PMC

Tsc2 but not Redd1 is required for growth factor signaling. Western blot analysis of Tsc2^+/+ and Tsc2^-/- MEFs (A) or Redd1^+/+ and Redd1^-/- MEFs (B) after serum deprivation for the indicated periods of time.

Down-regulation of mTOR function by hypoxia requires Redd1. (A) Western blot analysis of Tsc2^+/+ MEFs pretreated (30 min prior to initiation of the hypoxia time course) or not, with Actinomycin D and exposed to hypoxia for the indicated periods of time. Shown in parallel, analysis of Tsc2^+/+ cells treated with Actinomycin D for the indicated periods of time. Northern blot (B) and Western blot (C) analysis of Redd1^+/+ and Redd1^-/- MEFs treated with hypoxia for the indicated periods of time.

Tsc2 is both necessary and sufficient for the regulation of S6 phosphorylation by hypoxia. (A) Western blot analysis of Tsc2^-/- MEFs retrovirally transduced with either a Tsc2 expression vector or an empty vector and treated with hypoxia for the indicated periods of time. Tsc2^+/+ MEFs are included as controls. (B) Western blot analysis of HEK293 cells transfected with two different synthetic Tsc2 siRNAs (a and b) or a scrambled siRNA (Sc) and exposed to hypoxia for the indicated periods of time.

Tsc2 loss confers a proliferative advantage under hypoxic conditions. (A) Proliferation rates of Tsc2^-/- and Tsc2^+/+ MEFs under normoxic conditions. (B) Proliferation rates under hypoxic conditions of Tsc2^+/+ and Tsc2^-/- MEFs (treated or not with rapamycin). Error bars for A and B equal one standard deviation (n = 3). Note different Y-axis scales in A and B. (C) Western blot analysis of Tsc2^+/+ and Tsc2^-/- MEFs cultured in parallel under hypoxic conditions for the indicated number of days.

Tsc2 regulates mTOR in response to hypoxia. (A) Western blot analysis of Tsc2^+/+ and Tsc2^-/- MEFs. (Hif) Hif-1α and/or Hif-2α; (S6K-P) S6K phosphorylated T389; (S6-P) S6 phosphorylated on S235/236. Left panel shows MEF in 0.05% serum or following serum addition (10% serum for 45 min) pretreated or not with rapamycin (1.5 h prior to serum addition). Right panel shows MEFs exposed to hypoxia for the indicated periods of time. (B) Western blot analysis of Tsc1^+/+ and Tsc1^-/- mouse 3T3 cells treated with hypoxia for the indicated periods of time. (C) Western blot analysis of input (left) and ⁷mGTP-bound (right) proteins from extracts of Tsc2^+/+ and Tsc2^-/- MEFs exposed to hypoxia for the indicated periods of time. (D,E) Western blot analysis of extracts from Tsc2^+/+ and Tsc2^-/- MEFs exposed to hypoxia for the indicated periods of time. In E all the cells were treated with rapamycin for 26 h prior to lysis regardless of the duration of hypoxia.

Down-regulation of S6K1 phosphorylation by Redd1 and Redd2. (A) Western blot analysis of anti-HA immunoprecipitates (top) or whole-cell extracts (bottom) from HEK293 cells transfected with HA-tagged expression vectors encoding Redd1 (R1), Redd1 mutant lacking central domain (R1dC), Redd2 (R2), Redd1 and Redd2 (R1 + R2), or an empty vector (pcDNA3). HA-S6K1 (S6K) was cotransfected where indicated. Rapamycin was added several hours prior to cell harvest where indicated. (B) Western blot analysis of anti-HA immunoprecipitates (bottom) or whole-cell extracts (top) from HeLa cells exponentially growing under serum-rich conditions transfected with siRNAs (Sc, scrambled; Tsc2, b and a) and expression vectors as in A. Rapamycin was added several hours prior to cell harvest where indicated. Ig(H) indicates immunoglobulin heavy chain. (C) Western blot analysis of HA-Redd1 inducible U2OS cells transfected with the indicated siRNAs and stimulated to produce Redd1 with tetracycline for the indicated periods of time.

mTOR regulation by hypoxia is both AMPK and Lkb1 independent. (A) Western blot analysis of extracts of Tsc2^+/+ and Tsc2^-/- MEFs treated with AICAR for the indicated periods of time. (ACC-P) Acetyl-CoA carboxylase phosphorylated at S79; (AMPK-P) AMPK phosphorylated at T172. (B) Western blot analysis of Tsc2^+/+ MEFs after 4 h of treatment with either hypoxia or AICAR. (C) In vitro AMPK kinase assay of the same extracts used in B immunoprecipitated in antibody excess with a polyclonal anti-AMPK (α AMPK) antibody or normal rabbit IgG (IgG). Samples were normalized for protein concentration prior to immunoprecipitation. Error bars equal one standard deviation (n = 3). Also shown are background activities of immunoprecipitates incubated in the absence of substrate (SAMS peptide). (D) Western blot analysis of Tsc2^+/+ cells pretreated or not with the AMPK inhibitor compound C and exposed to either AICAR or hypoxia for the indicated periods of time. All cells treated with compound C were exposed to the drug for 9.5 h. (E,F) Western blot of Lkb1^+/+ or Lkb1^-/- MEFs treated with AICAR (E) or hypoxia (F) for the indicated periods of time.