SOX10 and S100B in Schwann cells

Figure 1. Expression pattern of Sox10 and other differentiation markers in Schwann cells. (A and B) Comparison of expression level between Sox10 and Sox9 in primary rat sciatic nerve Schwann cells and primary rat rib chondrocytes. (C) Time course of mRNA levels of Schwann cell differentiation markers determined by real-time RT-PCR analysis during rat perinatal stages. Figure 2. Modulation of S100B expression by SOX10 in Schwann cells. (A) mRNA levels of Sox10 (top) and S100b (bottom) in stable lines of primary rat Schwann cells retrovirally transfected with SOX10 or control GFP. (B) Protein level of S100B and Sox10 in stable lines of primary rat Schwann cells retrovirally transfected with SOX10 or control GFP. (C) Modulation of S100b expression by SOX10 in ROS cells. mRNA levels of Sox10 (top) and S100b (bottom) in stable lines of rat non-neurogenic ROS cells retrovirally transfected with SOX10 or control GFP. Figure 3. Suppression of S100B and Mpz expression by SOX10 insufficiency in Schwann cells. (A) mRNA levels of Sox10 (top), S100b (middle) and Mpz (bottom) in stable lines of primary rat Schwann cells retrovirally transfected with shRNA specific for Sox10 or GFP. (B) Protein levels of SOX10 and S100B in stable lines of primary rat Schwann cells retrovirally transfected with shRNA specific for SOX10 or control GFP. Figure 4. Identification of putative SOX10-response elements in S100B. (A) Luciferase activities after transfection of putative Schwann cell-related transcription factors into HeLa cells with a reporter construct containing a fragment (−1,000 to +200 bp) of the S100B gene. (B) Deletion analysis using luciferase-reporter constructs containing a series of deletion fragments of the S100B gene in HeLa cells transfected with SOX10 or control GFP. (C) Comparison of human (Hs), rat (Rn), and mouse (Mm) sequences in three putative SOX motifs in the S100B promoter and mutated sequences (Mut A, Mut B, and Mut C), used in the following mutagenesis analysis. (D) Site-directed mutagenesis analysis using luciferase-reporter constructs containing –334 to +200 bp of the S100B gene with mutations as in Figure 3C within the three SOX motifs in the cells above. Figure 5. Identification of putative response elements in S100B intron 1 by SOX10 and direct binding of SOX10 to the response elements. (A) Comparison of human (Hs), rat (Rn) and mouse (Mm) sequences in the putative SOX motif of the S100B intron 1 and mutated sequence (Mut D), used in the following mutagenesis analysis. (B) Deletion and site-directed mutagenesis analysis using luciferase-reporter constructs containing –334 to +200 bp of the S100B gene in HeLa cells transfected with SOX10 or control GFP. (C) ChIP assay performed using cell lysates of Schwann cells that were amplified by a primer set spanning the identified regions; sites A & B (top), site A (second row), site B (third row), and site D (fourth row), or not spanning the region (bottom) before (input) and after immunoprecipitation with antibodies to Sox10 (a-Sox10) or non-immune IgG (IgG). Genomic DNA was amplified as a positive control. Figure 6. Suppressed Schwann cell proliferation by SOX10-S100B signaling. Comparison of Sox10 and S100b mRNA levels between conditions of proliferation and differentiation in rat sciatic nerve Schwann cells. Figure 7. Enhanced proliferation by knockdown of Sox10 or S100b in Schwann cells. (A, B) BrdU labeling of stable lines of Schwann cells retrovirally transfected with SOX10 or shRNA specific for SOX10 and GFP (A). Ratio of BrdU-positive cells to total cells was quantified after 3 d culture of stable lines of Schwann cells transfected with Sox10 expressing vector, shRNA vector specific for Sox10, and control GFP vector (B). (C) Growth curves using the CCK-8 assay of stable lines of Schwann cells retrovirally transfected with sh-Sox10 or control GFP. (D, E) BrdU labeling of stable lines of Schwann cells retrovirally transfected with S100b or shRNA specific for S100b and GFP (D). Ratio of BrdU-positive cells to total cells were quantified after 3-day-old cultures of stable lines of Schwann cells were transfected with S100b expressing vector, shRNA vector specific for S100b, and control GFP vector (E). Figure 8. Enhanced proliferation by knockdown of S100b or Sox10 in C3H10T1/2 cells. (A) mRNA levels of S100b determined by real-time RT-PCR in stable lines of mouse mesenchymal C3H10T1/2 cells retrovirally transfected with S100b, shRNA for S100b, or control GFP. (B) mRNA levels of Sox10 determined by real-time RT-PCR in stable lines of mouse mesenchymal C3H10T1/2 cells retrovirally transfected with Sox10, shRNA for Sox10, or control GFP. (C and D) Growth curves using the CCK-8 assay of stable lines of C3H10T1/2 cells as mentioned above. Figure 9. Impaired myelination by knockdown of S100b. (A) Immunocytochemistry of neurons and stable lines of Schwann cells retrovirally transfected with shRNA specific for S100b or control GFP in DRG dissociated cultures. Staining of Tuj1 (red), MBP (green) and Hoechst (blue) in neurons, Schwann cells, and nuclei, respectively. (B) The number of MBP-positive Schwann cells in a high-power field of the immunocytochemistry as in Figure 9A.

图1 雪旺细胞（Schwann cells）中Sox10及其他分化标志物的表达模式。(A和B) 原代大鼠坐骨神经雪旺细胞与原代大鼠肋骨软骨细胞中，Sox10与Sox9的表达水平比较。(C) 大鼠围产期雪旺细胞分化标志物mRNA水平的时间进程，经实时RT-PCR分析测定。图2 雪旺细胞中SOX10对S100B表达的调控。(A) 经逆转录病毒转染SOX10或对照绿色荧光蛋白（GFP）的原代大鼠雪旺细胞稳定株中，Sox10（上方）与S100b（下方）的mRNA水平。(B) 经逆转录病毒转染SOX10或对照GFP的原代大鼠雪旺细胞稳定株中，S100B与Sox10的蛋白水平。(C) ROS细胞中SOX10对S100b表达的调控：经逆转录病毒转染SOX10或对照GFP的大鼠非神经源性ROS细胞稳定株中，Sox10（上方）与S100b（下方）的mRNA水平。图3 雪旺细胞中SOX10表达不足对S100B与Mpz表达的抑制作用。(A) 经逆转录病毒转染Sox10特异性短发夹RNA（shRNA）或GFP的原代大鼠雪旺细胞稳定株中，Sox10（上方）、S100b（中部）与Mpz（下方）的mRNA水平。(B) 经逆转录病毒转染SOX10特异性shRNA或对照GFP的原代大鼠雪旺细胞稳定株中，SOX10与S100B的蛋白水平。图4 S100B基因中推定SOX10应答元件的鉴定。(A) 将推定的雪旺细胞相关转录因子与含有S100B基因片段（-1000至+200 bp）的报告质粒共转染海拉细胞（HeLa）后的荧光素酶活性。(B) 在转染SOX10或对照GFP的海拉细胞（HeLa）中，使用含有一系列S100B基因缺失片段的荧光素酶报告质粒进行缺失分析。(C) 后续诱变分析中使用的S100B启动子区域3个推定SOX基序的人（Hs）、大鼠（Rn）、小鼠（Mm）序列比对，以及突变序列（Mut A、Mut B、Mut C）。(D) 采用上述突变的荧光素酶报告质粒（含S100B基因-334至+200 bp片段，且在3个SOX基序处带有图3C所示突变）进行定点诱变分析。图5 SOX10对S100B基因内含子1中推定应答元件的鉴定，以及SOX10与该应答元件的直接结合。(A) 后续诱变分析中使用的S100B内含子1推定SOX基序的人（Hs）、大鼠（Rn）、小鼠（Mm）序列比对，以及突变序列（Mut D）。(B) 在转染SOX10或对照GFP的海拉细胞（HeLa）中，使用含有S100B基因-334至+200 bp片段的荧光素酶报告质粒进行缺失与定点诱变分析。(C) 染色质免疫沉淀（ChIP）实验：以雪旺细胞裂解液为材料，使用跨越已鉴定区域（位点A与B（上方）、位点A（第二行）、位点B（第三行）、位点D（第四行））或不跨越该区域（下方）的引物对，在使用Sox10抗体（α-Sox10）或非免疫IgG进行免疫沉淀前后进行扩增；以基因组DNA扩增作为阳性对照，输入样本（input）为免疫沉淀前的对照。图6 SOX10-S100B信号通路对雪旺细胞增殖的抑制作用。比较大鼠坐骨神经雪旺细胞在增殖与分化条件下的Sox10与S100b mRNA水平。图7 敲低Sox10或S100b可增强雪旺细胞的增殖。(A、B) 经逆转录病毒转染SOX10、Sox10特异性shRNA与GFP的雪旺细胞稳定株的溴脱氧尿苷（BrdU）标记结果(A)。对转染Sox10表达载体、Sox10特异性shRNA载体与对照GFP载体的雪旺细胞稳定株培养3天后，定量统计BrdU阳性细胞占总细胞的比例(B)。(C) 采用细胞计数试剂盒-8（CCK-8）法测定转染sh-Sox10或对照GFP的雪旺细胞稳定株的生长曲线。(D、E) 经逆转录病毒转染S100b、S100b特异性shRNA与GFP的雪旺细胞稳定株的BrdU标记结果(D)。对转染S100b表达载体、S100b特异性shRNA载体与对照GFP载体的雪旺细胞稳定株培养3天后，定量统计BrdU阳性细胞占总细胞的比例(E)。图8 敲低S100b或Sox10可增强C3H10T1/2细胞的增殖。(A) 经逆转录病毒转染S100b、S100b特异性shRNA或对照GFP的小鼠间充质C3H10T1/2细胞稳定株中，经实时RT-PCR测定的S100b mRNA水平。(B) 经逆转录病毒转染Sox10、Sox10特异性shRNA或对照GFP的小鼠间充质C3H10T1/2细胞稳定株中，经实时RT-PCR测定的Sox10 mRNA水平。(C、D) 采用CCK-8法测定上述C3H10T1/2细胞稳定株的生长曲线。图9 敲低S100b可损害髓鞘形成。(A) 背根神经节（DRG）解离培养体系中，经逆转录病毒转染S100b特异性shRNA或对照GFP的神经元与雪旺细胞稳定株的免疫细胞化学染色结果：神经元采用神经元特异性Ⅲ类β微管蛋白（Tuj1）染色（红色），雪旺细胞采用髓鞘碱性蛋白（MBP）染色（绿色），细胞核采用Hoechst染色（蓝色）。(B) 如图9A所示的免疫细胞化学样本中，高倍视野下MBP阳性雪旺细胞的数量统计。