Cells were cultured as before in relevant media, and analyzed either at 70% confluency, or after two-days of differentiation as previously described. For the analysis, an RNEasy Mini Kit (Qiagen #74104, Hilden, Germany) was used to harvest DNA according to the manufacturer’s protocol. Next, iScript cDNA synthesis kits (Bio-Rad # 1708890, Hercules, CA, USA) were used to prepare cDNA with 1,000 ng of RNA for each sample, again according to the manufacturers’ instructions. Finally, qPCR was performed using the TaqMan Fast Universal PCR Master Mix without AmpErase UNG (ThermoFisher #4352042). Primers were: 18S (ThermoFisher #Hs03003631), Pax3 (ThermoFisher #Bt04303789), MyoD (ThermoFisher #Bt04282788), Myogenin (ThermoFisher #Bt03258929), and Myosin Heavy Chain (MHC) (ThermoFisher #Bt03273061). Reactions were performed using 2 uL of the prepared cDNA, according to the manufacturer’s instructions. Reactions were run on the Bio-Rad CFX96 Real Time System thermocycler (Hercules, CA, USA), and results were analyzed as 2−ΔΔct normalized to 18S expression and comparing relative expression between autocrine signaling cells and control cells.
Myogenin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Myogenin is a protein that plays a crucial role in the regulation of skeletal muscle development and differentiation. It is a member of the basic helix-loop-helix (bHLH) family of transcription factors, which are essential for the activation of muscle-specific gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Cfx96 real time system thermocycler, by Bio-Rad (4 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Taqman fast universal pcr master mix without amperase ung, by Thermo Fisher Scientific (3 mentions)
Myod1, by Thermo Fisher Scientific (3 mentions)
Myostatin, by Thermo Fisher Scientific (3 mentions)
Myosin heavy chain, by Thermo Fisher Scientific (2 mentions)
Pax 7, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Irdye 700, by LI COR (1 mentions)
Sc 32732, by Santa Cruz Biotechnology (1 mentions)
Odyssey image scanner system, by LI COR (1 mentions)
Image studio v3, by LI COR (1 mentions)
Tubulin t 9026, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Irdye 800, by LI COR (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Atp assay kit, by Abcam (1 mentions)
13 protocols using myogenin
1
Myogenic Differentiation Analysis
2
Western Blot Analysis of Cell Markers
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Western blot analysis was performed following standard protocols. Primary antibodies against CAP1 (sc-376286; dilution: 1:500, Santa Cruz), Tubulin (T9026; 1:5,000, Sigma), Flag (F7425; 1:1,000, Sigma), Myosin Heavy chain 1/2/4/6 (sc-32732; 1:1,000, Santa Cruz), GAPDH (5174S; 1:3,000, Cell Signaling), Myogenin (sc-12732; 1:1,000) and MyoD (MA1-41017; 1:1,000, Thermo Fisher Scientific) were incubated overnight at 4°C. Fluorophore-conjugated secondary antibodies IRDye 700 or IRDye 800 (1:15,000, LICOR Biosciences) were incubated for 1 h at room temperature. Imaging and quantifications were done using the Odyssey Image Scanner System with the software Image Studio V 3.1.4 (LI-COR Biosciences, Cambridge, United Kingdom), as described before (Werner et al., 2019 (link)).
3
Quantitative PCR Analysis of Myogenic Markers
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To further support immunostaining data, quantitative PCR was performed on sub-confluent and differentiated cells from P2 and P6. Briefly, RNA was harvested with the RNEasy Mini Kit (Qiagen #74104, Hilden, Germany) and cDNA was prepared using 200 ng of RNA for each reaction with the iScript cDNA synthesis kit (Bio-Rad # 1708890, Hercules, CA, USA) according to the manufacturers’ instructions. Next, qPCR was performed using the TaqMan Gene Expression Master Mix (ThermoFisher #4369016), with 18 S as a housekeeping control gene. Primers used were: 18 S (ThermoFisher #Hs03003631), Pax3 (ThermoFisher #Bt04303789), MyoD (ThermoFisher #Bt04282788), Myogenin (ThermoFisher #Bt03258929), and Myosin Heavy Chain (ThermoFisher #Bt03273061). Reactions were performed using 3.5 uL of the prepared cDNA, according to the manufacturer’s instructions. Reactions were run on the Bio-Rad CFX96 Real Time System thermocycler, and results were analyzed as 2−Δct normalized to 18 S expression.
4
G-Rf Modulates Muscle Cell Signaling
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G-Rf was purchased from Fleton Natural Products Co., Ltd. (Chengdu, China) and dissolved in purified water for use in animal experiments. For cell experiments, G-Rf was dissolved in dimethyl sulfoxide (DMSO). The ATP assay kit was obtained from Abcam (Cambridge, MA, USA). Antibodies against NRF-1, phospho-AMPK, and phospho-p38 and secondary anti-rabbit or anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). MHC type 3 (MHCIII), AMPK, p38, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). MyoD, myogenin, PGC-1α, and TFAM antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The specific inhibitors including compound c, LY294002, and SB203580 were obtained from Cayman Chemicals (Ann Arbor, MI, USA).
5
Immunofluorescence Analysis of Myogenic Markers
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At least three independent cultures were analyzed for each experiment. After growth times indicated in the text, cells were fixed with 4% paraformaldehyde (10 min at room temperature, RT), permeabilized with 0.5% Triton (10 min at RT), and blocked with 10% goat serum for 30 min at RT before incubation with antibodies. For MYH3 staining, primary antibodies were purchased from Santa Cruz Biotechnology, Inc., diluted 1:100 in blocking buffer, incubated at 4°C for 24 h, and visualized with Alexa-488 secondary antibody from Invitrogen diluted 1:500. For Myogenin staining, primary antibodies were purchased from Thermo Fisher, Inc., diluted 1:1000, incubated at 4°C for 24 h, and visualized with Alexa-594 secondary antibody, diluted 1:1000. Nuclear staining was with DAPI (Molecular Probe) diluted 1:1000 and incubated at RT for 30 min. Cells were imaged with Carl Zeiss 510 using 40× oil immersion objective. Images were composed and edited in LSM image software or Photoshop 7.0 (Adobe).
6
Immunostaining of Myofiber Constructs
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Cells encapsulated in the Fib/Mtr gel were fixed with 2% paraformaldehyde in PBS overnight at 4 °C. Following fixation, samples were washed with phosphate buffered saline (PBS) and then incubated in blocking solution (5% bovine serum albumin with 0.2% Triton-X 100) (Sigma-Aldrich) for 12 h30 (link),31 (link). The myofiber sheet with Fib/Mtr gel was treated with primary antibodies (sarcomeric α-actinin (1:500) (Abcam, Cambridge, MA, USA), laminin (1:500) (Abcam), type IV collagen (1:500) (Abcam), myosin heavy chain (1:200) (R&D Systems, Minneapolis, MN, USA), myogenin (1:500) (Thermo Fisher Scientific)) or AlexaFluor 568-conjugated phalloidin at 4 °C overnight. After washing with PBS, the tissue constructs were treated with fluorescently labeled secondary antibodies (1:800) (Thermo Fisher Scientific) for 2 h at 37 °C. For nuclei staining, tissue constructs were incubated with Hoechst33258 (Dojindo Laboratories, Kumamoto, Japan) for 5 min at RT. Images were acquired using a Zeiss 510 inverted confocal microscope and analyzed using LSM Image Software.
7
Quantifying BSC Gene Expression
8
Quantifying Muscle Stem Cell Differentiation
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Relative gene expression between proliferative and differentiated BSCs and iBSCs was assessed via quantitative PCR using the TaqMan Fast Universal PCR Master Mix without AmpErase UNG (ThermoFisher #4352042). Briefly, RNA was harvested at 70% confluency or after differentiation using the RNEasy Mini kit (Qiagen #74104, Hilden, Germany) according to the manufacturer's instructions. Next, cDNA was prepared using the iScript cDNA synthesis kit (Bio-Rad #1708890, Hercules, CA, USA) and 750 ng of RNA for each reaction. Finally, qPCR was performed using 2 uL of cDNA and primers for 18S (ThermoFisher #Hs03003631), Pax3 (ThermoFisher #Bt04303789), MyoD1 (ThermoFisher #Bt03244740), Myogenin (ThermoFisher #Bt03258929), Myosin Heavy Chain (ThermoFisher #Bt03273061), TERT (ThermoFisher #Bt03239211), and CDK4 (ThermoFisher #Bt03231354). Reactions were performed on a CFX96 Real Time System thermocycler (Bio-Rad, Hercules, CA, USA), and results were analyzed as 2 -Δct normalized to expression of the 18S housekeeping gene.
9
Quantitative RNA Expression Analysis
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Total RNA was isolated using miRNeasy Mini Kit (Qiagen) as previously described [16] (link). Gene expression was determined by real time PCR using TaqMan mRNA assays (Applied Biosystems, Foster City, CA). Target-specific PCR primers (Pax-7, MyoD, Myostatin, Myogenin, Atrogin 1, IGF-1) were obtained from Applied Biosystems. The cycle number at which the amplification plot crosses the threshold was calculated (CT), and the ΔΔCT method was used to analyze the relative changes in gene expression and normalized by β-actin.
10
Quantitative PCR Analysis of Myogenic Markers
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Myostatin, Myf5, myogenin and MYH2 expression levels were determined by quantitative (real-time) PCR on an ABI PRISM 7300 Sequence Detection System (Applied Biosystems) using a commercially available TaqMan PCR master mix. Spanning an exon junction, the TaqMan probes used for this analysis were as follows: Myostatin (Hs00976237_m1), Myf5 (Hs00271574_m1), myogenin (Hs01032275_m1), and MYH2 (Hs00430042_m1), all from Applied Biosystems. The 2−ΔΔCT method was used to quantify the relative expression levels of the genes. The mRNA expression levels for all samples were normalized to the housekeeping gene, GAPDH.
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