In mice, TSM phosphorylation of Olig2 is essential for tumor growth and resistance to genotoxic damage [6, 11]

In mice, TSM phosphorylation of Olig2 is essential for tumor growth and resistance to genotoxic damage [6, 11]. genotoxic damage, and to enhance resistance to temozolomide (TMZ) in glioma. Both SUMOylation and triple serine motif (TSM) phosphorylation of Olig2 are required for the antiapoptotic function. Olig2 SUMOylation enhances its genetic targeting ability, which in turn occludes p53 recruitment to promoter for DNA-damage responses. Our work uncovers a SUMOylation-dependent regulatory Rabbit Polyclonal to TUT1 mechanism of Olig2 in regulating cancer survival. (also known as promoter. Thus, SUMOylation is important for Olig2 to function as an antiapoptotic factor in genotoxic stress. Materials and methods Antibodies and reagents The following primary antibodies and reagents were used: mouse anti-Olig2 (MABN50, Millipore), mouse anti-SUMO1 (33-2400, Thermo Fisher Scientific), mouse anti-BrdU (sc-32323, Santa Cruz), mouse anti-p53 (sc-126, Santa Cruz), rabbit anti-p53 (ab32389, Abcam), mouse anti-Flag (F1804, Sigma), mouse anti-Myc (sc-40, Santa Cruz), rabbit anti-Olig2 (AB9610, Millipore), rabbit anti-SUMO1 (4940, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (9661, Cell Signaling Technology), rabbit anti-gamma H2AX (phospho S139) (ab2893, Abcam), rabbit anti-Ki67 (ab16667, Abcam), mouse anti-phosphoserine (ab6639, Abcam), rabbit anti-Olig2 (phospho S10?+?S13?+?S14) (ab183487, Abcam), rabbit anti-Histone H3 (4499, Cell Signaling Technology), rabbit anti-acetyl-p53 (Lys379) (2570, Cell Signaling Technology), rabbit anti-p21 (ab218388, Abcam), rabbit anti-HA (H6908, Sigma), and SUMOylation 1 Affinity Beads (ASM11, Cytoskeleton). Etoposide (ETO, E1383), temozolomide (TMZ, T2577), BrdU (B5002), and tamoxifen (TAM, T5648) were purchased from Sigma. CHIR-99021 (S2924) was purchased from Selleck. Cells and human GBM specimen HEK 293T, Neuro-2a, U87-MG, and HCT-116 cells were cultured in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum and maintained at 37?C in a 5% CO2 humidified incubator. Cells were transfected at 80C90% confluency using Lipofectamine 3000 (Invitrogen) according to the manufacturers instructions. Human GBM tissue was acquired from Ruijin Hospital (Shanghai, China) with the approval by Ethics Committee for Clinical Trial and Medical Devices of Ruijin Hospital. Informed consent was obtained from all subjects. Plasmids His-SUMO1 and HA-CBP plasmids were gifts from Jianxiu Yu and Zhaoyuan Hou (Shanghai Jiao Tong University School of Medicine, Shanghai, China), respectively [15, 16]. Mouse Olig2, Hdac1, p53, and Senp2 cDNA were amplified by PCR from mouse brain tissue and inserted into p3Flag-Myc-CMV24 (Sigma), pCDNA3.1/Myc-His(?) (Invitrogen), pEGFP-C1 (Clontech), and Dihydrofolic acid pCMV-HA (Clontech) vectors to obtain Flag-Olig2, Myc-Olig2, Myc-Hdac1, Flag-p53, GFP-Senp2, and HA-Senp2, respectively. HA-Sox10, HA-Olig1, and HA-Nkx2.2 were generated by inserting mouse Sox10, Olig1, and Nkx2.2 cDNA into pCDNA3 vector with an HA tag at the C-terminus, respectively. Myc-Sirt1 was generated by inserting human Sirt1 cDNA into pCDNA3.1/Myc-His(?) vector. Mutations Dihydrofolic acid of Flag/Myc-Olig2, His-SUMO1, and GFP-Senp2 were generated using PCR-directed mutagenesis and all mutations were confirmed by DNA sequencing. For luciferase reporter assay, human promoter fragment and mouse for 10?min, supernatant was collected as the cytoplasmic fraction. The pellet was washed with hypotonic buffer and then lysed with whole cell lysis buffer (20?mM Tris-HCl, pH 7.5, 150?mM NaCl, 1% Triton X-100, 0.1% SDS, 2?mM EDTA, 10% glycerol). After centrifugation at 13,000??for 10?min, supernatant was Dihydrofolic acid taken as the nucleus fraction. Protein concentration was quantitated using a BCA assay kit (Thermo Fisher Scientific). All buffers were supplemented with protease inhibitor cocktail, phosphatase inhibitor cocktail, 1?mM PMSF, and 0.5?mM dithiothreitol. Luciferase reporter assay Construction of the plasmids for luciferase reporter assay was described above. The luciferase plasmid, WT Flag-Olig2, 3KR Flag-Olig2, AQ Flag-Olig2, and/or His-SUMO1 into Neuro-2a cells using Lipofectamine 3000. At 24?h post transfection, cells were harvested and analyzed for the luciferase activity using a dual-luciferase reporter assay kit (Promega) following the manufacturers instructions. The luciferase activity for each group was normalized with luciferase activity. See also [5]. Lentivirus packaging Olig2-expressing lentiviral constructs were generated as described above. Lentivirus packaging was performed by OBiO Technology Co. Ltd (Shanghai, China). U87-MG cells were stably infected with Lenti-GFP (1.63??109?TU/mL), Lenti-Olig2WT (1.38??109?TU/mL), and Lenti-Olig23KR (1.17??109?TU/mL), respectively, [21]. TUNEL staining TUNEL staining was performed using an cell death detection kit (Roche) following the manufacturers instructions. Briefly, cells were fixed with 4% paraformaldehyde/4% sucrose and permeabilized in 0.1% Triton X-100 in phosphate-buffered saline (PBS). After washing with PBS, cells were then incubated in the reaction solution containing terminal deoxynucleotidyl transferase and nucleotide mixture at 37?C for 1?h. See also [22]. Immunofluorescence and confocal microscopy Immunofluorescence staining was performed as described previously [20]. Briefly, cells were fixed with 4% paraformaldehyde/4% sucrose, blocked in 5% goat serum and 0.1%.

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