7 and MDA-MB-468 cells determined by Western blot analysis before carrying out live cell imaging. (D) Quantification of Cy5p53Tet uptake in MCF7 and MDA-MB-468 cells via Nikon Element analysis. At least 200 cells per sample were measured by fluorescence microscopy. (E) Flow cytometry of MCF7 and MDA-MB-468 cells after incubation with 100 or 500 nM Cy5p53Tet for 2 h at 37 °C. FlowJo software was used to analyze the cytometric data. (F) Geometric MFI from the FACS experiments in (E). (G) MTT assay conducted in MCF7, MDA-MB-468, HCC70, SK-BR-3, and MCF10A cells to measure mitochondrial dehydrogenase activity in response to 500 nM Cy5p53Tet treatment for 24 h. Three independent experiments with biological replicates were performed for all ± SEM *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001.

Article Snippet: Human breast cancer cell lines MCF7, MDA-MB-468, MDA-MB-231, HCC70, and SK-BR-3 and normal human mammary epithelial cell MCF10A were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Live Cell Imaging, Staining, Incubation, Western Blot, Fluorescence, Microscopy, Flow Cytometry, Software, MTT Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Axin Inhibits Extracellular Signal-regulated Kinase Pathway by Ras Degradation via β-Catenin

doi: 10.1074/jbc.m611129200

Figure Lengend Snippet: FIGURE 5. Effect of Axin overexpression in cells either retaining wild-type or mutant -catenin gene. A, effect of Axin on Ras-ERK pathway activation in cells that retained a different genetic status of -catenin gene. Cells that retained wild-type -catenin gene (DLD-1 or SW-480 colorectal cancer cells, and Chang liver cells) or that retained mutated -catenin gene (HCT-116 or HepG2 cells) were transfected with the pCS2-MT vector or pCS2-MT-Axin. The levels of Axin, -catenin, p-Raf-1, p-MEK, p-ERK, ERK, -tubulin, and Pan-Ras were detected by Western blot analyses at 48 h after transfection. B, immunocytochemical analysis of Ras regulation by Axin. L929-GFP-Axin cells were grown in DMEM and infected with retroviral H-ras, and cells were induced 0.5 g/ml Dox for 0, 2, 8, and 24 h. Cells were incubated with anti-Pan-Ras antibody followed by labeling with anti-mouse rhodamine for Ras detection. GFP-Axin was visualized as GFP by confocal microscopic analysis. Cell nuclei were stained with DAPI. Bar, 10 m. C, effect of -catenin gene siRNA on Ras-ERK pathway regulation in HCT-116 cells that retained mutated -catenin gene. HCT116 cells were transfected with the pCS2-MT vector or pCS2-MT-Axin together with (or without) control or -catenin gene siRNA. The levels of Axin, -catenin, Pan-Ras, p-Raf-1, p-MEK, p-ERK, and total ERK were detected by Western blot analyses 48 h after transfection. D, effect of -catenin gene-S33Y, a non-degradable form of -catenin, on Ras-ERK pathway regulation in HCT-116 cells. HCT116 cells were transfected with the pCS2-MT vector or pCS2-MT-Axin together with (or without) the pcDNA3.0 vector or FLAG-S33Y--catenin gene-pcDNA3.0. The levels of Axin, -catenin, Pan-Ras, p-ERK, and total ERK were detected by Western blot analyses 24 h after transfection.

Article Snippet: After induction, the cells were examined under a phase-contrast fluorescence microscope (Nikon, TE-2000U, Japan) for visualization of morphology and expression of GFP or AxinGFP.DLD-1,HCT-116, and SW-480 colorectal cancer cells and Chang normal liver and HepG2 hepatoma cancer cells were obtained from the American Type Culture Collection and cultured as previously described (24).

Techniques: Over Expression, Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Infection, Retroviral, Incubation, Labeling, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Axin Inhibits Extracellular Signal-regulated Kinase Pathway by Ras Degradation via β-Catenin

doi: 10.1074/jbc.m611129200

Figure Lengend Snippet: FIGURE 6. Effect of Axin overexpression on Ras-ERK pathway activation by H-RasL61 and effect of EGFR knockdown on -catenin and Ras-ERK pathway regulation by Axin. A, effect of Axin on Ras-ERK pathway activation in cells that retained wild-type or overexpressed non-degradable mutant Ras. Chang liver (left) and HepG2 (right) cells were grown and transiently transfected with a combination of pCMV, pAxin, and pMT3-H- RasL61.TheGTPloadinganalysiswasperformedtodetectGTP-boundRas(Ras-GTP),asdescribedunder“Exper- imental Procedures.” The levels of Pan-Ras-GTP, p-ERK, Axin, -catenin, Pan-Ras, p-Akt, and -tubulin were detected by Western blot analyses. B, effect of EGFR siRNA on -catenin and Ras-ERK pathway regulation by Axin. L929-GFP-Axin cells were transfected with control or EGFR siRNA. The cells were treated with 0.5 g/ml Dox and/or 20 ng/ml EGF for 12 h and 10 min, respectively, before harvesting them in the required case. The levels of Axin, p-EGFR, Pan-Ras, p-Raf-1, p-MEK, p-ERK, ERK, -catenin, ABC (anti-active -catenin) (37), or p-p38 were detected by Western blot analyses. The p-EGFR blot was obtained by Western blot analyses with the samples immunoprecipitated by 2 g of anti-EGFR antibody against 600 g of total lysates with Protein A beads.

Article Snippet: After induction, the cells were examined under a phase-contrast fluorescence microscope (Nikon, TE-2000U, Japan) for visualization of morphology and expression of GFP or AxinGFP.DLD-1,HCT-116, and SW-480 colorectal cancer cells and Chang normal liver and HepG2 hepatoma cancer cells were obtained from the American Type Culture Collection and cultured as previously described (24).

Techniques: Over Expression, Activation Assay, Knockdown, Mutagenesis, Transfection, Western Blot, Control, Immunoprecipitation