Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 2. YAP1 and TAZ regulate expression of SLC38A1 and SLC7A5 in HCC cells. (A) Expression pattern of SLC family genes from microarray data. Amino acid transporters are shown in red. (B-E) The indicated cells were transfected with the indicated siRNAs. Cells were used for quanti- tative RT-PCR to measure SLC38A1 and SLC7A5. Expression level was normalized with siLuc samples. (F) Quantitative RT-PCR was done with Mst1/2 knockout samples to measure connective tissue growth factor, SLC38A1, and SLC7A5 messenger RNA expression level. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: CTGF, connective tissue growth factor; Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Microarray, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knock-Out, RNA Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 4. Amino acid transporters are important for proliferation of HCC cells. (A-D) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs, and cell proliferation rates were measured by an MTT assay at the indicated time points. Values shown were normalized to siLuc-treated cells and represent mean 6 standard deviation. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E) Recovered cell growth by exogenous SLC38A1 and SLC7A5 in YAP1/TAZ-depleted SK-Hep1 cells. Myc-tagged exogenous SLC38A1 and SLC7A5 were expressed in SK-Hep1 cells. Empty expression vector pCMV6 was used as a control. Cell proliferation rates were measured by the MTT assay at 72 hours after transfection of expression vectors. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (F,G) Tumor weight after treat- ment with siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in a subcutaneous xenograft model (F) or an orthotopic xenograft model (G) with SK-Hep1 HCC cells. At 6 weeks after siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine injection, mice were killed and tumor weights measured (n 5 10 per group). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: MTT Assay, Standard Deviation, Expressing, Plasmid Preparation, Control, Transfection, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 5. YAP1 and TAZ directly bind to the promoters of amino acid transporters. (A) Schematic representation of SLC38A1 promoter regions for ChIP assay (top) and ChIP assay results (bottom). (B) Schematic representation of SLC7A5 promoter regions for ChIP assay (top) and ChIP assay results (bottom). ChIP assay was done in SK-Hep1 cells with indicated antibodies. Recruitment of YAP1, TAZ, and TEAD1 proteins to the SLC38A1 or SLC7A5 promoter was analyzed using primers specific to the SLC38A1 or SLC7A5 promoter. Immunoglobulin G was used as an internal control. (C,D) ChIP samples were used for quantitative RT-PCR for quantification of binding in indicated cell lines. Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E,F) SK-Hep1 and SNU-449 cells were transi- ently transfected with indicated complementary DNA and reporter plasmids, and luciferase activity was measured by a luminometer (Promega). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: IgG, immunoglobulin G; IP, immunoprecipitation; TBS, TEAD binding site.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Control, Quantitative RT-PCR, Binding Assay, Transfection, Luciferase, Activity Assay, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 6. mTORC1 is regulated by SLC38A1 and SLC7A5 in HCC cells. (A) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs for 96 hours. Phosphorylation of S6K1 was assessed by western blotting with the indicated antibodies. (B) SK-Hep1 cells were treated with esterified glutamine (Gln, 4 mM) for 30 minutes after silencing of SLC38A1 with specific siRNA. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with indicated antibodies. (C,D) Xeno- grafted HCC tissues treated with the indicated siRNAs were used for western blot analysis with the indi- cated antibodies.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 7. YAP1 and TAZ activate mTORC1 through regulation of SLC38A1 and SLC7A5 (A,B). SK- Hep1 (A) and SNU-449 (B) HCC cells were transiently transfected with the indicated siRNA for 96 hours. Expression of YAP1 and TAZ and phosphorylation of S6K1 and S6 were assessed by western blot- ting with the indicated antibodies. (C) SK-Hep1 cells were transfected with SLC38A1 and SLC7A5 com- plementary DNA after silencing YAP1 and TAZ with specific siRNAs. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with the indicated antibodies. (D) Expres- sion of Mst1, Mst2, S6K1, and S6 and phosphorylation of S6K1 and S6 were assessed in liver tissues from wild-type and Mst1/2-/- mice (4 weeks old) by western blotting with indicated antibodies. Abbrevia- tion: Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Transfection, Expressing, Phospho-proteomics, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 8. Significance of YAP1/TAZ-mediated regulation of mTORC1 in human HCC and mouse HCC models. (A,B) Kaplan-Meier plots of overall survival of HCC patients in cohort 1. Patients were stratified according to expression level of SLC38A1 (A) or SLC7A5 (B). P values were calculated with the log-rank test. (C) Liver tumor numbers after rapamycin treatment in Mst1/2-/- mice. At 62 days after rapamycin treatment, mice were killed and tumors counted. Data are presented as mean 6 standard error of the mean. (D) Representative images of liver tissue treated with rapamycin or vehicle. (E) Expression and phosphorylation of mTOR1 downstream targets in HCC tissues assessed by western blotting with the indicated antibodies after rapamycin or vehicle treatment in Mst1/2-/-

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: SLCs contribute to endocrine resistance in breast cancer: role of SLC7A5 (LAT1)

doi: 10.1101/555342

Figure Lengend Snippet: Autophagy initiates but does not complete with LAT1 inhibition. A) markers for autophagy and the unfolded protein response show autophagic flux. B) knockdown of GRP78 shows an increase of LAT1 protein after 72 hours. C) The mRNA levels of LAT1 increase with GRP78 knockdown (one replicate is stronger than the other).

Article Snippet: SLC7A5 siRNA and plasmid DNA were obtained from OriGene.

Techniques: Inhibition