1. Volume 48, Issue 5, Pages 681-691 (December 2012)
HIF2α Acts as an mTORC1 Activator through the Amino Acid Carrier SLC7A5 
Ainara Elorza, Inés Soro-Arnáiz, Florinda Meléndez-Rodríguez, Victoria Rodríguez-Vaello, Glenn Marsboom, Guillermo de Cárcer, Bárbara Acosta-Iborra, Lucas Albacete-Albacete, Angel Ordóñez, Leticia Serrano-Oviedo, Jose Miguel Giménez-Bachs, Alicia Vara- Vega, Antonio Salinas, Ricardo Sánchez-Prieto, Rafael Martín del Río, Francisco Sánchez-Madrid, Marcos Malumbres, Manuel O. Landázuri, Julián Aragonés 
Molecular Cell 
Volume 48, Issue 5, Pages (December 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 48, 681-691DOI: (10.1016/j.molcel.2012.09.017)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Effects of HIF2α Activity on mTORC1 Activation in WT8 Cells
(A) Western blot analysis of HIF2α and tubulin protein in HIF2α (P-A)2, HIF2α (P-A)2bHLH∗, and WT8 control cells.
(B) Relative Oct4 gene expression in HIF2α (P-A)2, HIF2α (P-A)2bHLH∗, and WT8 control cells. The mean is shown; error bars represent SEM (n = 6, ∗∗∗p < 0.001).
(C) Cell lysates from control, HIF2α (P-A)2, and HIF2α (P-A)2bHLH∗ WT8 cells cultured for 72 hr in normal culture media (left panel) or in media with 50% of the normal amino acid content (right panel) were analyzed by western blot with antibodies as shown.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Contribution of SLC7A5 Expression to HIF2α-Dependent mTORC1 Activation
(A) Left panel, relative Slc7a5 gene expression in HIF2α (P-A)2, HIF2α (P-A)2bHLH∗, HIF1α (P-A)2, and WT8 cells. The mean is shown, and error bars represent SEM (n = 6, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Right panel, relative bNIP3 gene expression in HIF2α (P-A)2, HIF1α (P-A)2, and WT8 control cells (n = 4). The mean is shown; error bars represent SEM (n = 4, ∗p < 0.05, ∗∗∗p < 0.001).
(B) Western blot analyses of SLC7A5 and tubulin protein levels in HIF2α (P-A)2, HIF2α (P-A)2bHLH∗, and WT8 control cells.
(C) WT8 control cells were transfected with scrambled control small interfering RNA (siSCR), and HIF2α (P-A)2 WT8 cells were transfected with either siSCR or siRNA against Slc7a5 (siSLC7A5). The cells were subsequently cultured for 72 hr in media with 50% of the normal amino acid content. Whole-cell extracts were analyzed by western blot with antibodies indicated.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Effect of Endogenous HIF2α on mTORC1 Activity and SLC7A5 Expression in 786-O VHL-Deficient Cells
Western blot analysis with antibodies as shown in (A) HIF2α-silenced 786-O cells and their corresponding control siSCR-transfected 786-O cells 72 hr after transfection, (B) 786-O and their counterparts in which VHL expression was restored (786-O-VHL), and (C) SLC7A5-silenced 786-O cells and their corresponding control 786-O-shSCR cells. All these experiments were performed by culturing 786-O cells for 48 hr in media with 5% of the normal content of essential amino acids and glutamine.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 HIF2α Binds to the Slc7a5 Proximal Promoter
(A) Schematic representation of the human Slc7a5 gene indicating the positions of the proximal promoter and intron 1 and the nucleotide sequences corresponding to the putative hypoxia-response elements highlighted in bold.
(B and C) ChIP assay to assess the relative HIF2α binding activity to the human Slc7a5 proximal promoter or intron 1 in (B) HIF2α (P-A)2, HIF2α (P-A)2bHLH∗, and WT8 control cells or (C) 786-O and their counterparts in which VHL expression was restored (786-O-VHL). Representative gel showing DNA amplified in the ChIP assays is shown. The mean is shown; error bars represent SEM (n = 5, ∗p < 0.05). IgG, immunoglobulin G.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Role of the HIF2α-Dependent SLC7A5 Pathway in Cell Proliferation and Xenograft Growth
(A) Cell number fold induction in control, HIF2α (P-A)2, and HIF2α (P-A)2bHLH∗ WT8 cells cultured for 72 hr in normal media (left panel) or in media containing 5% of the normal amino acid concentration (right panel). Statistical significance was indicated only when HIF2α (P-A)2 proliferation differed significantly from that of both HIF2α (P-A)2bHLH∗ WT8 and control WT8 cells. The mean is shown; error bars represent SEM (n = 4, ∗p < 0.05, ∗∗p < 0.01.
(B) WT8 control cells were transfected with scrambled control siRNA (siSCR), and HIF2α (P-A)2 WT8 cells were transfected with either siSCR or siRNA for Slc7a5 (siSLC7A5). Twenty-four hours after transfection, cells were seeded at the same cell density in media containing 5% of the regular amino acid concentration, and the increase in cell number was analyzed after 72 hr. The mean is shown; error bars represent SEM (n = 4, ∗p < 0.05, ∗∗∗p < 0.001).
(C) WT8 control cells and HIF2α (P-A)2 WT8 cells were treated with rapamycin (20 nM) and cultured in media containing 5% of the regular amino acid concentration. The increase in cell number was analyzed after 72 hr. The mean is shown; error bars represent SEM (n = 8, ∗∗p < 0.01).
(D) Tumor-volume evolution (Vol) over a 45 day period of SLC7A5-silenced 786-O cells (white squares) and their corresponding control 786-O-shSCR cells (black squares) injected subcutaneously in the dorsal flanks of immunosuppressed severe combined immunodeficiency mice, as detailed in the Experimental Procedures section. The mean is shown; error bars represent SEM (n = 18, ∗p < 0.05, ∗∗p < 0.01).
See also Figure S4.
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8. Figure 6
Hypoxia and HIF2α-Dependent mTORC1 Activity and Slc7a5 Expression in Lung Tissue
(A) Wild-type mice were exposed to hypoxia (10% O2) (Hx10%) or normoxia (N) for 4 days. Protein extracts from the lungs were analyzed by western blots with antibodies as shown.
(B) Lung phospho-rpS6Ser235/6 immunostaining of mice exposed to normoxia or hypoxia (10% O2) for 4 days and in Vhlfl-UBC-Cre-ERT2, VhlflHIF2αfl-UBC-Cre-ERT2, and the corresponding control mice. Quantification of the number of bronchial epithelial cells positive for phospho-rpS6Ser235/6 staining in wild-type mice exposed to hypoxia (n = 4) or normoxia (n = 4) as well as Vhlfl-UBC-Cre-ERT2 (n = 4), VhlflHIF2αfl-UBC-Cre-ERT2 (n = 4), and the corresponding control mice (n = 3). Five airways were analyzed for each animal, and >1500 cells were analyzed for each group.
(C) Relative expression of Slc7a5 gene in the lungs of normoxic (n = 7) and hypoxic mice (n = 6), and Slc7a5 and Pgk1 in Vhlfl-UBC-Cre-ERT2 mice (n = 5), VhlflHIF2αfl-UBC-Cre-ERT2 mice (n = 5), and the corresponding controls (n = 6).
See also Figure S5. For (B) and (C), the mean is shown, and error bars represent SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
HIF2α-Dependent mTORC1 Activity and SLC7A5 Expression in VHL-Deficient Liver Tissue
(A) Protein extracts from the livers of Vhlfl-UBC-Cre-ERT2, VhlflHIF2αfl-UBC-Cre-ERT2, and the corresponding control mice were analyzed by western blots with the indicated antibodies.
(B) SLC7A5 immunostaining of liver tissue (hepatocytes) from Vhlfl-UBC-Cre-ERT2, VhlflHIF2αfl-UBC-Cre-ERT2, and corresponding control mice. Relative expression of Slc7a5 and Pgk-1 genes in the livers of Vhlfl-UBC-Cre-ERT2 (n = 7), VhlflHIF2αfl-UBC-Cre-ERT2 (n = 6), and corresponding control mice (n = 7).
The mean is shown; error bars represent SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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