Volume 44, Issue 3, Pages 491-501 (November 2011) ROS-Mediated p53 Induction of Lpin1 Regulates Fatty Acid Oxidation in Response to Nutritional Stress  Wissam Assaily, Daniel A. Rubinger, Keith Wheaton, Yunping Lin, Weili Ma, Wanli Xuan, Lauren Brown-Endres, Katsuya Tsuchihara, Tak W. Mak, Samuel Benchimol  Molecular Cell  Volume 44, Issue 3, Pages 491-501 (November 2011) DOI: 10.1016/j.molcel.2011.08.038 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 p53 Regulates Lpin1 Expression (A) Northern blot analysis of RNA isolated from parental DP16.1 cells and DP16.1/p53ts cells after incubation at 32°C for the times indicated. RNA (15 μg) was run in each lane and hybridized to Lpin1, p21WAF1 and GAPDH cDNA probes. RNA was visualized by autoradiography. (B) Western blot analysis of lipin-1 protein expression in parental DP16.1 cells and DP16.1/p53ts cells after incubation at 32°C for 0 or 18 hr. The ∗ points to a nonspecific protein band that lies immediately above lipin-1. (C) Northern blot analysis of RNA isolated from mouse Ba/F3 and Ba/F3 DD cells after γ-irradiation with a dose of 6 Gy at the times indicated. The blot was hybridized with the indicated cDNA probes. (D) Northern blot analysis of RNA isolated from the spleen, thymus, and bone marrow of wild-type (p53+/+) and p53−/− mice at the times indicated after whole body γ-irradiation with a dose of 6 Gy. The blot was hybridized with the indicated cDNA probes. (E) Diagram showing the 15 kb region of the mouse Lpin1 gene that was investigated for p53 binding by ChIP and position of the three interacting sequences, 1A, 1B, and 1C (top). ChIP-PCR detected enrichment of p53-bound Lpin1 gene DNA fragments in chromatin isolated from DP16.1/p53ts cells cultured at 32°C for 8 hr and parental p53-negative DP16.1 cells cultured at 32°C for 8 hr. Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Glucose Deprivation Induces Lpin1 Expression in a p53-Dependent Manner (A) Western blot analysis of p53 phosphorylation on Ser18 in C2C12 cells after glucose withdrawal for 24 hr. The blot was reprobed with an antibody against β-actin as a loading control. (B) Western blot analysis of p53 phosphorylation and lipin-1 expression in normal human fibroblasts (GM03491) expressing p53 shRNA or empty vector (pSuper) and cultured in 5 mM or 0 mM glucose for 24 hr. (C) Western blot analysis of p53 protein levels in C2C12 cells stably expressing p53 shRNA or empty vector (pSuper) and cultured in 25 mM or 1 mM glucose for 24 hr. (D) Northern blot analysis of Lpin1 expression in C2C12 cells (left panel) and in C2C12 cells stably expressing p53 shRNA or empty vector (right panel) using RNA isolated from cells cultured in 25 mM glucose or 24 hr after glucose withdrawal. The blots were reprobed with GAPDH cDNA as a loading control. Signal intensities were quantified by phosphorimage analyses and the relative abundance of Lpin1 mRNA was determined after normalizing to the level of GAPDH mRNA in each sample. The fold-difference in Lpin1 mRNA is expressed relative to the amount of Lpin1 in cells cultured in 25 mM glucose. Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Low Glucose-Induced p53 Phosphorylation on Ser18 Is Dependent on ATM and Not Dependent on AMPK (A) Activation of AMPK and ATM in C2C12 cells in response to low glucose. C2C12 cells were cultured for 24 hr in the presence of 25, 1, or 0 mM glucose. Cell lysates were analyzed for phospho-ACC (Ser79), phospho-AMPK (Thr172), and phospho-ATM (Ser1987) by western blotting. (B) C2C12 cells were cultured for 24 hr in 25 or 1 mM glucose in the presence or absence of the AMPK inhibitor, Compound C (40 μM), or the ATM inhibitor, KU-55933 (10 μM). The two inhibitors were added during the final 1.5 hr of the culture period. Cells grown under normal conditions (25 mM glucose) were treated with doxorubicin (200 ng/ml) for 24 hr in the presence or absence of KU-55933. Cell lysates were analyzed for phospho-p53 (Ser18) and phospho-ACC (Ser79) by western blotting. β-actin served as a loading control. (C) Western blot analysis of phospho-p53 (Ser18) and phospho-ATM (Ser1987) in C2C12 cells cultured for 30 hr in 1 mM glucose in the presence or absence of the ATM inhibitor KU-55933 (10 μM). KU-55933 was added in the final 2 hr of the culture period. (D) Western blot analysis of C2C12 cells expressing scrambled control siRNA or ATM siRNA. Cells were cultured in 25 mM or 1 mM glucose for 24 hr and protein extracts were immunoblotted with the indicated antibodies. (E) Western blot analysis of phospho-p53 (Ser15) in normal human BJ fibroblasts (AT +/+) and in the ATM-deficient human fibroblast strain GM2052B (AT−/−) cultured in 25 mM glucose or in 1 mM glucose for the indicated times. Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Glucose Deprivation Increases Intracellular ROS Levels Leading to ATM Activation (A) ROS levels in C2C12 cells cultured in 25 mM glucose or 1 mM glucose for 2 hr or 6.5 hr. NAC was added during the final 60 min of incubation in 1 mM glucose. Cells were treated with DCFDA and ROS levels were measured by flow cytometry. The results are expressed as the mean intensity of cell fluorescence. Error bars indicate the SEM (n = 4). (B) ROS levels in normal GM03491 human fibroblasts were cultured in 5 mM glucose or 0 mM glucose for 6.5 hr. ROS was measured as in (B). (C) GSH levels and GSH/GSSG ratio in C2C12 cells cultured in 25 mM glucose or 1 mM glucose for 2 hr. Error bars indicate the SEM (n = 3). The prooxidant, tert-butyl hydroperoxide (tBHP), was added as a positive control to ensure the assay could detect changes in cellular redox. (D) Western blot analysis of phospho-p53 (Ser18) in C2C12 cells cultured in 25 mM glucose or in 1 mM glucose for 18 hr in the presence or absence of 2 mM NAC. Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Lpin1 Induction in Response to Glucose Deprivation Is Dependent on ATM and Elevated ROS Northern blot analysis of Lpin1 expression in C2C12 cells and human fibroblasts (ATM+/+ and ATM−/−) using RNA isolated from cells cultured under the conditions indicated. C2C12 cells were cultured in 0 mM glucose for 18 hr before harvest and the human fibroblasts were cultured in 0 mM glucose for 8 hr before harvest. NAC was present throughout the duration of glucose withdrawal. Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Lipin-1 Regulates Fatty Oxidation in Response to Low Glucose (A) shRNA-mediated downregulation of lipin-1 in C2C12 cells. Cells stably expressing pBabe-Lpin1 or pBabe were transfected with Lpin1 shRNA and extracts were prepared 48 hr later. Samples were analyzed by western immunoblotting with the indicated antibodies. Lpin1 shRNA-1 and shRNA-2 are two independent shRNA sequences that target different regions in the Lpin1 mRNA transcript. The ∗ points to a nonspecific protein band that lies immediately above lipin-1. (B) C2C12 cells (control, C) or C2C12 cells stably expressing p53 shRNA (p53sh), Lpin1 shRNA-1 (Lsh1), Lpin1 shRNA-2 (Lsh2), or empty vector (V) were cultured in normal medium (25 mM glucose) or in medium containing 1 mM glucose for 24 hr before measuring fatty acid oxidation. Values shown represent the mean and SEM (n = 4). Molecular Cell 2011 44, 491-501DOI: (10.1016/j.molcel.2011.08.038) Copyright © 2011 Elsevier Inc. Terms and Conditions