Mal hepatocyte cell line (L02). Tubulin expression levels were used as internal controls. * indicates p , 0.05. The experiments were performed independently three times at least. doi:10.1371/journal.pone.0055615.g^2Klotho Suppresses Tumor Growth in HCC IIHC staining was quantitatively analysed using Axio-Vision MedChemExpress AKT inhibitor 2 computerized image analysis system assisted with the automatic measurement program (Carl Zeiss Jena Gmbh, Jena Germany). Briefly, the stained sections were evaluated at 2006 magnification and ten representative staining fields of each section were analysed to verify the mean optical density (MOD), which represented the strength of staining signals as measured per positive pixel. All the experiments were performed independently three times at least.performed with the use of Multi Cycle for Windows (Phoenix Flow Systems, San Diego, Calif.). Experiments were repeated in triplicate. Average values and standard deviation statistical analyses were computed.In Vivo Tumorigenesis AssayHep3B or SMMC-7721cells (56106 cells suspended in 100 ml PBS) transfected with vector, bKlotho or bKlotho plus myr-Akt were injected subcutaneously into the dorsal left flank of 4-weekold male Balb/c nude mice. Tumor diameter was measured every 2? days for 4 weeks. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor and calculated using the following formula: volume = 0.56(shortest diameter)26(longest diameter)[23]. Mice were sacrificed and the tumor weights were measured. All the experiments were performed independently three times at least.Western BlottingCells or tissues were lysed for total protein extraction in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 NP-40, 0.25 Na-deoxycholate, 1 mM EDTA, 1 mM NaF) together with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Proteins were separated by 10 SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride CB-5083 supplier membranes (Amersham Life Science, Piscataway, NJ). Membranes were incubated for an hour in a blocking buffer containing 5 nonfat dry milk and then probed with antibodies against bKlotho (Abcam, Cambridge, MA), phosphoAkt Ser473, total Akt, phospho-GSK-3b Ser9, total GSK-3b, total cyclin D1(Cell Signaling Technology, Inc, Danvers, MA) and tubulin (Sigma-Aldrich, St. Louis, MO) as indicated. The bound primary antibodies were then probed with respective secondary antibodies labeled with horseradish peroxidase. Immunolabeled proteins were detected by using the ECL system (Amersham Life Science, Piscataway, NJ). Band intensities were quantified using NIH ImageJ software. All the experiments were performed independently three times at least.Statistical AnalysisData were presented as the mean 6 SD error of the mean. Student’s t test was used for comparison among different groups. The correlation of bKlotho expression with various clinicopathologic parameters were calculated with x2 test. The difference in tumor growth rate between the two groups of nude mice was determined by repeated-measures analysis of variance. p , 0.05 was considered statistically significant.Colony Formation AssayCells were transfected with either vector or bKlotho. Two days following transfection, the Hep3B or SMMC-7721 cells were stripped and plated on 6-well culture dishes, and G418 (500 mg/ ml, Sigma-Aldrich, St. Louis, MO) was added to the culture media to select the transfected cells. Every 3 days the medium w.Mal hepatocyte cell line (L02). Tubulin expression levels were used as internal controls. * indicates p , 0.05. The experiments were performed independently three times at least. doi:10.1371/journal.pone.0055615.g^2Klotho Suppresses Tumor Growth in HCC IIHC staining was quantitatively analysed using Axio-Vision computerized image analysis system assisted with the automatic measurement program (Carl Zeiss Jena Gmbh, Jena Germany). Briefly, the stained sections were evaluated at 2006 magnification and ten representative staining fields of each section were analysed to verify the mean optical density (MOD), which represented the strength of staining signals as measured per positive pixel. All the experiments were performed independently three times at least.performed with the use of Multi Cycle for Windows (Phoenix Flow Systems, San Diego, Calif.). Experiments were repeated in triplicate. Average values and standard deviation statistical analyses were computed.In Vivo Tumorigenesis AssayHep3B or SMMC-7721cells (56106 cells suspended in 100 ml PBS) transfected with vector, bKlotho or bKlotho plus myr-Akt were injected subcutaneously into the dorsal left flank of 4-weekold male Balb/c nude mice. Tumor diameter was measured every 2? days for 4 weeks. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor and calculated using the following formula: volume = 0.56(shortest diameter)26(longest diameter)[23]. Mice were sacrificed and the tumor weights were measured. All the experiments were performed independently three times at least.Western BlottingCells or tissues were lysed for total protein extraction in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 NP-40, 0.25 Na-deoxycholate, 1 mM EDTA, 1 mM NaF) together with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Proteins were separated by 10 SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Amersham Life Science, Piscataway, NJ). Membranes were incubated for an hour in a blocking buffer containing 5 nonfat dry milk and then probed with antibodies against bKlotho (Abcam, Cambridge, MA), phosphoAkt Ser473, total Akt, phospho-GSK-3b Ser9, total GSK-3b, total cyclin D1(Cell Signaling Technology, Inc, Danvers, MA) and tubulin (Sigma-Aldrich, St. Louis, MO) as indicated. The bound primary antibodies were then probed with respective secondary antibodies labeled with horseradish peroxidase. Immunolabeled proteins were detected by using the ECL system (Amersham Life Science, Piscataway, NJ). Band intensities were quantified using NIH ImageJ software. All the experiments were performed independently three times at least.Statistical AnalysisData were presented as the mean 6 SD error of the mean. Student’s t test was used for comparison among different groups. The correlation of bKlotho expression with various clinicopathologic parameters were calculated with x2 test. The difference in tumor growth rate between the two groups of nude mice was determined by repeated-measures analysis of variance. p , 0.05 was considered statistically significant.Colony Formation AssayCells were transfected with either vector or bKlotho. Two days following transfection, the Hep3B or SMMC-7721 cells were stripped and plated on 6-well culture dishes, and G418 (500 mg/ ml, Sigma-Aldrich, St. Louis, MO) was added to the culture media to select the transfected cells. Every 3 days the medium w.
