Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 1. Schematic primary sequences of the STIM2 and STIM2-UI region, binding assay of GST-fused STIM1-UI protein with a triad sample, and co-immunoprecipitation of full-length STIM2 with SERCA1a. (a) The position of each domain in STIM2 is presented according to previous reports: the overall diagram92,93, and CAD/SOAR94. Numbers indicate the sequence of amino acids. UI means unidentified region. S, signal peptide; cEF, canonical EF-hand; hEF, non-functional hidden EF-hand; SAM, sterile α-motif; T, transmembrane domain; C, coiled-coil domain; CAD/SOAR, Ca2+ release-activated Ca2+ (CRAC)-activating domain/STIM1- Orai1-activating region; P, proline/serine-rich domain; K, lysine-rich domain. (b) Immobilized GST-fused STIM2-UI proteins on GST beads were separated on a 10% SDS-PAGE gel, and the gel was subjected to immunoblot with anti-GST antibody. GST or GST-fused STIM2-UI proteins are indicated by asterisks. (c) The bound proteins in the binding assay of GST-fused STIM2-UI protein with the triad sample from rabbit skeletal muscle were separated on a 10% SDS-PAGE gel, and the gel was stained with Coomassie Blue. GST was used as a negative control. GST-fused STIM2-UI proteins are indicated by an asterisk. The eight proteins that were bound to the GST-fused STIM2-UI protein are indicated on the right side of the figure (bands 1 to 8). (d) The triad sample obtained from rabbit skeletal muscle (30 μg of total proteins) was subjected to a co-immunoprecipitation assay with anti-STIM2 antibody, and the immunoprecipitant was subjected to immunoblot analysis with anti- STIM2 or anti-SERCA1a antibodies. Triad sample indicates a simple immunoblot of the triad sample. Without Ab indicates a reaction without anti-STIM2 antibody. Three independent experiments were conducted. IB, IP, or Ab means immunoblot, immunoprecipitation, or antibody, respectively. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full-length blots are presented in Supplementary Fig. 4. SERCA1a was successfully co-immunoprecipitated with full-length STIM2.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Binding Assay, Immunoprecipitation, Sequencing, Functional Assay, Sterility, SDS Page, Western Blot, Staining, Negative Control, Co-Immunoprecipitation Assay

Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 2. Knockdown of STIM2 in mouse primary skeletal myotubes. (a) Three different forms of siRNA were used to knock down STIM2 in mouse primary skeletal myotubes. The lysate of the STIM2-knockdown myotubes (50 μg of total protein) was subjected to immunoblot analysis with anti-STIM2 antibody (upper panel). α-actin and Coomassie Brilliant Blue staining were loading controls. #3 siRNA knocked down STIM2 more effectively compared with others (up to 90%). Untransfected control and scrambled siRNA-transfected myotubes were used as negative controls. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full-length blots are presented in Supplementary Fig. 5. Three independent experiments were conducted. (b) The STIM2-knockdown myotubes with #3 siRNA show normal myotube formations, and are indistinguishable from the untransfected or the scrambled siRNA-transfected control. ‘Immature myotubes on D3’ means untransfected immature myotubes on differentiation day 3 (left). ‘Myotubes on D5’ means untransfected, scrambled siRNA-transfected, or STIM2-knockdown myotubes on differentiation day 5. The bar represents 50 μm.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Knockdown, Western Blot, Staining, Control, Transfection

Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 3. Increases in 45Ca2+-uptake into the SR through SERCA1a and the amount of Ca2+ in the SR, and a decrease in the cytosolic Ca2+ level at rest by STIM2-knockdown. (a) Oxalate-supported 45Ca2+-uptake into the SR through SERCA1a using the homogenate of the STIM2-knockdown myotubes was measured at 70 nM or 1 μM of free 45Ca2+. Either an untransfected or a scrambled siRNA-transfected control was used as a negative control. The results are presented as the mean ± S.E. of three independent experiments (Table 3). *Significant difference compared with the untransfected control (p < 0.05). The STIM2-knockdown myotubes showed a significantly increased 45Ca2+-uptake through SERCA1a only at 1 μM of free 45Ca2+. (b) Cytosolic Ca2+ level at rest was examined in the STIM2-knockdown myotubes, and histograms are shown for the mean values of each. *Significant difference compared with the untransfected control (p < 0.05). The values are presented as the mean ± S.E. for the number of myotubes shown in the parentheses of Table 4. Cytosolic Ca2+ level at rest was decreased by STIM2-knockdown. (c) To measure the amount of Ca2+ in the SR, TG was applied to the STIM2-knockdown myotubes in the absence of extracellular Ca2+ to avoid extracellular Ca2+ entry. A representative trace for each group is shown, and the results are summarized as bar graphs in the right-hand panel. *Significant difference compared with the untransfected control (p < 0.05). The values are presented as the mean ± S.E. for the number of myotubes shown in the parentheses of Table 4. The amount of Ca2+ in the SR was significantly increased by STIM2-knockdown.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Knockdown, Transfection, Control, Negative Control

Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 4. Decreased SOCE by STIM2-knockdown. The Ca2+ in the SR of the STIM2-knockdown myotubes was depleted by the treatment of TG (2.5 μ M) in the absence of extracellular Ca2+. Extracellular Ca2+ (2 mM) was applied to the myotubes to induce SOCE. (a) A representative trace for each group is shown. The results are summarized as bar graphs for the area under the peaks (b) or the slope at the rising phase of the peaks (c). *Significant difference compared with the untransfected control (P < 0.05). The values are presented as the mean ± S.E. for the number of myotubes shown in the parentheses of Table 4. SOCE was significantly decreased by STIM2-knockdown.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Knockdown, Control

Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 5. Enhanced intracellular Ca2+ release through RyR1 in response to caffeine or KCl by STIM2- knockdown. Caffeine (a specific agonist of RyR1) (a), or KCl (a membrane depolarizer) (b), was applied to the STIM2-knockdown myotubes, and the intracellular Ca2+ release from the SR to cytosol through RyR1 in the myotubes was measured. Histograms are shown for the normalized peak amplitude to the mean value of those from the untransfected control. The results are presented as the mean ± S.E. for the number of experiments in the parentheses in Table 4. *Significant difference was compared with the untransfected controls (P < 0.05). Intracellular Ca2+ releases in response to either caffeine or KCl is significantly increased by STIM2-knockdown.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Knockdown, Membrane, Control

Journal: Scientific reports

Article Title: STIM2 regulates both intracellular Ca 2+ distribution and Ca 2+ movement in skeletal myotubes.

doi: 10.1038/s41598-017-18256-3

Figure Lengend Snippet: Figure 6. Decreased protein content level of TRPC6, JP1, or CaM1 by STIM2-knockdown. (a) Lysate of the STIM2-knockdown myotubes was subjected to an immunoblot assay with one of the antibodies against thirteen proteins that mediate or regulate skeletal muscle functions. α-Actin was used as a loading control. Three independent experiments per each protein were conducted. JP, junctophilin; CSQ, calsequestin. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full- length blots are presented in Supplementary Figs 6 to 19. Among them, the expression level of TRPC6, JP1, or CaM1 was significantly decreased (indicated by asterisks). (b) The protein content level in (a) is presented as bar graphs. Bar graphs are presented as the mean ± S.E. for three independent experiments. *Significant difference compared with the scrambled siRNA control (P < 0.05). The protein content level of TRPC6, JP1, or CaM1 in the STIM2-knockdown myotubes was significantly decreased.

Article Snippet: Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862).

Techniques: Knockdown, Western Blot, Control, Expressing