1. Volume 50, Issue 2, Pages 200-211 (April 2013)
The B55α Subunit of PP2A Drives a p53-Dependent Metabolic Adaptation to Glutamine Deprivation 
Michael A. Reid, Wen-I Wang, Kimberly Romero Rosales, Meng Xu Welliver, Min Pan, Mei Kong 
Molecular Cell 
Volume 50, Issue 2, Pages (April 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 50, 200-211DOI: (10.1016/j.molcel.2013.02.008)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 α4 Expression Protects Cells from Glutamine Deprivation
(A) 3T3 MEFs expressing vector (Vec) or Flag-α4 were treated with indicated medium conditions, and viability was assessed by propidium iodide exclusion at indicated time points.
(B) 3T3 MEFs expressing vector or Flag-α4 were cultured in glutamine (Gln)-free medium for 72 hr, lysed, and immunoblotting was performed using the indicated antibodies.
(C) FL5.12 cells expressing vector or Flag-α4 were subjected to glutamine depletion, and viability was assessed by propidium iodide exclusion at indicated time points (upper panel). Cells were lysed, and immunoblotting was performed using indicated antibodies (lower panel).
(D) HT1080 cells were transiently transfected with either LPC-vector control or LPC-Flag-α4. Glutamine deprivation was initiated 24 hr after transfection, and viability was assessed by trypan blue exclusion (upper panel). Cells were lysed, and immunoblotting was performed using indicated antibodies (lower panel). Data represent at least two independent clones for each cell type and are shown as the average of three experiments ± SEM. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The PP2A Regulatory Subunit B55α Is Specifically Induced upon Glutamine Deprivation
(A) 3T3 MEFs were cultured in glutamine-free medium for 24 hr, and quantitative real-time PCR was performed to determine mRNA expression of all PP2A subunits relative to 18S. Data are mean ± SEM of three independent experiments performed in duplicate.
(B) 3T3 MEFs were starved in glutamine-free medium for 24 hr, and immunoblotting was performed using indicated antibodies. Data represent at least three independent experiments.
(C–E) 3T3 MEFs were treated with glutamine-free medium for the time indicated (C) or indicated medium conditions (EAA, essential amino acids; Leu, leucine; FBS, fetal bovine serum) for 24 hr (D and E), and quantitative real-time PCR was performed to determine Ppp2r2a mRNA expression relative to 18S and normalized to the complete medium condition. The data presented are mean ± SEM of three independent experiments performed in duplicate.
(F) 3T3 MEFs were treated with glutamine-free medium or glucose-free medium for 24 hr, and cells were lysed, immunoprecipitated (IP), and immunoblotted with indicated antibodies.
(G) 3T3 MEFs were cultured in glutamine-free medium for the indicated time followed by DCDFA treatment and ROS measurement by flow cytometry. Data represent mean ± SEM of two independent experiments with ROS levels normalized to the complete medium condition.
(H) 3T3 MEFs were treated with control or glutamine-free medium for 24 hr, upon which cellular GSH levels were measured. Data represent mean ± SEM of four independent experiments with GSH levels normalized to the complete medium condition.
(I) 3T3 MEFs were treated with complete medium, glutamine-free medium, or glutamine-free medium supplemented with NAC (20 mM) or GSH-MEE (10 mM) for 16 hr, and ROS levels were measured by flow cytometry. Data represent mean ± SEM of two independent experiments performed in duplicate with ROS levels normalized to the complete medium condition.
(J and K) 3T3 MEFs were treated with complete medium, glutamine-free medium, or glutamine-free medium supplemented with NAC (20 mM) or GSH-MEE (10 mM), and cells were lysed after 24 hr for mRNA expression and 48 hr for protein expression. Quantitative real-time PCR was performed to determine Ppp2r2a mRNA expression relative to 18S, and immunoblots were probed with the specified antibodies. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005, Student’s t test. See also Figure S2.
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5. Figure 3
α4 Promotes PP2A-B55α Complex Formation and Activity, which Are Required for α4-Mediated Cell Survival
(A) 3T3 MEFs expressing Flag-α4 were infected with retrovirus containing shRNA against B55α or scrambled shRNA. Lysates were prepared from two clones for shB55α and bulk cells for scrambled controls, and immunoblotting was performed with indicated antibodies.
(B) Two clones expressing shRNA against B55α (C18, C20) in Flag-α4 MEFs and 3T3 MEFs with vector control or Flag-α4 expressing scrambled shRNA were subjected to glutamine depletion, and viability was assessed by trypan blue exclusion. Data represent mean ± SEM of three independent experiments.
(C) 3T3 MEFs expressing vector or Flag-α4 were cultured in complete or glutamine-free medium for 24 hr, then lysed, immunoprecipitated, and immunoblotted with the indicated antibodies.
(D) 3T3 MEFs expressing vector or Flag-α4 were cultured in complete or glutamine-free medium for 24 hr, lysed, and immunoprecipitated with the indicated antibodies, upon which phosphatase activity was measured. Data represent mean ± SEM of three independent experiments. ∗p < 0.05 and ∗∗p < 0.01, Student’s t test.
(E) Tamoxifen-inducible α4fl MEFs expressing Cre-ER fusion protein were treated with or without tamoxifen (4-HT) (200 nM) for 48 hr, then starved of glutamine for 24 hr, lysed, and immunoblotting was performed with the indicated antibodies. For (C) and (E), input lane represents control cells in complete medium. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
α4- and B55α-Mediated Cell Survival upon Glutamine Deprivation Is p53 Dependent
(A) 3T3 MEFs expressing either vector or Flag-α4 were treated with glutamine-free medium for 24 or 48 hr, and immunoblotting was performed using indicated antibodies.
(B) 3T3 p53 wild-type (p53+/+) or knockout (p53−/−) MEF clones were generated using retrovirus expressing vector control (Vec) or Flag-α4. Immunoblotting was performed using indicated antibodies.
(C) p53+/+ or p53−/− MEFs with or without Flag-α4 expression were treated with glutamine-free medium, and viability was assessed by trypan blue exclusion at time points indicated. Data are mean ± SEM of two independent experiments performed in triplicate.
(D) Two clones expressing shRNA against B55α (C18, C20) in Flag-α4 MEFs and 3T3 MEFs with vector control or Flag-α4 expressing scrambled shRNA were treated with glutamine (No Gln) or glucose (No Glc)-free medium for 24 hr, and immunoblotting was performed using indicated antibodies.
(E) Flag-α4 MEFs expressing scrambled or B55α shRNA were treated with glutamine-free medium for 24 hr, and quantitative real-time PCR was used to determine mRNA expression of p53 target genes relative to 18S and normalized to the complete medium condition. Data represent mean ± SEM of three independent experiments.
(F and G) Flag-α4 MEFs were treated with complete medium, glutamine-free medium, or glutamine-free medium supplemented with NAC (20 mM) or GSH-MEE (10 mM), cells were lysed after 24 hr, and immunoblots were probed with the specified antibodies.
(H) Flag-α4 MEFs were treated with complete medium, glutamine-free medium, or glutamine-free medium supplemented with NAC (20 mM) or GSH-MEE (10 mM) for 48 hr, and viability was assessed by propidium iodide exclusion. Data represent mean ± SEM of three independent experiments performed in duplicate. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005, Student’s t test. See also Figure S4.
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7. Figure 5
B55α Interacts with EDD to Promote p53 Activation and Cell Survival
(A) Silver-stained gel of eluates from 293T cells expressing vector or Flag-B55α. The bands unique to the Flag-B55α eluates were identified by mass spectrometric analysis.
(B and C) 293T cells transiently overexpressing Flag-B55α (B) or Flag-EDD (C) were lysed and immunoprecipitated with anti-Flag antibody. Immunoblots were probed with the specified antibodies.
(D) HT1080 cells stably expressing Flag-B55α were treated with glutamine-free medium for 24 hr, lysed, and immunoprecipitated with anti-Flag antibody. Immunoblots were probed with the specified antibodies.
(E) 3T3 MEFs were treated with glutamine-free medium for 24 hr, lysed, and immunoprecipitated with anti-EDD antibody. Immunoblots were probed with the specified antibodies.
(F) 3T3 MEFs expressing Flag-α4 and either scrambled or B55α shRNA were lysed and immunoprecipitated with anti-EDD antibody (upper panels) or anti-phospho-serine antibody (lower panels). Immunoblots were probed with the specified antibodies.
(G) 3T3 MEFs expressing Flag-α4 and either scrambled or B55α shRNA were transiently transfected with either scrambled or EDD siRNA (20 nM), treated with glutamine-free medium for 24 hr, and immunoblotting was performed using indicated antibodies.
(H) 3T3 MEFs expressing Flag-α4 and either scrambled or B55α shRNA were transiently transfected with either scrambled or EDD siRNA (20 nM), treated with glutamine-free medium for 24 hr, and quantitative real-time PCR was performed to determine indicated gene mRNA expression relative to 18S and normalized to the complete medium condition. Data presented are mean ± SEM of two independent experiments performed in triplicate.
(I) 3T3 MEFs expressing Flag-α4 and either scrambled or B55α shRNA were transiently transfected with either scrambled or EDD siRNA (20 nM), treated with glutamine-free medium for 72 hr, and assessed for viability by trypan blue exclusion at indicated time points. Data represent mean ± SEM of three independent experiments. ∗p < 0.05 and ∗∗p < 0.01, Student’s t test. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
B55α Is Induced upon Glutamine Deprivation in Cancer Cells and Is Required for Cell Survival
(A) Cancer cells (as indicated) were cultured in complete medium, lysed, and immunoblotting was performed with indicated antibodies.
(B) Indicated cancer cells were treated with glutamine-free medium for 24 hr, and quantitative real-time PCR was performed to determine PPP2R2A mRNA expression relative to 18S and normalized to the complete medium condition. Data presented are mean ± SEM of two independent experiments performed in duplicate.
(C) Cancer cells (as indicated) were cultured in glutamine-free medium for 24 hr, lysed, and immunoblotting was performed with indicated antibodies.
(D and E) HT1080 cells were infected with retrovirus containing shRNA against B55α or scrambled shRNA. B55α protein levels were shown by immunoblotting (D). Cells were treated with glutamine-free medium, and viability was assessed by trypan blue exclusion at indicated time points. Data are mean ± SEM of two independent experiments performed in triplicate (E).
(F) HCT116 cells with wild-type (p53+/+) or knockout (p53−/−) p53 were treated with glutamine- or glucose-free medium for 72 hr, and viability was assessed by trypan blue exclusion. The data presented are mean ± SEM of two independent experiments performed in duplicate.
(G) HT1080 cells expressing scrambled or B55α shRNA were treated with glutamine-free medium for 24 hr, and quantitative real-time PCR was used to determine mRNA expression of p53 targets relative to β-ACTIN and normalized to the complete medium condition. Data represent mean ± SEM of two independent experiments performed in duplicate. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005, Student’s t test. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Glutamine Limitation-Induced B55α Is Required for Tumor Growth In Vivo
(A) Nude mice were injected subcutaneously on one side with 2 × 106 HT1080 cells expressing either scrambled or B55α shRNA, and tumor growth was measured over time. Graph represents mean tumor volume ± SD from two independent experiments (seven animals per experiment) and a total of 14 mice per group.
(B) Representative image of scrambled and B55α shRNA xenograft tumors.
(C) Schematic of how peripheral and core tumor samples were obtained.
(D and E) Glutamine and GSH concentrations were determined for peripheral and core samples from xenograft tumors. Data represent mean ± SD of three individual tumors per genotype normalized to scramble control peripheral samples.
(F) Quantitative real-time PCR was performed to determine PPP2R2A mRNA expression for peripheral and core tumor regions. Data represent mean ± SEM of triplicate quantitative real-time PCRs from individual tumors as indicated.
(G) Samples from peripheral (P) or core (C) regions of individual tumors as indicated were lysed, and immunoblotting was performed with indicated antibodies.
(H) Schematic showing molecular mechanism by which cells survive glutamine deprivation. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005, Student’s t test. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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