Journal: The Journal of biological chemistry

Article Title: Human p300 protein is a coactivator for the transcription factor MyoD.

doi: 10.1074/jbc.271.15.9009

Figure Lengend Snippet: FIG. 2. p300 associates MyoD in vivo. Immunoblot with an anti- MyoD antibody (Santa Cruz) following immunoprecipitation of whole cell extracts prepared from C2C12 myoblast cells in lysis buffer con- taining either 250 mM salt (lanes 2 and 3) or 150 mM salt (lanes 1, 4, and 5) with an anti-p300 polyclonal antiserum (5 ml). NRbS, controls for immunoprecipitation with normal rabbit serum. WCL, whole cell ex- tract without immunoprecipitation.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T buffer (containing 20 mM Tris-Cl, pH 7.6, 137 mMNaCl, and 0.5% Tween 20) for 1 h, incubated with anti-MyoD antibodies (Santa Cruz) diluted in TBS-Twith 3% the drymilk for 1 h, washed in TBS-T, further incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase in TBS-T, and finally washed in TBS-T.

Techniques: In Vivo, Western Blot, Immunoprecipitation, Lysis

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Sequences of all primers.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Effects of MY on the expressions of related genes in the tibia muscle of different groups. mRNA expression of AMPK (A) , Ldh (B) , Lcad (C) , Mcad (D) , Tfam (E) , Myod (F) , Murf1 (G) , Chrna1 (H) , Chrnd (I) , Rapsyn (J) , Agrin (K) , and IL-1β (L) . * p < 0.05, compared with the control group; # p < 0.05, compared with the OA group.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Effects of MY on the expressions of related proteins in the tibia muscle of different groups. (A) Protein bands visualized by western blotting. Protein expression of AMPK (B) , Myod (C) , Tfam (D) , Chrna1 (E) , Ldh (F) , and Mcad (G) . * p < 0.05, compared with the control group; # p < 0.05, compared with the OA group.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Western Blot, Expressing, Control

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 1. IL-4 treatment promoted myoblast differentiation and increased the expression of myogenic regulatory factors and myomerger in C2C12 cells. (A) Representative immunofluorescence images of MyHC (red) in the differentiated C2C12 cells 72 h after IL-4 application. The nuclei of C2C12 cells were counterstained with DAPI (blue). Scale bars: 50 µm. (B,C) The number of myonuclei per myotube and the differentiation index of IL-4-treated C2C12 cells. N = 3 per group. (D) Representative images of EdU (green) and counterstained nuclei (Hoechst 33342; blue) after 24 h IL-4 treatment in GM. (E) The number of EdU-positive cells significantly decreased in the IL-4-treated C2C12 cells. N = 4 per group. (F) Fold changes in the mRNA levels of MyoD, myogenin, myomaker, and myomerger in the IL-4-treated C2C12 myoblasts. Myoblasts were maintained in DM with 10 ng/mL IL-4. After 72 h incubation, the cells were harvested for qRT-PCR. N = 5 per group. Data are presented as mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 3. Reduction of IL-4/IL-4Rα signaling suppressed the increased expression of MyoD, myo- genin, and myomerger, but did not affect myomaker expression. (A) Representative immunofluores- cence images of myogenin (red) in the IL-4RαKD C2C12 myoblasts. Cell nuclei were counterstained with DAPI (blue). C2C12 cells transfected with control siRNA (Ctrl) or IL-4Rα siRNA (IL-4RαKD) were grown in GM for 24 h and maintained in DM with or without recombinant IL-4 (10 ng/mL) for 72 h. Scale bars: 50 µm. (B) The percentage of myogenin-positive cells was significantly sup- pressed by IL-4Rα knockdown. N = 5 per group. (C–E) Reduction of IL-4/IL-4Rα signaling by IL-4Rα knockdown significantly suppressed the increased expression of MyoD, myogenin, and my- omerger, whereas the expression of myomaker was not affected. Cells were treated as described in (A). (C,E) show fold changes in mRNA and protein levels, respectively. N = 6 per group. Representative western blot analysis is shown in (D). Data represent mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Transfection, Control, Recombinant, Knockdown, Western Blot

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 4. Stimulation of IL-4/IL-4Rα signaling increased the expression of myogenic regulatory factors and myomerger, but not myomaker, even in GM. (A) Representative immunofluorescence images of myogenin (red) in the IL-4RαKD C2C12 myoblasts. The nuclei were counterstained with DAPI (blue). C2C12 cells were treated as in Figure 3 except that cells were maintained in GM, instead of DM, for 24 h. Scale bars: 50 µm. (B) The percentage of myogenin-positive cells significantly increased by IL-4 treatment, which was suppressed by IL-4Rα knockdown. N = 4 per group. (C) Fold change in the mRNA levels of MyoD, myogenin, myomerger, and myomaker. N = 6 per group. Data represent as mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Knockdown

Journal: BMC Genomics

Article Title: MicroRNA-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1

doi: 10.1186/s12864-023-09431-0

Figure Lengend Snippet: miR-668-3p inhibits myogenic differentiation. A , C The mRNA levels of MyHC, MyoD and MyoG were determined after overexpression and knockdown of miR-668-3p. B , D The protein levels of MyHC, MyoD, and MyoG after treatment with miR-668-3p mimics or inhibitor were measured by Western blotting and quantified by ImageJ. The membrane was cleaved prior to hybridization with the antibody, and all protein bands in Figure B and protein bands in Figure D were from the same sample, respectively. Full-length blots/gels are presented in Supplementary Fig. . E , F MyHC immunofluorescence staining and differentiation index after overexpression and knockdown of miR-668-3p. The data represent the mean ± SD from at least three independent experiments. (*, P < 0.05; **, P < 0.01)

Article Snippet: Twenty micrograms of protein was electrophoresed on 10% SDS polyacrylamide gels, and the protein was transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010), Then, the membranes were blocked with 5% BSA at 4 °C for 2 h and incubated with antibodies (1:1000) against cyclin E (Santa, sc-377,100), cyclin D (Abways, CY5404), p27 (Santa, sc-1641), p21 (Abways, CY5088), CDK4 (Abways, CY5827), PCNA (Abways, CY1245), MyHC (R&D Systems, MAB4470), MyoG (Novus Biologiacals, NB100-56510), MyoD (Novus Biologiacals, NBP1-54153), Appl1 (Abways, CY8185), p38α MAPK (Abways, CY5262), Phospho-p38α MAPK (Abways, CY6390), β-Tubulin (Abways, AB0039), and GAPDH (Abways, AB0036) at 4 ℃ overnight.

Techniques: Over Expression, Knockdown, Western Blot, Membrane, Hybridization, Immunofluorescence, Staining

Journal: BMC Genomics

Article Title: MicroRNA-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1

doi: 10.1186/s12864-023-09431-0

Figure Lengend Snippet: Appl1 promotes myoblast proliferation and differentiation. A The mRNA expression of Cyclin E, PCNA, Cyclin D, p21 and p27 at 24 h after overexpression of Appl1 was measured using RT‒qPCR. B The protein expression of Appl1, cyclin D and p27 was determined using Western blotting and quantified by ImageJ. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure B are from the same membrane. Full-length blots/gels are presented in Supplementary Fig. A. C EdU staining after transfection with the Appl1 overexpression vector. Quantification the ratio of EdU-positive cells/total cells. D The mRNA expression of MyHC, MyoD and MyoG after overexpression of Appl1 was measured using RT‒qPCR. E The protein expression of Appl1, MyHC and MyoG was determined using Western blotting and quantified by ImageJ. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure E are from the same membrane. Full-length blots/gels are presented in Supplementary Fig. B. F MyHC immunofluorescence staining and differentiation index after Appl1 overexpression. The data represent the mean ± SD from at least three independent experiments. (*, P < 0.05; **, P < 0.01)

Article Snippet: Twenty micrograms of protein was electrophoresed on 10% SDS polyacrylamide gels, and the protein was transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010), Then, the membranes were blocked with 5% BSA at 4 °C for 2 h and incubated with antibodies (1:1000) against cyclin E (Santa, sc-377,100), cyclin D (Abways, CY5404), p27 (Santa, sc-1641), p21 (Abways, CY5088), CDK4 (Abways, CY5827), PCNA (Abways, CY1245), MyHC (R&D Systems, MAB4470), MyoG (Novus Biologiacals, NB100-56510), MyoD (Novus Biologiacals, NBP1-54153), Appl1 (Abways, CY8185), p38α MAPK (Abways, CY5262), Phospho-p38α MAPK (Abways, CY6390), β-Tubulin (Abways, AB0039), and GAPDH (Abways, AB0036) at 4 ℃ overnight.

Techniques: Expressing, Over Expression, Western Blot, Membrane, Hybridization, Staining, Transfection, Plasmid Preparation, Immunofluorescence

Journal: BMC Genomics

Article Title: MicroRNA-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1

doi: 10.1186/s12864-023-09431-0

Figure Lengend Snippet: miR-668-3p inhibits myogenesis mainly by targeting Appl1 and inhibiting MAPK signalling. A EdU staining experiments were performed after cotransfection of miR-668-3p and Appl1. Quantification of the ratio of EdU-positive cells/total cells. B The mRNA expression levels of the proliferation genes CDK4 and Cyclin D after cotransfection of miR-668-3p and Appl1. C The protein expression levels of the proliferation genes CDK4 and Cyclin D after cotransfection of miR-668-3p and Appl1. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure C are from the same sample. Full-length blots/gels are presented in Supplementary Fig. A. D The mRNA expression levels of the differentiation genes MyHC, MyoD and MyoG after cotransfection of miR-668-3p and Appl1. E The protein expression levels of the differentiation genes MyHC and MyoG after cotransfection of miR-668-3p and Appl1. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure E are from the same membrane. Full-length blots/gels are presented in Supplementary Fig. B. F MyHC immunofluorescence staining and differentiation index after cotransfection of miR-668-3p and Appl1. G The levels of phosphorylated p38 MAPK after overexpression of miR-668-3p. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure G are from the same sample. Full-length blots/gels are presented in Supplementary Fig. C. H The levels of phosphorylated p38 MAPK after miR-668-3p knockdown. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure H are from the same sample. Full-length blots/gels are presented in Supplementary Fig. D. I The levels of phosphorylated p38 MAPK after overexpression of Appl1. The membrane was cleaved prior to hybridization with the antibody, and the protein bands in Figure I are from the same sample. Full-length blots/gels are presented in Supplementary Fig. E. The data represent the mean ± SD from at least three independent experiments. (*, P < 0.05; **, P < 0.01)

Article Snippet: Twenty micrograms of protein was electrophoresed on 10% SDS polyacrylamide gels, and the protein was transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010), Then, the membranes were blocked with 5% BSA at 4 °C for 2 h and incubated with antibodies (1:1000) against cyclin E (Santa, sc-377,100), cyclin D (Abways, CY5404), p27 (Santa, sc-1641), p21 (Abways, CY5088), CDK4 (Abways, CY5827), PCNA (Abways, CY1245), MyHC (R&D Systems, MAB4470), MyoG (Novus Biologiacals, NB100-56510), MyoD (Novus Biologiacals, NBP1-54153), Appl1 (Abways, CY8185), p38α MAPK (Abways, CY5262), Phospho-p38α MAPK (Abways, CY6390), β-Tubulin (Abways, AB0039), and GAPDH (Abways, AB0036) at 4 ℃ overnight.

Techniques: Staining, Cotransfection, Expressing, Membrane, Hybridization, Immunofluorescence, Over Expression, Knockdown

Journal: BMC Genomics

Article Title: MicroRNA-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1

doi: 10.1186/s12864-023-09431-0

Figure Lengend Snippet: The primer sequences for real-time qPCR

Article Snippet: Twenty micrograms of protein was electrophoresed on 10% SDS polyacrylamide gels, and the protein was transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010), Then, the membranes were blocked with 5% BSA at 4 °C for 2 h and incubated with antibodies (1:1000) against cyclin E (Santa, sc-377,100), cyclin D (Abways, CY5404), p27 (Santa, sc-1641), p21 (Abways, CY5088), CDK4 (Abways, CY5827), PCNA (Abways, CY1245), MyHC (R&D Systems, MAB4470), MyoG (Novus Biologiacals, NB100-56510), MyoD (Novus Biologiacals, NBP1-54153), Appl1 (Abways, CY8185), p38α MAPK (Abways, CY5262), Phospho-p38α MAPK (Abways, CY6390), β-Tubulin (Abways, AB0039), and GAPDH (Abways, AB0036) at 4 ℃ overnight.

Techniques: