SOX10 and S100B in Schwann cells

<p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> <p>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).</p> <p>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.</p> <p>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.</p

Suppression of S100B and Mpz expression by SOX10 insufficiency in Schwann cells.

<p>(A) mRNA levels of <i>Sox10</i> (top), <i>S100b</i> (middle) and <i>Mpz</i> (bottom) in stable lines of primary rat Schwann cells retrovirally transfected with shRNA specific for <i>Sox10</i> or <i>GFP</i>. All experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus <i>GFP</i> or sh-<i>GFP</i>. (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.</p

Suppressed Schwann cell proliferation by SOX10-S100B signaling.

<p>Comparison of <i>Sox10</i> and <i>S100b</i> mRNA levels between conditions of proliferation and differentiation in rat sciatic nerve Schwann cells. All experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus proliferation condition.</p

Enhanced proliferation by knockdown of <i>Sox10</i> or <i>S100b</i> in Schwann cells.

<p>(A, B) BrdU labeling of stable lines of Schwann cells retrovirally transfected with <i>SOX10</i> or shRNA specific for <i>SOX10</i> and <i>GFP</i> (A). Ratio of BrdU-positive cells to total cells was quantified after 3 d culture of stable lines of Schwann cells transfected with <i>Sox10</i> expressing vector, shRNA vector specific for <i>Sox10</i>, and control <i>GFP</i> vector (B). (C) Growth curves using the CCK-8 assay of stable lines of Schwann cells retrovirally transfected with sh-<i>Sox10</i> or control <i>GFP</i>. Experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus control. (D, E) BrdU labeling of stable lines of Schwann cells retrovirally transfected with <i>S100b</i> or shRNA specific for <i>S100b</i> and <i>GFP</i> (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 <i>S100b</i> expressing vector, shRNA vector specific for <i>S100b</i>, and control <i>GFP</i> vector (E). Experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus control.</p

Impaired myelination by knockdown of <i>S100b</i>.

<p>(A) Immunocytochemistry of neurons and stable lines of Schwann cells retrovirally transfected with shRNA specific for <i>S100b</i> or control <i>GFP</i> 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 Fig. 9A. Experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus control.</p

Identification of putative SOX10-response elements in S100B.

<p>(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 <i>S100B</i> gene. *<i>P<</i>0.05 versus GFP. (B) Deletion analysis using luciferase-reporter constructs containing a series of deletion fragments of the <i>S100B</i> 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 <i>S100B</i> gene with mutations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115400#pone-0115400-g003" target="_blank">Fig. 3C</a> within the three SOX motifs in the cells above. *<i>P<</i>0.05 versus wild-type (WT) with SOX10. All experiments were repeated independently three times with data shown as the mean ± SEM.</p

Enhanced proliferation by knockdown of <i>S100b</i> or <i>Sox10</i> in C3H10T1/2 cells.

<p>(A) mRNA levels of <i>S100b</i> determined by real-time RT-PCR in stable lines of mouse mesenchymal C3H10T1/2 cells retrovirally transfected with <i>S100b</i>, shRNA for <i>S100b</i>, or control <i>GFP</i>. (B) mRNA levels of <i>Sox10</i> determined by real-time RT-PCR in stable lines of mouse mesenchymal C3H10T1/2 cells retrovirally transfected with <i>Sox10</i>, shRNA for <i>Sox10</i>, or control <i>GFP</i>. (C and D) Growth curves using the CCK-8 assay of stable lines of C3H10T1/2 cells as mentioned above. All experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus control.</p

Expression pattern of <i>Sox10</i> and other differentiation markers in Schwann cells.

<p>(A and B) Comparison of expression level between <i>Sox10</i> and <i>Sox9</i> 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. All experiments were repeated independently three times with data shown as the mean (bars) ± SEM (error bars).</p

Identification of putative response elements in S100B intron 1 by SOX10 and direct binding of SOX10 to the response elements.

<p>(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 <i>S100B</i> gene in HeLa cells transfected with SOX10 or control GFP. *<i>P<</i>0.05 versus wild-type (WT) with SOX10. All experiments were repeated independently three times with data shown as the mean ± SEM. (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 (α-Sox10) or non-immune IgG (IgG). Genomic DNA was amplified as a positive control.</p

Modulation of S100B expression by SOX10 in Schwann cells.

<p>(A) mRNA levels of <i>Sox10</i> (top) and <i>S100b</i> (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 <i>S100b</i> expression by SOX10 in ROS cells. mRNA levels of <i>Sox10</i> (top) and <i>S100b</i> (bottom) in stable lines of rat non-neurogenic ROS cells retrovirally transfected with SOX10 or control GFP. Experiments were repeated independently three times with data shown as the mean ± SEM. *<i>P<</i>0.05 versus control.</p