Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: VLDL induces ER stress and inflammation. Mouse C2C12 myotubes were incubated in the presence (black bars) or absence (control, Ct, white bars) of 300 μg/ml VLDL for 24 h. (a) mRNA abundance of Bip, Chop and Nqo1. mRNA levels are normalised to Aprt (n = 8–10, five independent C2C12 cultures were used). (b) BiP, phospho-eIF2α (Ser51), TRB3, CHOP and β-actin protein levels. (c), mRNA abundance of Il6, Mcp1, Tnfα, IκBα and Socs3. (d) IκBα, p65 and β-actin protein levels. (e) Phospho-STAT3 (Tyr705), SOCS3 and β-actin protein levels. The graphs show quantification expressed as a percentage of control samples. Data are means ± SD of five independent experiments and were compared by Student’s t test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Incubation, Control

Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: VLDL reduces PGC-1α and AMPK levels and induces insulin resistance. Mouse C2C12 myotubes were incubated in the presence (black bars) or absence (control, Ct, white bars) of 300 μg/ml VLDL for 24 h. (a) Pgc1α, Pparα (Ppara), Pparβ/δ (Pparb/Ppard), Acox and Mcad mRNA levels (n = 8–10, five independent C2C12 cultures were used). (b) PGC-1α, NRF1, phospho-AMPK (Thr172), phospho-ACC (Ser79) and β-actin protein levels. (c) NQO1, NRF2 and β-actin protein levels. (d) IRβ, phospho-IRS-1 (Ser307), and β-actin protein levels. (e) Phospho-Akt (Ser473) protein levels. Where indicated, cells were incubated with 100 nmol/l insulin (Ins, I) for the last 10 min. The graphs show quantification expressed as a percentage of control samples. Data are means ± SD of five independent experiments and compared by Student’s t test (a–d) or two-way ANOVA followed by Tukey post hoc test (e). **p < 0.01 and ***p < 0.001 vs control; †††p < 0.001 vs control cells incubated with insulin

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Incubation, Control

Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: ERK1/2 inhibition and knockdown prevents the effects of VLDL. (a) C2C12 myotubes (MT) and isolated skeletal muscles (SM) were in-cubated in the presence (black bars) or absence (control, Ct, white bars) of 300 μg/ml VLDL (myotubes) or 500 μg/ml VLDL (muscle) and the protein levels of phospho-ERK1/2 (Thr202/Tyr204) were analysed. (b, c) C2C12 myotubes were incubated in the presence (black bars) or absence (control, white bars) of 300 μg/ml VLDL for 24 h; 10 μmol/l U0126 was added to control (light grey bars) or VLDL-treated (dark grey bars) myotubes and the mRNA abundance of Bip, Chop, Nqo1, Il6, Mcp1 and Tnfα (b) and Pgc1α, Pparα, Pparβ/δ, Acox and Mcad (c) was evaluated. (d, e) C2C12 cells were transfected with control siRNA or ERK1/2siRNA and incubated in the presence or absence of 300 μg/ml VLDL. The mRNA abundance of Bip, Chop, Il6, Mcp1 and Tnfα (d) and Pgc1α, Pparα, Acox and Mcad (e) was evaluated. White bars, control siRNA; light grey bars ERK1/2 siRNA; black bars VLDL + control siRNA; dark grey bars VLDL+ERK1/2 siRNA. The graphs show quantification expressed as a percentage of control. Data are means ± SD of five independent experiments and were compared by Student’s t test (a) or two-way ANOVA followed by Tukey post hoc test (b–e). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control; †††p < 0.001 vs VLDL-exposed cells

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Inhibition, Knockdown, Isolation, Muscles, Control, Incubation, Transfection

Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: ApoCIII activates ERK1/2 and induces ER stress, inflammation and insulin resistance. C2C12 myotubes were incubated in the presence (black bars) or absence (control, Ct, white bars) of 100 μg/ml apoCIII for 24 h. (a) Phospho-ERK1/2 (Thr202/Tyr204) protein levels. (b) BiP, phospho-eIF2α (Ser51), TRB3 and β-actin protein levels. (c) mRNA abundance of Pgc1α, Pparα and Pparβ/δ. (d) PGC-1α, NRF1 and β-actin protein levels. (e) Autoradiograph of EMSA performed with a 32P-labelled PPAR nucleotide and crude nuclear protein extract (NE) from C2C12 myotubes. One main specific complex (Compl I) based on competition with a molar excess of unlabelled probe is shown. The supershift assay performed by incubating NE with an antibody (Ab) directed against PPARβ/δ shows a reduction in the band, whereas the band is unchanged by an unrelated antibody against Oct1. (f) IRβ, phospho-IRS-1 (Ser307) and β-actin protein levels. (g) Phosphorylated Akt (Ser473) protein levels. Where indicated, cells were incubated with 100 nmol/l insulin (Ins, I) for the last 10 min. The graphs show quantification expressed as a percentage of control. Data are means ± SD of five independent experiments and were compared by Student’s t test (a–f) or two-way ANOVA followed by Tukey post hoc test (g). *p < 0.05, **p < 0.01 and***p < 0.001 vs control; †††p < 0.001 vs control cells incubated with insulin

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Incubation, Control, Autoradiography

Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: ERK1/2 inhibition prevents the effects of apoCIII on ER stress and inflammation. (a–e) C2C12 myotubes were incubated in the presence (black bars) or absence (control, Ct, white bars) of 100 μg/ml apoCIII for 24 h; 10 μmol/l U0126 was added to control myotubes (light grey bars) or apoCIII-treated myotubes (dark grey bars). (a) mRNA abundance of Bip, Chop, Socs3, Il6, Mcp1 and Tnfα. (b) BiP, phospho-eIF2α (Ser51), phospho-ERK1/2 (Thr202/Tyr204) and β-actin protein levels. (c) IκBα, NRF2, phospho-STAT3 (Tyr705) and β-actin protein levels. (d) mRNA abundance of Pgc1α, Pparα, Pparβ/δ, Acox and Mcad. (e) PGC-1α, phospho-AMPK (Thr172), phospho-ACC (Ser79) and β-actin protein levels. (f, g) C2C12 myotubes were transfected with control or ERK1/2 siRNA and incubated in the presence or absence of 100 μg/ml apoCIII for 24 h. The mRNA abundance of Bip, Chop, Il6, Mcp1 and Tnfα (f) and Pgc-1α, Pparα, Pparβ/δ, Acox and Mcad (g) was evaluated. White bars, control siRNA; light grey bars ERK1/2 siRNA; black bars, apoCIII+ control siRNA; dark grey bars, apoCIII+ERK1/2 siRNA The graphs show quantification expressed as a percentage of control. Data are means ± SD of five independent experiments and were compared by two-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control; ††p < 0.01 and †††p < 0.001 vs apoCIII-exposed cells

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Inhibition, Incubation, Control, Transfection

Journal: Diabetologia

Article Title: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells

doi: 10.1007/s00125-017-4401-5

Figure Lengend Snippet: TLR2 mediates the effects of apoCIII on ERK1/2, ER stress and inflammation. Mouse C2C12 myotubes were incubated in the presence or absence (control, Ct, white bars) of 100 μg/ml apoCIII for 24 h; 50 μg/ml of IgG was added to apoCIII-treated myotubes (black bars) or 50 μg/ml of the neutralising antibody against TLR2 (TLR2NAb) was added to the control (light grey bars) or apoCIII-treated (dark grey bars) myotubes. (a) Phospho-ERK1/2 (Thr202/Tyr204) protein levels. (b) mRNA abundance of Bip, Chop, Il6, Mcp1 and Tnfα. (c) BiP, phospho-eIF2α (Ser51), IκBα, and β-actin protein levels. (d) IRβ, phospho-IRS-1 (Ser307) and β-actin protein levels. (e) PGC-1α, NRF1 and β-actin protein levels. (f) mRNA abundance of Pgc1α, Pparα, Acox and Mcad mRNA. The graphs show quantification expressed as a percentage of control. Data are means ± SD of five independent experiments and were compared by two-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control; ††p < 0.01 and †††p < 0.001 vs apoCIII-exposed cells

Article Snippet: Cell culture Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described [ 15 ].

Techniques: Incubation, Control

Journal: Pharmacological research

Article Title: A positive feedback loop between AMPK and GDF15 promotes metformin antidiabetic effects.

doi: 10.1016/j.phrs.2022.106578

Figure Lengend Snippet: Fig. 4. Metformin increases GDF15 levels via AMPK activation. (a) Gdf15 mRNA levels, and (b) GDF15 secreted into the culture medium in C2C12 myotubes exposed to different concentrations of metformin (Met) for 24 h. (c) Gdf15 mRNA levels in C2C12 myotubes exposed to 0.5 mM metformin (Met) for 24, 48 or 72 h or (d) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (e) Gdf15 mRNA levels in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or ATF3 siRNA and treated with 2 mM metformin for 24 h. (f) Atf3 mRNA levels in C2C12 myotubes exposed to different concentrations of metformin (Met) for 24 h or (g) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (h) GDF15 mRNA levels in Huh-7 human hepatoma cells exposed to different concentrations of metformin (Met) for 24 h. (i) ATF3 mRNA levels in Huh-7 cells exposed to different concentrations of metformin (Met) for 24 h. (j) Gdf15 mRNA levels in mouse primary hepatocytes exposed to different concentrations of metformin (Met) for 24 h or (k) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (l) Gdf15 mRNA levels in mouse primary culture of hepatocytes exposed to 2 mM metformin (Met) for 24 h in the presence or absence of 30 µM compound C (Comp C). (m) Immunoblot analysis of GDF15, and total and phospho-AMPK in C2C12 in mouse primary culture of hepatocytes exposed to 2 mM metformin (Met) for 24 h in the presence or absence of 30 µM compound C (Comp C). (a,b,d-h) n = 5 per group. (c,i-l) n = 4 per group. Data are presented as the mean ± SEM. * *p < 0.01 and * **p < 0.001 vs. control. ###p < 0.001 vs. Ct siRNA + Met or Met.

Article Snippet: Mouse C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin.

Techniques: Activation Assay, Transfection, Control, Western Blot

Journal: Pharmacological research

Article Title: A positive feedback loop between AMPK and GDF15 promotes metformin antidiabetic effects.

doi: 10.1016/j.phrs.2022.106578

Figure Lengend Snippet: Fig. 5. GDF15 stimulates the insulin signaling pathway in cultured myotubes. (a) mRNA levels of Cpt1b, Acadm, Acox, Pgc1a, Tnfa, Il6 and Trb3 in C2C12 myotubes exposed to 100 ng/mL of recombinant GDF15 (rGDF15) for 24 h. (b) Immunoblot analysis of PGC-1α, PPARα, and IRβ in C2C12 myotubes exposed to 100 ng/mL of recombinant GDF15 (rGDF15) for 24 h. (c) Immunoblot analysis of total and phospho-Akt (Ser473) in C2C12 myotubes exposed to 100 ng/mL of recombinant GDF15 (rGDF15) for 24 h. Where indicated, cells were incubated with 100 nM of insulin (Ins) for the last 10 min n ≥5 per group. Data are presented as the mean ± SEM. *p < 0.05, * *p < 0.01 and * **p < 0.001.

Article Snippet: Mouse C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin.

Techniques: Cell Culture, Recombinant, Western Blot, Incubation

Journal: Pharmacological research

Article Title: A positive feedback loop between AMPK and GDF15 promotes metformin antidiabetic effects.

doi: 10.1016/j.phrs.2022.106578

Figure Lengend Snippet: Fig. 6. Gdf15 knockdown prevents metformin-mediated AMPK activation. (a) Immunoblot analysis of GDF15, and total and phospho-AMPK in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or GDF15 siRNA and treated with 2 mM metformin for 24 h. (b) mRNA levels of Pgc1a, Acox and Mcp1 in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or GDF15 siRNA and treated with 2 mM metformin for 24 h. (c) mRNA levels of Pgc1a and Il6 in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or GDF15 siRNA and exposed to 0.5 mM palmitate or palmitate plus 2 mM metformin for 24 h. (d) Immunoblot analysis of total and phospho-AMPK, PPARα, IRβ, IκBα and PGC-1α in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or GDF15 siRNA and exposed to 0.5 mM palmitate or palmitate plus 2 mM metformin for 24 h. (a,b) n = 5 per group. (c,d) n = 4–5 per group. Data are presented as the mean ± SEM. *p < 0.05, * *p < 0.01 and * **p < 0.001.

Article Snippet: Mouse C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin.

Techniques: Knockdown, Activation Assay, Western Blot, Transfection, Control

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: C26 CM induced myotube atrophy and activated DRP1 expression. (A) C2C12 myotubes visualized by a biologic microscopy at ×200 magnification and. (B) Analysis of myotubes’ mean diameters. (C) DRP1 protein levels of the HS/CM group by western blotting. Data were shown as mean ± SD. ***P<0.01. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 CM (conditional medium); DRP1, dynamin-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Expressing, Microscopy, Western Blot, Cell Culture

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: Regulation of DRP1 levels modulates myotube wasting during C26 CM-induced muscle atrophy. (A) Fluorescence images of eGFP expression and. (B) DRP1 protein levels of KD/OE/Dc group. (C) C2C12 myotubes visualized by a biologic microscopy and. (D) Analysis of myotubes’ mean diameters of KD/OE/Dc group. Data were shown as mean ± SD. ***P<0.01 vs. control. eGFP, enhanced green fluorescent protein. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 CM (conditional medium); Dc, myotube infected with control lentivirus in CM; OE, myotube infected with overexpressed lentivirus in CM; KD, myotube infected with knockdown lentivirus in CM; DRP1, dynamin-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Fluorescence, Expressing, Microscopy, Control, Cell Culture, Infection, Knockdown

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: Transcriptome analysis of C2C12 myotubes. (A) MA plot (M-values: log-intensity ratios/A-values: log-intensity averages) based on the mean value of gene expression and the fold change, red represents up-regulated genes and blue represents down-regulated genes. (B) Heatmap for clustering analysis of differential expressed genes, blue represents high-expressed genes and red represents low-expressed genes. (C) Scatter plot of enrichment KEGG pathways based on differential expressed genes, the most prominent 30 pathways were selected. Q-values are numbers transferred from adjusted Q-values by hypergeometric test. The green degree of enrichment represents the ratio: the differential expressed genes to the total annotation genes in this pathway. (D&E) Enrichment of KEGG pathway for ribosome and oxidative phosphorylation, red represents up-regulated genes, blue represents down-regulated genes. CM, myotube cultured in DMEM of 33% C26 conditional medium; Dc, myotube infected with control lentivirus in C26 conditional medium; KD, myotube infected with knockdown lentivirus in C26 conditional medium.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Gene Expression, Phospho-proteomics, Cell Culture, Infection, Control, Knockdown

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: C26 CM reduced the marker of ribosomal biogenesis at mRNA levels. Gene expression levels of Pol-I, c-Myc, Ubf, Nop56, Ncl from C2C12 myotubes in KD/Dc/CM/HS groups using a real-time PCR method (n=4). Data were shown as mean ± SD. *P<0.05. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 conditional medium; Dc, myotube infected with control lentivirus in C26 conditional medium; KD, myotube infected with knockdown lentivirus in C26 conditional medium; Pol-I, RNA polymerase I; c-Myc, MYC proto-oncogene; Ubf, upstream binding factor; Nop56, nucleolar protein 56; Ncl, nucleolin; PCR, polymerase chain reaction.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Marker, Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Infection, Control, Knockdown, Binding Assay, Polymerase Chain Reaction

Journal: Frontiers in Physiology

Article Title: The role and regulation of erythropoietin (EPO) and its receptor in skeletal muscle: how much do we really know?

doi: 10.3389/fphys.2013.00176

Figure Lengend Snippet: Detectability of the EPO-R in skeletal muscle .

Article Snippet: Wang et al., , Mouse , C2C12 myoblasts , Yes (RT-PCR) , Yes (WB) , EPO-R antibody (Santa Cruz Biotechnology).

Techniques: Gene Expression, Expressing, Northern Blot, Immunohistochemistry, In Situ Hybridization, Isolation

Journal: bioRxiv

Article Title: Myosin Heavy Chain-embryonic is a crucial regulator of skeletal muscle development and differentiation

doi: 10.1101/261685

Figure Lengend Snippet: MyHC-emb (A) and MyHC-slow (A') RNA expression detected by qPCR, starts at around E10.5, increasing gradually and peaking at E15.5, declining thereafter until birth (note the Y-axis scale difference between MyHC-emb and -slow, indicating that MyHC-emb transcripts are expressed many folds higher than MyHC-slow). The transcript levels for both MyHC-emb and -slow were normalized to E9.5 expression. MyHC-emb (B, B') and -slow (C, C') proteins are expressed in the somitic region (boxed region in B and C are magnified in B' and C' respectively) in the mouse embryo at E10.5, in the limb muscle fibers during embryonic myogenesis at E13.5 (D, D', E, E', where D' and E' magnifications of the boxed region in D and E respectively), and in the limb muscle fibers during fetal myogenesis at E16.5 (F, F', G, G', with F' an G' magnifications of the boxed region in F and G respectively). The protein expression levels of MyHC-emb and MyHC-slow were calculated over the course of development by analyzing the fluorescence intensity relative to E12.5, with MyHC-emb exhibiting a minor peak at E14.5 and a major peak at E16.5, declining thereafter, whereas MyHC-slow levels were relatively constant until E16.5, peaking at E17.5 following which it started declining (H). During C2C12 mouse myogenic cell differentiation, MyHC-emb (I) and MyHC-slow (J) RNA expression detected by qPCR increases as differentiation progresses, with MyHC-emb levels peaking by day 7 of differentiation and MyHC-slow levels peaking by day 6 of differentiation (note the Y-axis scale difference between MyHC-emb and -slow, indicating that MyHC-emb transcripts are expressed many folds higher than MyHC-slow). MyHC-emb (K, L) and -slow (K, M) proteins are expressed by day 5 of C2C12 cell differentiation, with MyHC-emb levels peaking by day 7 (L), and MyHC-slow levels increasing until the thirteenth day of differentiation (M). (Scale bars in C, E, and G is 100 microns; scale bars in C’, E’, and G' is 25 microns).

Article Snippet: C2C12 mouse myoblasts (ATCC; Cat# CRL-1722) were cultured and maintained according to ATCC guidelines in growth medium containing DMEM-Dulbecco’s Modified Eagle Medium (Gibco; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma; Cat# F2442) and 2% penicillin-streptomycin (Gibco; Cat# 15140122).

Techniques: RNA Expression, Expressing, Fluorescence, Cell Differentiation

Journal: bioRxiv

Article Title: Myosin Heavy Chain-embryonic is a crucial regulator of skeletal muscle development and differentiation

doi: 10.1101/261685

Figure Lengend Snippet: At postnatal day 0, the number of myofibers are significantly increased in the EDL muscle in the hind limbs of MyH3 Δ/Δ animals compared to MyH3 +/+ animals (A), while no significant difference in the number of myofibers is observed in the case of the soleus muscle (B). MyHC-slow positive fibers are increased in P0 MyH3 Δ/Δ animal hind limb cross sections (D, D’), compared to MyH3 +/+ control animals (C, C’), where the sections are labeled by immunofluorescence for MyHC-slow (Red), Laminin (Green) and DAPI (Blue). C’’ and D’’ are magnified regions of the boxed area of MyH3 +/+ and MyH3 Δ/Δ P0 hind limb cross sections from C’ and D’ respectively. The elevated number of MyHC-slow+ fibers in P0 MyH3 Δ/Δ hind limb cross sections compared to MyH3 +/+ controls was quantified normalized to total area and is statistically significant (G). The myofiber size was found to be smaller in the soleus muscle of MyH3 Δ/Δ animals (F) as compared to MyH3 +/+ animals (E). The myofiber area of myofibers of the EDL and soleus muscles at P0 were quantified, grouping myofibers into 3 categories by area, 0-50 μm 2 , 50-150 μm 2 and above 150 μm 2 . It was found that the number of myofibers in the 0-50 μm 2 group was increased significantly in the soleus muscle of MyH3 Δ/Δ animals compared to controls (I), but was unchanged in the EDL (H). Protein levels of MyHC-slow and MyHC-IIa are about 5-fold upregulated in the MyH3 Δ/Δ animals compared to MyH3 +/+ animals by western blots (J), which is quantified by densitometry (K and L). MyHC-emb protein levels are significantly reduced upon treatment of C2C12 cells with MyH3 siRNA compared to control siRNA over 9 days of differentiation as detected by western blots and densitometry (M and N), while protein levels of MyHC-slow are significantly increased from day 5 onwards reaching a peak of more than 6-fold increase by day 7 upon MyHC-emb knockdown as detected by western blots and densitometry (M and O). Knockdown of MyHC-emb results in a significantly reduced fusion index at days 5 and 7 of C2C12 differentiation, compared to control siRNA treated cells (P). (Scale bar in D′ is 200 microns, D′′ is 40 microns and F is 20 microns respectively).

Article Snippet: C2C12 mouse myoblasts (ATCC; Cat# CRL-1722) were cultured and maintained according to ATCC guidelines in growth medium containing DMEM-Dulbecco’s Modified Eagle Medium (Gibco; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma; Cat# F2442) and 2% penicillin-streptomycin (Gibco; Cat# 15140122).

Techniques: Control, Labeling, Immunofluorescence, Muscles, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Myosin Heavy Chain-embryonic is a crucial regulator of skeletal muscle development and differentiation

doi: 10.1101/261685

Figure Lengend Snippet: Volcano plots depicting results from RNA sequencing experiment comparing P0 MyH3 +/+ muscle samples to MyH3 Δ/Δ samples for quadriceps, tibialis anterior, gastrocnemius, and diaphragm muscles respectively (A-D). The adjusted P values are on a log10 scale, and candidates that are significantly up or down regulated are marked as dark spots on the volcano plot (A-D). Selected candidate genes from the RNA sequencing experiment were validated for up or down regulation by qPCR on MyH3 +/+ samples compared to MyH3 Δ/Δ samples for the P0 quadriceps muscle (E) or P0 diaphragm muscle (F). 6 candidates from the RNA sequencing experiment were tested for mis-regulation in day 5 MyH3 siRNA treated C2C12 cells compared to control siRNA treated cells, and 3 were found to be significantly mis-regulated (G). Venn diagram depicting number of candidates obtained and degree of overlap in the RNA sequencing results comparing P0 MyH3 +/+ muscle samples to MyH3 Δ/Δ samples for quadriceps, tibialis anterior, gastrocnemius, and diaphragm muscles (H).

Article Snippet: C2C12 mouse myoblasts (ATCC; Cat# CRL-1722) were cultured and maintained according to ATCC guidelines in growth medium containing DMEM-Dulbecco’s Modified Eagle Medium (Gibco; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma; Cat# F2442) and 2% penicillin-streptomycin (Gibco; Cat# 15140122).

Techniques: RNA Sequencing, Muscles, Control

Journal: bioRxiv

Article Title: Myosin Heavy Chain-embryonic is a crucial regulator of skeletal muscle development and differentiation

doi: 10.1101/261685

Figure Lengend Snippet: MyHC-emb knockdown in C2C12 cells by siRNA leads to a reduction in the reserve cell population (B, B′) compared to control siRNA treated cells (A, A′), by immunofluorescence for Myosin heavy Chain (Red), Phalloidin (Green), and DAPI (Blue), on day 5 of differentiation, with the white arrows marking some reserve cells in A′ and B′ which are magnified areas taken from A and B respectively. Quantification of the number of reserve cells in control siRNA treated cells compared to MyH3 siRNA treated cells at day 5 of differentiation indicates that there is a significant, 6-fold decrease in reserve cell number normalized to total area in the MyH3 siRNA treated cells (C). No significant difference in the fraction of dividing, phospho-histone H3+ cells were observed in the MyH3 siRNA treated C2C12 cells compared to control siRNA treated cells at days 3, 5 and 7 of differentiation (D). Upon knockdown of MyHC-emb in C2C12 cells, MyoD the protein level increases significantly by 2-fold on early days (days 3 and 5) of C2C12 cell differentiation while the levels decrease significantly to about a quarter of normal levels on later days (days 7 and 9) of differentiation as detected by western blots and densitometry (E and F). The protein levels of Myogenin decreases to a quarter of the normal level on day 3, increases significantly by more than 2-fold by day 5 of C2C12 cell differentiation and again decreases significantly on later days (days 7 and 9) of differentiation as detected by western blots and densitometry upon knockdown ofMyHC-emb (E and G). Cell death as determined by levels of cleaved Caspase3 was significantly elevated in the MyH3 siRNA treated C2Cl2 cells compared to control siRNA treated cells at day 1 of differentiation, whereas it was significantly less in the MyH3 siRNA treated cells compared to control cells at days 5, 7 and 9 of differentiation, with no significant difference on day 3 (E), which is quantified by densitometry (H). (Scale bar in B is 100 microns and B′ is 25 microns).

Article Snippet: C2C12 mouse myoblasts (ATCC; Cat# CRL-1722) were cultured and maintained according to ATCC guidelines in growth medium containing DMEM-Dulbecco’s Modified Eagle Medium (Gibco; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma; Cat# F2442) and 2% penicillin-streptomycin (Gibco; Cat# 15140122).

Techniques: Knockdown, Control, Immunofluorescence, Cell Differentiation, Western Blot

Journal: bioRxiv

Article Title: Effects of HMGCR deficiency on skeletal muscle development

doi: 10.1101/2024.05.06.591934

Figure Lengend Snippet: shRNA mediated knockdown of Hmgcr in C2C12 myoblasts. (A) qPCR was performed in triplicate for each sample with n=5 experiments. Transcript levels were normalized against Gapdh with fold change calculated as Hmgcr knockdown versus scrambled control. Unpaired t-test results are shown. ***, p < 0.0002. (B) shRNA mediated knockdown of Hmgcr in C2C12 myoblasts. Successful shRNA transfection was confirmed by detecting GFP positive cells.

Article Snippet: C2C12 myoblasts were transfected with either a cocktail of three different shRNAs targeting mouse- Hmgcr or scrambled shRNA control ( GeneCopoeia ) using Lipofectamine 3000 ( Invitrogen ).

Techniques: shRNA, Knockdown, Control, Transfection

Journal: bioRxiv

Article Title: Effects of HMGCR deficiency on skeletal muscle development

doi: 10.1101/2024.05.06.591934

Figure Lengend Snippet: The oxygen consumption rate (OCR) of Hmgcr shRNA versus scrambled shRNA C2C12 myoblasts was determined using the RESIPHER system at (A) 24 hours, (B), 48 hours, and (C) 72 hours. The OCR of the Hmgcr knockdown cells was increased compared to scrambled controls at 24 and 48 hours. (D) Rotenone treatment at 72 hours confirms that the increased oxygen consumption occurred via mitochondrial respiration. (E) A A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 72 hours shows that the total cellular metabolic activity is higher in Hmgcr knockdown cells compared to scrambled controls. Paired t-tests were performed. *, p <0.05. Error bars show standard error of the mean (SEM).

Article Snippet: C2C12 myoblasts were transfected with either a cocktail of three different shRNAs targeting mouse- Hmgcr or scrambled shRNA control ( GeneCopoeia ) using Lipofectamine 3000 ( Invitrogen ).

Techniques: shRNA, Knockdown, MTT Assay, Activity Assay

Journal: Cell and Tissue Research

Article Title: Elevation of phosphate levels impairs skeletal myoblast differentiation

doi: 10.1007/s00441-020-03254-1

Figure Lengend Snippet: a , b mRNA expression profile of C2C12 myoblasts after differentiation in phosphate supplemented media . Increased phosphate concentrations significantly decreased marker gene expression after 6 days of treatment. Myogenin expression was reduced by 2.0 mM phosphate at day 1 (d1) and day 2 (d2) of treatment ( c ). ERK1/2 inhibition attenuated high-dosage phosphate effects on Myogenin expression as shown by NTC to treatment ratios ( d ). Values are shown as mean + SD (* p < 0.05; ** p < 0.01;*** p < 0.001)

Article Snippet: Murine C2C12 (ATCC® CRL 1772TM) myoblast cells (ATCC, Manassas, USA) were cultured according to the manufacturer’s instructions.

Techniques: Expressing, Marker, Gene Expression, Inhibition

Journal: Cell and Tissue Research

Article Title: Elevation of phosphate levels impairs skeletal myoblast differentiation

doi: 10.1007/s00441-020-03254-1

Figure Lengend Snippet: a-c Protein expression and cell signaling in phosphate-treated C2C12 myoblasts. Phosphate supplementation during differentiation significantly decreased the number of marker protein expressing nuclei ( a , b ). Phosphate directly induces ERK-phosphorylation 15 min after treatment in differentiated C2C12 cells ( c ). MHC protein expression was reduced after 6 days of 2.0 mM phosphate treatment as shown by MHC:b-Actin ratios ( d ). Values are shown as mean + SD (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Murine C2C12 (ATCC® CRL 1772TM) myoblast cells (ATCC, Manassas, USA) were cultured according to the manufacturer’s instructions.

Techniques: Expressing, Marker, Phospho-proteomics