任杰2草稿.doc

Supplementary. Fig. 1. (a) Sirt1 and mutant HTT (detected by HTT 81-90 antibody) 补充。图1。（一）与突变高温热处理（即抗体检测81 - 90）protein levels were detected by Western blotting in cerebral cortex of N171-82Q mice. (b) 蛋白水平，免疫印迹检测大脑皮层n171-82q小鼠。（二）Sirt1 and mutant HTT (detected by 1C2 antibody) protein levels were detected by Western 与突变高温热处理（检测12抗体）蛋白水平检测西方blotting in cerebral cortex of BACHD mice.在大脑皮层的bachd小鼠印迹。Supplementary Fig. 2. Sirt1 improves glucose tolerance and regulates energy 补充图2。SIR T1的改善糖耐量和调节能量metabolism in N171-82Q HD mice. (a) Blood glucose levels were measured after an 代谢n171-82q高清小鼠。（一）血糖水平测定后overnight fast in 14-week-old mice. Mean S.E.M., n=10. (b-c) Mice were fasted 隔夜快14周大的老鼠。平均扫描，10例。（b - c）小鼠禁食overnight and then administered D-glucose (1g/kg, i.p); blood samples were taken before 一夜之间，然后管理葡萄糖（克/千克，知识产权）；血液样本被带到之前and at indicated times after glucose administration. The absolute values (b) and area 在表示时间后血糖管理。绝对值（二）和面积under curve (AUC) (c). n=12. Mean S.E.M. (d-f) Effects of Sirt1 overexpression on 在曲线（联合自卫军）（丙）。12例。平均扫描电镜（东风）影响，SIR T1的表达body weight (d), food intake (e), energy expenditure (f) measured by an Oxymax （四）体重，食物摄入量，能源支出（电子）（女）衡量一个oxymaxmetabolic system at 16 weeks old mice. Mean S.E.M. n=12. *p0.01 compared with 代谢系统在16周大的老鼠。平均扫描。12例。* 0.01相比the value of wild type (WT); *p0.05 compared with the value of HD.价值的野生型（野生）；* * 0.05相比的价值，高清。Supplementary Fig. 3 Transgene Sirt1 is highly expressed in the brain and barely 补充图3基因SIR T1高表达在大脑和勉强detectable in the pancreas by immunofluorescent staining in Sirt1 transgenic mice. 检测胰腺中的免疫荧光染色在转基因小鼠中。(a) Representative pictures of immunofluorescent staining of transgene Sirt1 (HA-tagged, （一）代表的照片，免疫荧光染色的基因SIR T1（医管局标记，green), NeuN staining (red), and DAPI in different brain regions from a Sirt1 trangenic 绿色），抗原染色（红色），与不同脑区从一个SIR T1转基因mouse (Sirt1) and a wild type control mouse (WT). Scale bar 100 m. (b) Representative 小鼠（中）和野生型（野生）控制鼠标。规模100米（乙）代表pictures of immunofluorescent staining of transgene Sirt1 (HA, green), insulin (red), and 图片的免疫荧光染色的基因SIR T1（哈哈，绿），胰岛素（红色），和DAPI (blue) in pancreatic islets of Langerhans from a Sirt1 transgenic mouse (Sirt1) and 吲哚（蓝色）胰腺胰岛从SIR T1转基因小鼠（SIRT 1）和a wild type littermate control (WT). Scale bar 50 m.野生型窝控制（野生）。规模50MSupplementary Fig. 4. Sirt1 has no effects on mutant HTT aggregates in N171-82Q 补充图4。SIR T1没有影响突变高温热处理总量在n171-82qHD mice. (a) Diagram shows the areas of the cortex that were evaluated for HTT 小鼠高清。（一）图显示该地区的皮层，被评价为高温热处理aggregates. (b) Average number of cells with mutant HTT aggregates in the cerebral cortex 集料。（二）平均人数细胞突变高温热处理总量在大脑皮层of mice in each microscope field. (c) Representative images from striatum immunostained 小鼠在每个显微镜。（三）代表图像从纹状体染色with EM48 antibody. Scale bars 50 m. (d) Average number of cells with mutant HTT 与em48抗体。规模酒吧50米（四）平均人数细胞突变高温热处理aggregates in the striatum of mice in each microscope field. Mean S.E.M. from 5 mice per 聚集在小鼠纹状体在每个显微镜。平均扫描电镜从5个小鼠每group. (e) N-terminal mutant HTT (N63-148Q-myc) expression was induced by withdrawal 集团。（五）N -末端突变高温热处理（n63-148q-myc）表达诱导撤出of doxycycline from the medium, and Sirt1 overexpression was introduced by retrovirus at 强力霉素的媒介，并介绍了逆转录病毒在SIR T1的表达the same time as mutant HTT induction. Cells were fixed for immunofluorescent staining at 同时突变高温热处理感应。细胞是固定的，免疫荧光染色48 h after induction of mutant HTT expression. Mutant HTT was labeled with myc antibody 48小时后诱导表达的突变高温热处理。突变高温热处理标记基因抗体(myc-tagged N63-148Q). Mean S.E.M. from three independent experiments.（基因n63-148q）。平均扫描电镜从三个独立的实验。Supplementary Fig. 5. Sirt1 increases neuronal resistance to mutant HTT toxicity. (a) 补充图5。SIR T1增加神经元抗突变高温热处理毒性。（一）Primary cortical neurons were cultured from Sirt1 transgenic mice or wild type (WT) control 初级皮层神经元培养SIR T1转基因小鼠和野生型（野生）控制mice. Levels of Sirt1 in primary cortical neurons were detected by Western blotting. (b) 小鼠。水平对初级皮层神经元的免疫印迹检测。（二）Neurons overexpressing Sirt1 are more resistant to mutant HTT-induced neurotoxicity. 神经过度SIR T1的抗突变高温热处理引起的神经毒性。Cortical neurons were cultured from Sirt1 transgenic mice or wild type control mice, and 皮层神经元培养SIR T1转基因小鼠或野生型小鼠，以及transfected with mutant HTT (Htt-N63-148Q) or normal HTT (Htt-N63-16Q) fragment at 转染突变高温热处理（htt-n63-148q）或正常的高温热处理（htt-n63-16q）片段DIV5, and neuronal survival was assessed at 48 h after transfection by nuclear DNA div5，与神经元生存进行了评估在48小时后转染核脱氧核糖核酸morphology. The numbers of surviving neurons in transfected neurons were measured. 形态。存活神经元数量在转染细胞测定。*p0.05 vs the value of WT neurons transfected with HTT-N63-148Q.* 0.05与价值型细胞转染htt-n63-148q。Supplementary Fig. 6. Effects of Foxo3a and Sirt1 on mutant HTT-induced ATP 补充图6。影响及意义在突变高温热处理引起的三磷酸腺苷deficits in STHdh 赤字sthdhQ111/Q111q111/ q111cells. (a) HPLC chromatograms showing adenosine 细胞的。（一）的液相色谱显示腺苷nucleotides ATP, ADP, and AMP in wild-type STHdh 三磷酸腺苷二磷酸腺苷，磷酸腺苷核苷酸，与野生型sthdhQ7/Q77/7and quantification data 量化数据showing decreased ATP levels as well as ATP/ADP ratio in mutant STHdh 显示减少三磷酸腺苷水平以及三磷酸腺苷/二磷酸腺苷比突变sthdhQ111/Q111q111/ q111cells. 细胞的。(b) Increase in Foxo3a had similar protective effects as Sirt1 on mutant HTT-induced （二）增加提供了类似的保护作用，如SIR T1突变高温热处理引起的ATP deficits in STHdh 三磷酸腺苷赤字sthdhQ111/Q111q111/ q111cells. (c) Effects of overexpression of Foxo3a and Sirt1 细胞的。（三）表达的影响及意义中on mutant HTT-induced energy deficits in STHdh 突变高温热处理引起的能源赤字sthdhQ111/Q111q111/ q111cells. Mean S.E.M from 细胞的。平均s.e.m从three independent experiments. *p0.05 vs the values of pcDNA vector-transfected group 三个独立的实验。* 0.05与价值的重组载体转染组by Students t-tests.学生的t检验。Supplementary Fig. 7. Foxo3a regulates levels of DARPP32 and BDNF levels in cells 补充图7。转录因子调节水平darpp32和脑源性神经营养因子在细胞水平expressing mutant HTT (STHdh Q111/Q111表达突变高温热处理（sthdh q111/ q111cells). (a) Knockdown of Foxo3a by siRNA 细胞）。（一）击倒的转录因子的表达decreased DARPP32 levels in STHdh 在sthdh darpp32水平下降Q111/Q111q111/ q111cells. Foxo3a siRNA or scramble controls 细胞的。转录因子表达或争夺控制were transfected into STHdh 转染sthdhQ111/Q111q111/ q111cells and DARPP32 levels were determined at 24 h 细胞和darpp32水平确定在24小时after transfection. *p 0.01 vs scramble RNA transfected group. (b-c) Overexpression of 转染后。* 0.01队争夺转染组。（b - c）过度Foxo3a increased levels of DARPP32 (b) and BDNF (c) in STHdh 提供更高水平的darpp32（乙）和脑源性神经营养因子（丙）在sthdhQ111/Q111q111/ q111cells. Foxo3a 细胞的。转录因子cDNA or vector control (pc DNA) was transfected into cells, and levels of DARPP32 and 基因或矢量控制（电脑脱氧核糖核酸）转染细胞，和水平的darpp32和BDNF were determined at 48 h after transfection. *p0.05 vs pc DNA vector group by 脑源性神经营养因子被确定在48小时后转。* 0.05与电脑的基因载体组Students t-tests.学生的t检验。Supplementary Methods 辅助方法Mutant HTT aggregates immunostaining Brain sections were cut at 40 m and 突变高温热处理总量染色的脑切片削减40米immunostained with an EM48 antibody (mouse monoclonal antibody 1:100. a gift from S. 染色的em48抗体（小鼠单克隆抗体1 : 100。从美国的礼物Li and X. Li at Emory University), which preferentially recognizes mutant HTT 李和X .李，埃默里大学）优先承认突变高温热处理aggregates. The cell numbers with mutant HTT inclusions in the striatum and cortex were 集料。细胞数与突变高温热处理包裹在纹状体和皮层counted by a person blinded to the groups. N63-148Q PC12 cells were transduced with 由数人失明的团体。n63-148q PC 12细胞转导Sirt1 retrovirus, 48 h after transduction, cells were fixed by 4% paraformaldehyde and 逆转录病毒转导的骨髓，48小时后，细胞固定的4%甲醛和immunofluorescent staining was carried out with following antibodies to c-myc (for 免疫荧光染色进行了以下抗c - myc（为mutant HTT, as N63-148Q is myc tagged, 1:300, Oncogene), Sirt1 ( 1:1000, Upstate Inc.). 突变高温热处理，如n63-148q是基因标记，1 : 300，SIR T1基因），（1 : 1000，北部股份有限公司。）。Pictures were taken and percentage of cells with mHTT aggregates was calculated. 照片是采取和百分比细胞mhtt总量计算。Foxo3a overexpress/siRNA STHdh cells were planted in 6-well plates at the day before 转录因子过度/干扰sthdh细胞种植在6孔板的前一天transfection. Cells were then transfected with Lipofectamine TM 2000 (Invitrogen) 转染。转染细胞与脂质体转染2000（公司）according to the manufacturers instructions. Briefly, 2 g Foxo3a pcDNA plasmid 根据制造商的指示。简单地说，2克提供重组质粒(Addgene) and 4 l Lipofectamine TM 2000 were diluted in 250 l of Opti-MEM medium （addgene）和4我ofectamine2000被稀释250升opti-mem介质respectively. For Foxo3a siRNA (Sigma), 200 pmol/well RNA and 4 l/well 分别为。为提供小干扰RNA（西格马），为200和4好/好Lipofectamine TM 2000 were diluted in 250 l of Opti-MEM medium, respectively. After 脂质体转染2000被稀释250升opti-mem介质，分别。后incubation for 5 min, DNA or siRNA and Lipofectamine TM 2000 were mixed and 潜伏期为5分钟，或干扰和脂质体转染2000混合incubated for 20 min at room temperature. 500 L of the mixture of DNA or RNA and 培养20分钟室温。500我的混合物核酸和LipofectamineTM2000 were added to cultures. The maintenance medium DMEM lipofectaminetm2000被添加到文化。维持培养基containing FBS was replaced at 4 h after transfection. 含胎牛血清替代在4小时后转染。Coimmunoprecipitation for detecting mutant HTT and Sirt1 interaction BACHD 共免疫沉淀检测突变高温热处理和bachd SIR T1的相互作用mouse brain tissues were lysed in RIPA buffer (cell signaling) containing protease 小鼠脑组织被溶解在帕缓冲（细胞信号）含有蛋白酶inhibitors (Sigma) on ice for 30 min. After adding 2 times volume of PBS, tissue lysates 抑制剂（西格马）冰上30分钟后，加入2倍体积的磷酸盐缓冲液，组织裂解物were immunoprecipitated using antibody to Sirt1 (1:1000, Upstate,) overnight, then 进行免疫沉淀使用抗体（1 : 1000，SIR T1的北部，一夜之间，然后）washed with RIPA/PBS (1:2) buffer, and membranes were blotted with MW1 antibody 洗帕/磷酸盐缓冲液（1 : 2）缓冲区，和膜的剥离，与mw1抗体(1:10000, gift from Dr. Paul H. Patterson) and antibody to Sirt1 (1:10000, Upstate).（1 : 10000，礼物从保罗博士阁下帕特森）和抗体（1 : 10000，SIR T1北部）。Immunofluorescent staining in pancreas Animals were anesthetized using inhalation 免疫荧光染色胰腺动物麻醉使用吸入isoflurane (1ml/430cm3异氟醚（毫升/430cm3) before euthanization by decapitation. The pancreas was ）在euthanization斩首。胰腺collected from each animal and fixed in formalin for 8-10 hours and then transferred into 收集每个动物和福尔马林固定为8 - 10小时，然后转移到phosphate buffered saline (PBS). Subsequently, the pancreata were embedded in paraffin 磷酸盐缓冲液