Both p38 MAPK and Akt mediated the effect of TRAF6 during myogenic differentiation.

<p>(A–C) C2C12 cells were transfected with the GFP-siRNA or TRAF6-siRNAs with or without various cDNA expression vectors as indicated. (A). Cells were harvested 24 hours after transfection. (B, C) Twenty-four hours after transfection, cells were induced to differentiate in DM for another 36 hours before harvest. WCEs from (A–C) were subjected to SDS-PAGE and Western blot analysis for various proteins as indicated. MKK6EE, MEK1ca and Aktca are the constitutively active mutants of MKK6, MEK1, and Akt respectively. xp: Xpress-tag.</p

Muscle regeneration was accelerated in STAT1−/− mice after cardiotoxin injection.

<p>Tibialis anterior muscles from 2-month-old wild-type and STAT1−/− mice were injected with 30 ul of 10 nM cardiotoxin. The TA muscles were harvested at different time points as indicated after injury. (A) The muscles were fixed in 4% paraformaldehyde/PBS (pH 7.4) for 1 hour at 4°C, infiltrated sequentially with 10%, 20% and 30% sucrose, and embedded in O. C. T. solution. Six micrometers of cryosections were stained with hematoxylin and eosin. Bars: 300 um. (B) The diameter of the regenerated fibers and the percentage of un-repaired region from three independent experiments were measured 10 days post-injury. Data were presented as mean ± SD. Asterisk: p<0.05. (C) Tibialis anterior muscles were homogenized in protein lysis buffer. Equal amounts of protein were separated by SDS-PAGE followed by western blotting with different antibodies as indicated.</p

TRAF6 was required for myogenic differentiation.

<p>(A, B) C2C12 cells were transfected with a GFP-siRNA, two different TRAF6-siRNAs, or two different TRAF2-siRNAs as indicated. (C) C2C12 cells were transfected in triplicate with either the GFP-siRNA or TRAF6-siRNA together with different luciferase reporter constructs as indicated. Twenty-four hours (h) after transfection, cells were induced to differentiate for 24 h or 48 h before harvest. For (A, B), 60 µg of whole cell extracts (WCE) were subjected to SDS-PAGE and Western blot (WB) analysis for various proteins indicated on the left side of the panels. For (C), cells were harvested after 24 h in DM and WCE were prepared and subjected to luciferase assays. Fold change was calculated as the ratio of the luciferase activity of the TRAF6-siRNA transfected cells over that of the GFP-siRNA transfected cells. The results were presented as mean+s.d. (D–G) C2C12 cells (D, E) or primary myoblasts (F, G) were transfected with the GFP-siRNA or TRAF6-siRNA. Cells were fixed at DM 48 h and subjected to immunostaining for myosin heavy chain (MHC) or MyoD. The nuclei were counterstained with DAPI. In (E, G), the percentage of MHC-positive cells was calculated as the ratio of the number of nuclei in MHC-positive cells over that of DAPI-positive nuclei (E) or MyoD-positive nuclei (G). Cells from five different microscopic fields were counted and the results were presented as mean+s.d.</p

The E3 ligase activity of TRAF6 was required for myogenic differentiation.

<p>(A) C2C12 cells were transfected with either the GFP-siRNA or the TRAF6-siRNA together with an empty vector or a vector encoding human TRAF6 as indicated. (B) C2C12 cells were transfected with either an empty vector (pcDNA) or an expression vector encoding either the wild-type (WT) or an E3 ligase-defective mutant TRAF6. (C) C2C12 cells were transfected in triplicate with G133-<i>luc</i> together with pcDNA or an expression vector encoding the wild-type or the mutant human TRAF6. Twenty-four hours after transfection, cells were switched to DM for another 24 hours. WCEs were prepared and subjected to luciferase assays. Fold change was calculated as the ratio of the luciferase activity in TRAF6-transfected cells over that in pcDNA-transfected cells. The results were presented as mean+s.d. (D) C2C12 cells were transfected with the GFP-siRNA or TRAF6-siRNA together with pcDNA or an expression vector encoding the wild type or mutant human TRAF6. For (A), (B), and (D), cells were harvested at DM 36 h and WCE were subjected to SDS-PAGE and Western blot analysis for various proteins as indicated.</p

Loss of STAT1 in the bone marrow-derived cells accelerated muscle regeneration.

<p>Donor bone marrow cells were freshly prepared from the femurs of donor mice and filtered through a sterile 40 µm nylon Cell Strainer to remove debris. The cells were suspended in DMEM without serum prior transplantation. The recipient mice were given 8 Gy Gamma-irradiation and each injected with 2×10⁶ donor bone marrow cells through retro-orbital vein. Six to eight weeks after bone marrow transplantation, tibialis anterior muscles of the recipient mice were injected with 30 ul of 10 nM cardiotoxin. (A) The muscles were fixed in 4% paraformaldehyde/PBS (pH 7.4) for 1 hour at 4°C, infiltrated sequentially with 10%, 20% and 30% sucrose, and embedded in O. C. T. solution. Six micrometers of cryosections were stained with hematoxylin and eosin. Bars: 300 um. (B) The tails and peripheral blood cells from the recipient mice were harvested, and genomic DNA was extracted. PCR was used for genotyping. The upper and lower bands indicate genotypes of STAT1−/− and wild-type respectively. (C) The percentage of regenerated region 12 days post-injury and unrepaired region 15 days post-injury from three independent experiments were measured. Data were presented as mean ± SD. Asterisk: p<0.05.</p

STAT1−/− and WT mice have similar extent of macrophage infiltration, but distinct profile of cytokines, after cardiotoxin injection.

<p>(A) Tissues from injured tibialis anterior, Gastrocnemius and Soleus muscles were digested twice in DMEM containing 0.2% Collagenase B and 0.2% Trypsin-EDTA at 37°C for 45 min. The digested tissues were applied to Ficoll-Paque centrifugation and the interface band was collected. For each staining reaction, 10⁶ cells were stained with PE-Cy5-anti-CD11b and PE-anti-Mac2 in 100 ul system and subjected to flow cytometry analysis. (B) The percentage of macrophages from three independent experiments was analyzed. Data were presented as mean ± SD. (C–F) Total RNA was isolated from tibialis anterior muscles and reverse transcribed. Triplicate samples were subjected to quantitative PCR. GAPDH was used as an internal control. The relative abundance of genes of interest was calculated after normalization to GAPDH. Data from three independent experiments were presented as mean ± SD. Asterisk: p<0.05.</p

Knockdown of TRAF6 impaired the CTX-induced muscle regeneration.

<p>(A–D) Tibialis anterior (TA) muscles of adult mice were injected with 30 µl of 10 µM CTX. One day after CTX injection, the GFP-siRNA and TRAF6-siRNA mixed with liposome were injected into the left and right injured TA muscles respectively once per day. TA muscles were collected at day (D) 3 and day 5 after CTX injection. The total RNA, muscle sections, or WCEs were prepared and subjected to RT-qPCR (A), hematoxylin-eosin staining (B), immunostaining (C), or SDS-PAGE and Western blot analysis (D). The number of myofibers with central nuclei (B) or the number of eMHC-positive myofibers (C) was calculated from five different microscopic fields. The results were presented as mean+s.d. CTX: cardiotoxin. eMHC: embryonic MHC.</p

TAK1 was required for myogenic differentiation.

<p>(A, B) C2C12 cells were transfected with various siRNAs as indicated. Cells were harvested at the indicated time points and WCEs were subjected to SDS-PAGE and Western blot analysis. (C–F) C2C12 cells (C, D) or primary myoblasts (E, F) were transfected with either the GFP-siRNA or the TAK1-siRNA. Forty-eight hours after induction of differentiation, cells were fixed and subjected to immunostaining for MHC or MyoD. The nuclei were counterstained with DAPI. In (D, F), the percentage of MHC-positive cells was calculated the same way as that described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034081#pone-0034081-g001" target="_blank">Fig. 1(E, G)</a>. (G, H) C2C12 cells were co-transfected with an empty vector or a TRAF6-expressing vector together with the GFP-siRNA or TAK1-siRNA as indicated. Twenty-four hours after induction of differentiation, cells were harvested for subsequent analysis: WCE were subjected to SDS-PAGE and Western blot analysis (G), while total RNA was prepared for RT-qPCR analysis (H) of relative myogenin gene expression. 1, 2, 3 in (G) and (H) denote the same set of samples.</p

Additional file 1 of Association between the triglyceride-glucose index and impaired cardiovascular fitness in non-diabetic young population

Additional file1: Table S1. Definition of physical activity. Table S2. Definition of smoking status. Table S3. The measurement methods of laboratory indicators. Table S4.The weights used in the analysis of this study. Table S5. The collinearity assessment outcomes for Model 3. Table S6. Comprehensive details of the Survival Cohort Study Design for NHANES 1999-2004. Table S7. Baseline characteristics of the study population with accessible survival outcomes. Table S8. Hazard ratios for all-cause mortality and cardiovascular mortality according to TyG Index. Figure S1. The association between TyG index and CVF impairment. Figure S2. The association between TyG index and all-cause mortality. Figure S3 The association between TyG index and cardiovascular mortality. Figure S4. Sensitivity analysis 1. Figure S5. Sensitivity analysis 2.Don

Additional file 1 of The metabolic score for insulin resistance in the prediction of major adverse cardiovascular events in patients after coronary artery bypass surgery: a multicenter retrospective cohort study

Additional file 1: Table S1. Baseline characteristics between excluded and included participants. Table S2. Sensitivity analysis for the association between the METS-IR and MACE. Table S3. Subgroup and interaction between the METS-IR (Per SD) and non-fatal MI and across various subgroups