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doi: 10.1038/nrc2501. and NuRD (nucleosome remodeling and deacetylase) complex in A431 cells. In addition, we identified PHF14 (the herb homeodomain finger-14) as a novel hypoxia-sensitive epigenetic regulator that plays a key role in cell cycle progress and protein synthesis. Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14ARF, p15INK4b, and p16INK4a, which are responsible for G1-S phase transition and decrease of AKT-mTOR-4E-BP1/pS6K signaling pathway, a grasp regulator of protein synthesis, in response to environmental cues. Analysis of Domperidone TCGA colon cancer (n=461) and skin cancer (n=470) datasets revealed a positive correlation between PHF14 expression and protein translation initiation factors, eIF4E, eIF4B, and RPS6. Significance of PHF14 gene was Domperidone further exhibited by mouse xenograft model using PHF14 KD cell lines. protein translation in the early hypoxia response, we employed quantitative pulsed stable isotope labeling with amino acids in cell culture (pSILAC) method to discriminate the newly synthesized proteins from pre-existing ones before hypoxia stress [16] and directly quantify protein translation events of A431 squamous carcinoma cells in response to hypoxia or serum starvation. Study of synthesized or translationally suppressed proteins under environmental stress revealed key molecules responsible for metabolic shift, malignant transformation, Domperidone or epigenetic regulation in cancer cells. More importantly, our approach has discovered a novel pathway of hypoxia-driven cell cycle arrest via epigenetic regulation. We identified PHF14 (the herb homeodomain (PHD) finger-14) as a novel key cell cycle regulator. PHF14, a relatively understudied epigenetic reader, was initially identified as a histone-binding protein through PHD finger motif [17C19]. In this report, we investigated the association between PHF14 and cell cycle arrest in cancer cells. By genetic depletion of PHF14 protein, hypoxic cancer cells increased the expression of CDK inhibitors, p15INK4b and p16INK4a, and p53-dependent cell cycle regulator, p14ARF, and consequently inhibited G1-to-S phase transition [20, 21]. In addition, PHF14 knockdown was associated with inhibition of AKT-mTOR-4E-BP1/S6K phosphorylation, which implicated that hypoxia-mediated suppression of PHF14 may regulate protein synthesis through AKT-mTOR signaling pathway. RESULTS Quantitative proteomic analysis of hypoxia-responsive proteins using pSILAC method To investigate the early cellular response DPP4 to hypoxic stress, we employed pSILAC-based quantitative proteomic approach to detect synthesis of proteins and translational dynamics. The workflow for pSILAC labeling scheme and proteomic analysis is described in Figure ?Physique1A1A and the Materials and Methods section. Briefly, A431 cells grown in light medium, made up of unlabeled [12C6, 14N2]-Lys and [12C6]-Arg, were switched to heavy medium, containing labeled [13C6, 15N2]-Lys and [13C6]-Arg for 24 hr. The incorporation of the stable isotopes labeled heavy lysine and arginine in the proteins allowed us to differentiate newly synthesized proteins from pre-existing proteins (Physique ?(Figure1A).1A). Proteome profiles were acquired from two biological replicates and further analyzed to select target protein groups. The Spearman’s rank correlation coefficients between two biological replicates from normoxic or hypoxic cell proteomes were respectively 0.883 and 0.853, confirming Domperidone a high reproducibility of dataset (Supplementary Physique 1). Key regulated proteins were selected when they appeared in both dataset and further validated by RT-qPCR or western blot analysis to confirm their expression changes. Open in a separate window Physique 1 Quantitative pSILAC based proteomic analysis of A431 cells(A) Protein labeling and evaluation scheme for pSILAC-LC-MS. A431 cells grown in light medium (L, R0K0) were transferred to heavy medium (H, R6K8) and cultured for 24 hr under either normoxia or hypoxia. Pre-existing protein was fully labeled R0K0 and newly synthesized protein was labeled R6K8. Protein synthesis ratio was determined by heavy/light labeled peptide. (B) Summary of proteins identified by pSILAC-LC-MS/MS in A431 cells under normoxia (NxSF) or hypoxia (HxSF). (C) Distribution of protein synthesis ratio (log2[H/L]). It clearly indicated the suppression of protein synthesis under hypoxia. (D) Summary of ribosomal proteins identified by pSILAC-LC-MS. (E) Cellular protein synthesis ratio of ribosomal proteins under normoxia or hypoxia (synthesized proteins [heavy/light (H/L) ratios] between normoxic and hypoxic A431 cells are presented in Figure ?Physique1B1B and ?and1C.1C. A total of 3475 proteins and 3452 proteins were identified with at least two unique tryptic peptides using Proteome Discoverer v2.2 (Thermo Fisher Scientific Inc., Waltham, MA, USA) from A431 cells grown under either normoxia or hypoxia for 24 hr in heavy medium, Domperidone respectively (Supplementary Data 1 and 2). Normoxic A431 cells (NxSF) sustained their protein.