In summary, using DNA microarrays, we have described the pattern of expression of genes which are induced during transdifferentiation of pre-myoblastic C2C12cells during 24 h of treatment with rhBMP2 and rhBMP7. A total of 1,318 genes were found to be induced by both rhBMPs which could be involved in osteoblastic differentiation, twenty-four of which were confirmed through RT-qPCR after 4, 8, 12 and 24h of treatment, resulting in 13 transcription-related genes (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1) which were induced between 4 and 24 h after treatment. Four of the genes identified (Lrp6, Dvl1, Ecsit and PKCδ) are implicated in cell signalling, displaying a more heterogeneous pattern of expression, and seven are ECM-related genes (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10), which were preferentially activated after 12 h of treat- ment. Our results revealed some genes which were already known to be activated by BMP2. However, we found that many of them were also upregulated by treatment with rhBMP7, thus representing a novel sub- set of genes related with BMP7-induced differentiation of precursor cells into osteoblasts: Hoxc8, Glis1, Glis3, Ecsit, PKCd, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10. Fifteen genes were upregulated in the early period (during the first 12 h) while nine genes were upregulated at a later period (between 12 and 24 h). Among the downregulated genes, MyoD showed a decreased abundance in mRNA relative expression (data not shown), which is explained by the fact that, in the absence of BMPs, this muscular satellite cell line would be programmed to differentiate into myoblasts . Currently, we do not know whether the increased expression of genes at later stages was merely due to the indirect downstream effects of BMP/SMAD signalling as a result of earlier gene (for example, Ids) expression changes. Subsequent studies may elucidate whether the products of these upregulated early genes are responsible for inducing the expression of later genes.
These observations demonstrated that PLL-BNNTs were internalized by the cells, but provided no information on the nature of the mechanism responsible for this up-take. Sodium azide was exploited to investigate the energy-dependence of the up-take mechanism of PLL-BNNTs. The chosen incu- bation time was 30 min, as optimal compromise between effective ATP blocking by sodium azide, and reduced cyto- toxic effects resulting from a prolonged energy deficiency. Figure 2b shows a confocal image of cells incubated for 1 h in a medium containing 5 µ g/mL of Qdot-labelled BNNTs: internalization occurred as shown in the previous tests. An image of C2C12cells analogously incubated but following pre-treatment with sodium azide is shown in Figure 2c. Here a clear inhibition of cell up-take was evidenced by a negli- gible amount of internalized BNNTs and a massive BNNT accumulation on the cell membrane. This result demonstrates that the internalization of PLL-BNNTs by C2C12 is energy- dependent, as already noticed for PEI-coated BNNTs in other cell lines. 18
Methods: The mouse myoblasts, C2C12cells, were seeded in 6-well plastic plates and cultured in DMEM media. After differentiation, in a pilot study, C2C12 myotubes were treated with different concentrations of Res and ATRA for 12 h. The best result was obtained by treatment of 1and 25 μ M of Res and 1 μ M of ATRA. Then the main study was continued by single and combined treatment of these compounds at chosen concentration. After treatments, total RNA was extracted from C2C12cells. Complementary DNA (cDNA) was generated by the cDNA synthesis kit and FNDC5 mRNA expression was evaluated by the real-time PCR method.
dystrophy . Activation of SIRT1 promotes cell proliferation both in the liver  and colon , and SIRT1 has been proven to correlate with decrease of muscle mass, skeletal muscle strength  and oxidative damage in mdx mice . However, overexpression of SIRT1- induced proliferation of muscle cells in vivo and in vitro has not been reported yet. In this study, we have shown that SIRT1 regulates skeletal muscle regeneration in C2C12cells model, and that the stimulating effect is both approximate- ly in dose-dependent manner and time-depen- dent manner. And in order to study the mecha- nisms and feature of SIRT1-inducing prolifera- tion in muscle cells, we have test several mus- cle regeneration-related signaling pathways such as mTOR, myostatin and Wnt signaling pathways. And our results showed that Wnt sig- naling pathway plays a significant role in SIRT1- inducing proliferation in muscle cells, which has not been reported yet elsewhere.
HepG2 and C2C12cells were cultured in 6-well plate (5×10 3 and 3 × 10 5 cells/mL, respectively), When cells reached con ﬂ uence, the mediums were replaced by DMEM/RPMI 1640 supplemented with R. anatolicus extract at various concentrations (200, 100, 50, 25 µg/ mL and DMSO as solvent control). After 24-h incubation, cells harvested for glycogen and total protein measure- ment. In order to prevent spontaneous glycogenolysis, the cells were thawed at the moment of glycogen extrac- tion. Glycogen was extracted from the cells and measured according to M. Rousset et al method. 35 The relative levels of glycogen content of HepG2 and C2C12cells were normalized to the protein content by the Bradford reagent. The value obtained was indicated as µg of glycogen per milligram of protein.
, and PDGF receptor α from cell extracts from IGF-1-stimulated cells (Figure 4B, top panels) confirm the decreased expression after IGF-1 treatment identified from the mass spectroscopic analysis. As a control, West- ern blots for two proteins whose expression remained constant during the 8 hour IGF-1 treatment (actin and Hsp27) are shown (Figure 4B, bottom panels). When quantified, data from western blots indicate Rho-GDI expression levels decrease 5.1-fold, cofilin levels drop 8.3- fold, and PDGF receptor α levels decrease by 4-fold over the 8 hour time course. These observed decreases in pro- tein expression are similar to those observed in the 2D gels. Averaged analysis of the spots on the 2D gels indi- cates a 4.3-fold decrease for Rho-GDI, a 6-fold decrease for cofilin, and a 4-fold decrease for PDGF receptor α. Fig- ure 5A shows regions of 2D gels in which expression of other proteins including Rad50, enolase, IκBKb, and Hsp70 increased after treatment with IGF-1. Western blots for these proteins confirmed the change in expression IGF-1 treatment of C2C12cells activates MAP kinase and PI
This study examined the role of CSN3 in skeletal muscle differentiation and proliferation. CSN3 is subunit 3 of CSN complex, a highly conserved multifunctional com- plex composed of 8 subunits (CSN1-CSN8) present in several organisms [2, 8, 44]. The mammalian CSN com- plex is involved in numerous molecular and cellular processes including protein degradation and phosphoryl- ation, gene transcription, cell cycle regulation, subcellular localization, apoptosis and development [39, 45–47]. Some of the individual subunits were shown to have an additional role(s) independent of the complex . Tissue specific roles of individual CSN subunits are not well characterized. We previously reported that CSN3 binds to β1D integrin, an isoform of β1 integrin expressed in differentiated cardiac and skeletal muscle. In fact, CSN3 localized to integrin adhesions during muscle cell differentiation in C2C12cells and Z-bands iso- lated from adult mice . This work is the first study to examine the role of CSN3 in skeletal muscle proliferation and differentiation.
To conclude, our study highlights the antioxidant role of 3,4-DHPEA-EA(P) in C2C12cells, as it is able to reduce intracellular levels of ROS resulting in inhibition of cell death and atrophy, conditions observed under increased oxidative stress. Future studies should be carried out to better clarify the effects of 3,4-DHPEA-EA(P) at cellular level and in vivo models to develop new therapeutic strategies for the treatment of muscular diseases related to the increase of oxidative stress.
phosphorylated-active form of JNK and of its downstream target c-Jun. We demonstrated, by Western blot analysis, that 3,4-DHPEA-EA(P) was efficient in inhibiting the phospho-active form of JNK. This data suggests that the growth arrest and cell death of C2C12 proceeds via the JNK/c-Jun pathway. Moreover, we demonstrated that 3,4-DHPEA-EA(P) affects the myogenesis of C2C12cells; because MyoD mRNA levels and the differentiation process are restored with 3,4-DHPEA-EA(P) after treatment. Overall, the results indicate that 3,4-DHPEA-EA(P) prevents ROS-mediated degenerative process in a genomic and epigenomic manner by functioning as an efficient antioxidant.
Micropatterned C2C12 myotubes develop more sarcomeres than those grown on other substrates In this study, we compared C2C12 myotubes cultured on three different substrates. The first was a micropatterned gelatin hydrogel with 10 μm-wide grooves as previously described (see “Methods” section) . The second was the same gelatin hydrogel but lacking grooves. The third was either plastic (molecular experiments) or collagen- coated glass (morphological experiments) to mimic the predominant cell culture methods used by the skeletal muscle research community. Cells were analyzed at day 4 of differentiation (D4) and day 7 of differentiation (D7) (Fig. 1). Some analyses also used primary myoblasts from C57Bl6/J gastrocnemius  as a reference, also differen- tiated for 4 or 7 days on micropatterned gelatin hydrogels. Because one defining property of skeletal muscle in vivo is the presence of sarcomeres yet most C2C12 myo- tube cultures do not yield robust sarcomeres, we examined sarcomere formation in vitro. To facilitate these studies, we generated a stable C2C12 cell line ex- pressing eGFP-tagged α-actinin-2 (ACTN2). Cells were FACS sorted to select low expressors, to mitigate poten- tial unwanted effects of high α-actinin-2 overexpression. Representative images of eGFP-ACTN2 C2C12cells at D7 on each substrate are shown (Fig. 2a – c, e). Primary myotubes at D7 on patterned gelatin hydrogels served as a reference (Fig. 2d, f ). The proportion of myotubes
450a-1, a non-myogenic miRNA which does not have overlapping predicted targets with miR-1/206. Luciferase quantification showed that this miRNA did not cross- react with the selected SR 3′ UTRs or with 3′ UTRs car- rying tandem copies of the complete reverse comple- ment of either miR-206 (2x206) or miR-1 (2x1) (data not shown). However, it efficiently downregulated the positive control construct 2x450a-1, constructed simi- larly to 2x206 and 2x1 (Additional file 3: Figure S3). To determine the sensitivity of the assay, we next assessed the activity of miR-1/206 on the 2x206 and 2x1 con- structs as well as on the 3′ UTR of Ccnd1, previously identified as a miR-206 target in C2C12cells . We also monitored promoter interference causing potential transcriptional repression of the reporter construct using a miRless construct, which expresses the Renilla lucifer- ase RNA without miRNA target sites. Figure 1a shows the result of this analysis with each luciferase signal measured in the presence of miR-1 or miR-206 normal- ized to the same constructs co-transfected with the con- trol miRNA, miR-450a-1 (see “Methods”). The data, presented as fold change versus miR-450a-1 control, show that miRless expression did not change appreciably in the presence of miR-1 or miR-206 and that miR-1 and miR-206 efficiently targeted 2x1 and 2x206 along with the positive control Ccnd1 (Fig. 1a). Furthermore, both miR-1 and miR-206 also reduced luciferase activity by targeting the 3′ UTRs of Srsf9 and Tra2b fused to the reporter gene while they did not have any effect on the Srsf3 construct (Fig. 1b). While statistically significant, the negative regulation of the Tra2b 3′ UTR was modest in magnitude. Thus, we focused our next set of experi- ments only on Srsf9 activity and its potential role in muscle differentiation. We substantiated the specific miR-1/206 targeting of the Srsf9 3′ UTR by MRE muta- genesis. When we reversed the orientation of the pre- dicted MRE in the Srsf9 3′ UTR, which preserves positioning of any unrecognized flanking elements, this mutant construct (termed Srsf9 MRE Rev) restored re- porter gene activity to levels measured in the presence of the control miR-450a-1 (Fig. 1c). Moreover, endogen- ous Srsf9 mRNA expression decreased in myoblasts when we transfected miR-1 or miR-206 overexpression plasmids (Additional file 4: Figure S4 A). Taken to- gether, these results establish that expression of the miR-1/206 family can directly modulate the level of Srsf9 in C2C12 myoblasts.
Dietary supplemental herbs with many beneficial effects have long been considered to enhance health status and physical strength as well as to improve abnormal status among the elderly . Yams are commonly use in medications because of their various pharmaceutical functions, which include enhancement of the digestive process in the stomach and intestines, immune regulation, and antiaging, antiinflammation and antioxidation effects. In traditional medicine, yams are known as a nourishing herb that alleviates yin deficiency in the spleen, lung, and kidney by providing a supplementary energy, qi, therefore, it is used to treat metabolic syndromes such as obesity, diabetes, and hypothyroidism . In addition, yams have been used to prevent the aging process (e.g., muscle weakness) because they control muscle function by spleen control . However, there is little known about the medicinal effects of yams on muscle function. In the present study, we investigated whether yam extract and its active compound, allantoin could help enhance the muscle function in myotubes. The results revealed that yam extract and allantoin significantly increased myoblast differentiation into myotubes in C2C12cells and mitochondrial biogenesis through upregulation of the mitochondrial transcription factors, PGC1α, TFAM, NRF1, and SIRT1 via activation of the AMPK/ACC signaling pathway.
SHME upregulates HO-1 and Nrf2 protein expression Because HO-1 is an important component of cellular defenses against oxidative stress, we assessed whether non-cytotoxic concentrations of SHME would affect HO-1 protein expression. As shown in Figure 5, C2C12cells exposed to the SHME showed concentration- and time-dependent increases in HO-1 expression com- pared with that in the control group. Several studies have reported that Nrf2 is an important upstream con- tributor to the mechanism of HO-1 expression; thus, we examined whether the SHME could induce Nrf2 expres- sion in C2C12cells. After exposure to SHME, C2C12
Cell viability was evaluated by a standard colorimetric assay as previously described . Briefly, C2C12cells (0.4 × 10 4 cells/100 μL) were seeded in a 96-well micro- plate and treated with amygdalin at concentrations of 0– 25 μM for 24 or 48 h. At the end of the treatment, the cell monolayers were incubated with 3-[4,5-dimethyl- thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) so- lution (0.5 mg/mL) in phosphate-buffered saline for 4 h. The formation of purple formazan was measured in rela- tion to the absorbance at 570 nm by a microplate reader (Bio-Rad, USA). The cell viability was presented as a per- centage relative to that of vehicle-treated control cells.
Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) ph- thalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myob- lasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin in- creases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12cells were ex- posed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.
Figure 4. Cellular localization and delayed degradation of p27 after CLB treatment. (A-B) CLB increases p27 in the nucleus and depletes it from the cytoplasm, based on Western blotting (A) and immunofluorescence (B). In (A), nuclear and cytosolic p27 were measured in C2C12cells incubated with 100 µM CLB for 1 h. H3 and GAPDH were used as loading controls for nuclear and cytosolic fractions, respectively. Results are mean ± SD from triplicate experiments. **, p < 0.01; ***, p < 0.001. In (B), synchronized cells were treated with 100 µM CLB for 12 h, probed with anti-p27 antibody, and stained with Alexa 594-conjugated secondary antibody (red). DAPI was used to label the nucleus (blue). Scale bar, 50 µm. Only p27 in the nucleus was quantified. Data are mean ± SD (n = 3). (C) CLB delays p27 degradation. C2C12cells were incubated with 0 or 100 µM CLB for 1 h and then treated with cycloheximide according to indicated times. p27 stability was examined by quantitative Western blot, results are mean ± SD from two independent experiments. **, p < 0.01.
indicative of apoptotic cell death, was checked after cells were loaded with PA or with PA and unsaturated fatty acids (Figure 2A). Treatment of C2C12 with PA induced obviously DNA cleavage, and AA but not ETYA pre- vented more effectively PA-caused DNA laddering than did POA or OA. Apoptosis is associated with a wide set of biochemical and physical changes in cytoplasm, nu- cleus and plasma membrane. However, the alteration in the mitochondrial permeability transition precedes cellu- lar apoptosis, that is, mitochondrial opening induces depolarization of the transmembrane potential with con- comitant release of apoptogenic factors and loss of oxi- dative phosphorylation. In this presentation, changes in mitochondrial potential of C2C12cells exposed to PA were measured by using JC-1 (Figure 2B). As apoptotic progression undergoes, the electrochemical gradient across the mitochondrial membrane collapses and is aptly monitored by JC-1 dye. PA caused a decrease in mito- chondrial membrane potential, which was indicated by the increased ratio of fluorescence (485 nm/530 nm). Fur- thermore, PA-induced mitochondrial dysfunction was re- versed to the control level by AA. In contrast, ETYA did
In present study, we discovered the expression of miR-696 decreased during C2C12 cell proliferation and differentiation (Figure 1F and G), which indicated that miR-696 might have a negative function in skel- etal muscle myogenesis. Through Edu and cell cycle test, we testified that miR-696 could repress the pro- liferation of C2C12cells (Figure 2B-2E and Figure 2H-2J). According to the flow cytometry results, de- spite the tiny difference between miR-696 mimics group and NC group, it could be of biological signif- icance. The similar results to our study were also re- ported in several previous studies [17, 49]. Besides, the genes involved in cell cycle regulation, such as cyclin D1, cyclin E and Cdk4, were down-regulated or up-regulated when miR-696 was over-expressed or inhibited (Figure 2F and K). Moreover, some studies showed that a decreased expression of those cell pro- liferation related genes induced cell proliferation re- pression by arresting muscle cells in the G0/G1 stage [43, 50]. Therefore, it can be concluded that miR-696 could play a negative role in muscle cell proliferation. Previous researches indicated that many miR- NAs could influence both the proliferation and dif- ferentiation process of skeletal muscle. Many studies also reported that some miRNAs had opposite roles in the process of proliferation and differentiation, such as miR-133 and miR-29, which repressed myoblast proliferation and promoted cell differentiation [39, 51]. On the other hand, some miRNAs were con- firmed to have the same effect on the muscle cell pro- liferation and differentiation, such as miR-214 and miR-203 [19, 52].
Materials and Methods: Curcumin was extracted using alcohol and chloroform from turmeric powder.TLC chromatography was used to confirm purity of Curcumin extracted. Mouse C2C12 myoblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM 1.5 g/l glucose) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. After differentiating of C2C12cells during 4 days to myotubes, cells were treated separately in the presence of insulin (100 nM), Curcumin (25 µM) and co-treatment for 24h.RNA extraction from C2C12cells was performed and GLUT4 expression levels were examined by semi-quantitative RT-PCR.
Here, we concluded that the 13 upregulated miRNAs might play a key role in the commitment of C2C12cells into osteoblastic lineage. To evaluate the biological effect of the 13 upregulated miRNAs on Runx2-induced oste- ogenic differentiation, anti-miRs and miRNA mimics for these miRNAs were used in a functional screening. Finally, the screening identified miR-690 as a candidate regulator of Runx2-induced osteogenic differentiaition. Previous study has reported that miR-690 might play an important role in glucose regulation of β-cell func- tion . MiR-690 is highly overexpressed in functional myeloid-derived suppressor cells (MDSCs) and manipu- lates MDSC activity by repressing transcription factor CCAAT/enhancer-binding protein α in inflammatory diseases as well as cancer [34, 35]. MiR-690 can also regulate fibroblast migration and dermal wound repair by directly targeting Versican . In addition, miR-690 was found to be involved in granulopoiesis , gluta- mate-induced excitotoxicity , and regulating testos- terone signaling in liver . However, the function of miR-690 in regulating osteogenic differentiation was not clear. In this study, data obtained from in vitro experi- ments revealed that miR-690 overexpression enhances Runx2-induced osteogenic differentiation of C2C12cells, whereas knockdown of miR-690 leads to the opposite effect (Fig. 2b–d). These findings suggest that miR-690 is Fig. 6 Proposed model for the regulation and function of miR-690