Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-324-5p Inhibits C2C12 cell Differentiation and Promotes Intramuscular Lipid Deposition through lncDUM and PM20D1

doi: 10.1016/j.omtn.2020.09.037

Figure Lengend Snippet: Overexpression of miR-324-5p Inhibits the Differentiation of C2C12 Myoblasts (A) The efficiency of miR-324-5p overexpression after agomiR-324-5p transfection compared with that of the negative control (NC) on day 6 of differentiation. (B) Immunofluorescence staining of muscle myosin heavy chain (MyHC) in C2C12 myoblasts. Scale bars, 300 μm. (C) The fusion index was assessed as the ratio of MyHC-positive cells to the total number of nuclei. (D) The RNA level of myogenesis-related genes, including Myo d , Myog , and Myhc , detected by quantitative real-time PCR after overexpression of miR-324-5p. (E) Western blot analysis of myogenesis-related proteins after overexpression of miR-324-5p. (F) Quantification of protein levels. The data are expressed as mean ± SD; n = 5. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Membranes were then incubated overnight with primary antibodies as follows: anti-MYOD (Santa Cruz, Dallas, TX, USA), anti-MYOG (Novus Biologicals, CO, USA), anti-MyHC (Abcam, Cambridge, UK), anti-OCT4 (Santa Cruz), anti-ELOVL6 (Cell Signaling Technology), anti-ACC (Cell Signaling Technology), anti-p-ACC (Cell Signaling Technology), and anti-CPT1 (Santa Cruz Biotechnology).

Techniques: Over Expression, Transfection, Negative Control, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-324-5p Inhibits C2C12 cell Differentiation and Promotes Intramuscular Lipid Deposition through lncDUM and PM20D1

doi: 10.1016/j.omtn.2020.09.037

Figure Lengend Snippet: Inhibition of miR-324-5p Promotes C2C12 Myoblast Differentiation (A) The efficiency of miR-324-5p inhibition after antagomiR-324-5p transfection compared with that of the negative control (NA) on day 6 of differentiation. (B) Immunofluorescence staining of MyHC in C2C12 myoblasts. Scale bars, 300 μm. (C) The fusion index was assessed as the ratio of MyHC-positive cells to the total number of nuclei. (D) Quantitative real-time PCR was used to detect the expression of myogenesis-related genes, including Myo d , Myog , and Myhc , after inhibition of miR-324-5p. (E) Western blot analysis of myogenesis-related proteins after inhibition of miR-324-5p. (F) Quantification of protein levels. The data are expressed as mean ± SD; n = 5. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Membranes were then incubated overnight with primary antibodies as follows: anti-MYOD (Santa Cruz, Dallas, TX, USA), anti-MYOG (Novus Biologicals, CO, USA), anti-MyHC (Abcam, Cambridge, UK), anti-OCT4 (Santa Cruz), anti-ELOVL6 (Cell Signaling Technology), anti-ACC (Cell Signaling Technology), anti-p-ACC (Cell Signaling Technology), and anti-CPT1 (Santa Cruz Biotechnology).

Techniques: Inhibition, Transfection, Negative Control, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-324-5p Inhibits C2C12 cell Differentiation and Promotes Intramuscular Lipid Deposition through lncDUM and PM20D1

doi: 10.1016/j.omtn.2020.09.037

Figure Lengend Snippet: Summary Diagram Showing That miR-324-5p Inhibits C2C12 Myoblast Differentiation and Promotes Oleate-Induced Lipid Deposition in C2C12 Myoblasts DAG, diacylglycerol; TG, triglyceride; MyoG, myogenin; MyoD, myoblast determination protein; Dppa2, developmental pluripotency associated 2.

Article Snippet: Membranes were then incubated overnight with primary antibodies as follows: anti-MYOD (Santa Cruz, Dallas, TX, USA), anti-MYOG (Novus Biologicals, CO, USA), anti-MyHC (Abcam, Cambridge, UK), anti-OCT4 (Santa Cruz), anti-ELOVL6 (Cell Signaling Technology), anti-ACC (Cell Signaling Technology), anti-p-ACC (Cell Signaling Technology), and anti-CPT1 (Santa Cruz Biotechnology).

Techniques:

Journal: Journal of thermal biology

Article Title: Effects of heat stress exposure on porcine muscle satellite cells.

doi: 10.1016/j.jtherbio.2023.103569

Figure Lengend Snippet: Fig. 4. Expression of myogenic transcription factors in PMSCs exposed to heat stress. (A) Immunostaining images of transcription factors in PMSCs. Cell nuclei were stained with DAPI (blue), Pax7 (green), and MyoD (red). (B, C) Changes in Pax7, MyoD, and MyoG protein levels in PMSCs by heat stress. (D) Gene expression of Pax7, MyoD, and MyoG in PMSCs by heat treatment. Values are presented as the mean ± SE. a-c Different letters represent statistically significant differences among treatment groups (p < 0.01). Scale bar, 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: These membranes were blocked with TBST buffer containing 5% skim milk for 1.5 h at room temperature, and then incubated with primary antibodies against GAPDH (1:5000, monoclonal, MA5-15738, Invitrogen), Bax (1:1000, monoclonal, sc-7480, Santa Cruz), Bcl-2 (1:1000, monoclonal, sc-7382, Santa Cruz), caspase3 (1:1000, monoclonal, NB100-56708, Novusbio), Pax7 (1:1000, monoclonal, DSHB), MyoD (1:1000, polyclonal, 18943-1-AP, Proteintech), MyoG (1:1000, J.

Techniques: Expressing, Immunostaining, Staining, Gene Expression

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: SelK is upregulated in during SCs differentiation and C2C12 myoblast differentiation. (A) Immunofluorescence staining of Pax7 (red) and SelK (green) in TA muscle of wild-type mice without 1.2% BaCl 2 injection. Scale bar = 50 μm. (B) Immunofluorescence staining of Pax7 (red) and SelK (green) in TA muscle of wild-type mice at 5 d postinjury. Scale bar = 50 μm. (C) Immunofluorescence staining of MyoG (red) and SelK (green) in TA muscles of wild-type mice at 5 d postinjury. Scale bar = 50 μm. (D) Western blot analysis of SelK protein levels in uninjured and 5 d-injured TA muscle. N = 3 mice in each group for A-D. (E) Immunofluorescence staining of SelK in C2C12 myoblasts cultured in growth medium for 1 d or in differentiation medium for 2 d. Scale bar = 50 μm. (F) qRT-PCR analysis of SelK mRNA levels in C2C12 myoblasts cultured in growth medium for 1 or 2 d (G1 or G2) or in differentiation medium for 1, 3, 5 or 7 d (D1, D3, D5 or D7). (G) and (H) Western blotting analysis of SelK protein levels in C2C12 myoblasts cultured in growth or differentiation medium for 1 or 2 d (G1 or G2) or in differentiation medium for 1, 3, 5 or 7 d (D1, D3, D5 or D7). N = 3 in each group for E-H. The results are presented as means ± S.D. ***p < 0.001, values significantly different from the corresponding control by unpaired t -test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Immunofluorescence, Staining, Injection, Western Blot, Cell Culture, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: Ablation of SelK impairs skeletal muscle regeneration. (A) A schematic illustration of the knockout of SelK using CRISPR/Cas-mediated genome engineering. (B) Western blotting analysis of SelK protein levels in the TA muscle of wild-type (Ctrl) mice and SelK KO mice at 5 d postinjury (N = 3). (C) Average overall body weight of adult wild-type mice and SelK KO mice (N = 5). (D) Relative uninjured TA muscle wet weight of adult wild-type and SelK KO mice (N = 5). (E) Immunohistochemistry analysis of SCs markers (Pax7 and MyoG) and SelK in the TA muscle of SelK KO mice at 5 d postinjury (N = 3). Scale bar = 50 μm. (F) H&E staining of TA muscle in adult Ctrl and SelK KO mice at 0, 3, 5 and 7 d postinjury (N = 3). Scale bar = 50 μm. (G) Rate of myofibers containing two or more centrally located nuclei per field and (H) the average cross-sectional area (CSA) of myofibers calculated from TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (I) Distribution of regenerative myofiber CSAs of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (J) Immunofluorescence staining of MyHC (green) in TA muscles of Ctrl and SelK KO mice at 5 d postinjury (N = 3) and (K) the percentage of MyHC-positive (MyHC + ) fibers in TA muscle of Ctrl and SelK KO mice at 5 d postinjury. Nuclei were labelled using DAPI staining. Scale bar = 50 μm. The results are presented as means ± S.D. *p < 0.05, **p < 0.005, values significantly different from the corresponding control by unpaired t -test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Knock-Out, CRISPR, Western Blot, Immunohistochemistry, Staining, Immunofluorescence

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: Inactivation of SelK inhibits the myogenic differentiation progression of SCs. (A) Immunofluorescence staining of MyoD (green) and Pax7 (red) in TA muscle of Ctrl and SelK KO mice at 3 d postinjury (N = 3). Nuclei were labelled using DAPI staining. Scale bar = 20 μm. (B) Quantification of the percentages of Pax7 + MyoD − , Pax7 + MyoD + and Pax7 − MyoD + cell populations in Ctrl and SelK KO TA muscles at 3 d postinjury. (C) Immunofluorescence staining of MyoD (green) and MyoG (red) in TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). Nuclei were labelled using DAPI staining. Scale bar = 20 μm. (D) Quantification of the percentages of MyoD + MyoG − , MyoD + MyoG + and MyoD − MyoG + cell populations in Ctrl and SelK KO TA muscles at 5 d postinjury. (E) qRT-PCR analysis of myogenic transcription factors (MyoD1, MyoG, MyH1, MyH2 and MyH3) mRNA levels in TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (F) and (G) Western blotting analysis of MyoD, MyoG and MyHC protein levels in uninjured and 5 d-postinjured TA muscle of Ctrl and SelK KO mice (N = 3). The results are presented as means ± S.D. *p < 0.05, **p < 0.005, ***p < 0.001, values significantly different from the corresponding control by unpaired t -test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: The differentiation ability of C2C12 myoblast cells is reduced by SelK silencing. (A) and (B) Western blotting analysis of SelK protein levels in siSelK-transfected C2C12 myoblast cells cultured in differentiation medium for 1, 3 and 5 d (N = 3). (C) qRT-PCR analysis of SelK mRNA levels in siSelK-transfected C2C12 myoblast cells cultured in differentiation medium for 0, 1, 3 and 5 d (N = 3). (D) Immunofluorescence staining of MyHC in control siRNA (siNC) and SelK siRNA (siSelK) C2C12 myoblast cells cultured in differentiation medium for 5 d (N = 3). Scale bar = 100 μm. (E) Quantitative analysis of the percentage of MyHC + C2C12 myoblast cells. (F) Immunofluorescence staining of MyoG in siNC and siSelK C2C12 myoblast cells cultured in differentiation medium for 5 d (N = 3). Scale bar = 50 μm. (G) Quantitative analysis of the percentage of MyoG + C2C12 myoblast cells. (H) and (I) Western blotting analysis of MyoD, MyoG and MyHC protein levels in siNC and siSelK C2C12 myoblast cells cultured in differentiation medium for 0, 1, 3 and 5 d (N = 3). The results are presented as means ± S.D. **p < 0.005, ***p < 0.001, values significantly different from the corresponding control by unpaired t -test.

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Western Blot, Transfection, Cell Culture, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: Silencing of SelK enhances oxidative stress in SCs and C2C12 myoblast cells during myogenic differentiation. (A) Activities of antioxidant enzymes (SOD and CAT) and the contents of oxidative stress markers (H 2 O 2 , MDA and T-AOC) in TA muscles of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (B) and (C) DCFH-DA staining and quantification of ROS generation in C2C12 myoblast cells cultured in differentiation medium for 0, 1, 3 and 5 d (N = 3). (D) Immunofluorescence staining of MyHC and MyoG and bright field microscopy in siNC and siSelK C2C12 myoblast cells treated with NAC or PBS after 5 d in differentiation medium (N = 3). (E) Quantitative analysis of the percentage of MyHC + cells and (F) the percentage of MyoG + cells treated with NAC or PBS after 5 d in differentiation medium. (G) Immunofluorescence staining of MyHC and MyoG and bright field microscopy in siNC and siSelK C2C12 myoblast cells treated with SF or DMSO after 5 d in differentiation medium (N = 3). (H) Quantitative analysis of the percentage of MyHC + cells and (I) the percentage of MyoG + cells treated with SF or DMSO after 5 d in differentiation medium. (J) and (K) Western blotting analysis of MyoD, MyoG and MyHC protein levels in siNC and siSelK C2C12 myoblast cells treated with NAC or PBS after 5 d in differentiation medium (N = 3). (L) and (M) Western blotting analysis of MyoD, MyoG and MyHC protein levels in siNC and siSelK C2C12 myoblast cells treated with SF or DMSO after 5 d in differentiation medium (N = 3). The results are presented as means ± S.D. *p < 0.05, **p < 0.005, ***p < 0.001, values significantly different from the corresponding control by unpaired t -test. Bars that do not share the same letters are significantly different (p < 0.05) from each other by one-way ANOVA.

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Staining, Cell Culture, Immunofluorescence, Microscopy, Western Blot

Journal: Redox Biology

Article Title: Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation ☆

doi: 10.1016/j.redox.2022.102255

Figure Lengend Snippet: Silencing SelK aggravates ER stress in SCs and C2C12 myoblast cells during myogenic differentiation. (A) and (B) Western blotting analysis of ER stress-related genes (GRP78, ATF6, p-IRE1, p-PERK, p-eIF-2α and CHOP) protein levels in the TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (C) qRT-PCR analysis of ER stress-related genes mRNA levels in the TA muscle of Ctrl and SelK KO mice at 5 d postinjuy (N = 3). (D) and (E) Western blotting analysis of ER stress-related genes protein levels in siNC and siSelK C2C12 myoblast cells treated with 4-PBA or DMSO after 5 d in differentiation medium (N = 3). (F) Quantification of DCFH-DA stained ROS generation in siNC and siSelK C2C12 myoblast cells treated with 4-PBA or DMSO after 3 d in differentiation medium. (G) and (H) Western blotting analysis of ER stress-related genes protein levels in siNC and siSelK C2C12 myoblast cells treated with TUDCA or H 2 O after 5 d in differentiation medium (N = 3). (I) Quantification of DCFH-DA stained ROS generation in siNC and siSelK C2C12 myoblast cells treated with TUDCA or H 2 O after 3 d in differentiation medium (N = 3). (J) Immunofluorescence staining of MyoG and MyHC and bright field microscopy in siNC and siSelK C2C12 myoblast cells treated with 4-PBA or DMSO after 5 d in differentiation medium (N = 3). (K) Quantitative analysis of the percentage of MyHC + cells and (L) the percentage of MyoG + cells treated with 4-PBA or DMSO after 5 d in differentiation medium. (M) Immunofluorescence staining of MyoG and MyHC and bright field microscopy in siNC and siSelK C2C12 cells treated with TUDCA or H 2 O after 5 d in differentiation medium (N = 3). (N) Quantitative analysis of the percentage of MyHC + cells and (O) the percentage of MyoG + cells C2C12 cells treated with TUDCA or H 2 O after 5 d in differentiation medium. The results are presented as means ± S.D. *p < 0.05, **p < 0.005, values significantly different from corresponding control by unpaired t -test. Bars that do not share the same letters are significantly different (p < 0.05) from each other by one-way ANOVA.

Article Snippet: The primary antibodies used for immunohistochemistry were as follows: mouse anti-Pax7 (cat. No. sc-81648, Santa Cruz, 1:50), rabbit anti-SelK (cat. No. ab139949, Abcam, 1:50), and mouse anti-MyoG (cat. No. GB14118, Servicebio, 1:50).

Techniques: Western Blot, Quantitative RT-PCR, Staining, Immunofluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model

doi: 10.3389/fcell.2024.1429759

Figure Lengend Snippet: Evolution of marker protein expression along terminal myogenesis indicates differentiation of skeletal myotubes from hiPSC . hiPSC were determined and further differentiated for up to 60 days. (A-A′) Upper panels: schematic protocol timeline for hiPSC determination towards skeletal myogenic fate (A) and hiPSC-derived myoblast terminal differentiation (A′) . Lower panels: representative brightfield images of cell populations at various determination and differentiation timepoints. Presomitic mesoderm cells, premyogenic progenitors and mixed cell population consisting of myoblasts and myotubes were imaged at d-45, d-40 and d-22, respectively. hiPSC-derived myoblasts were obtained after 50 days of hiPSC determination (A) and were further induced to terminally differentiate into myotubes after 6 days of proliferation and 4 days of differentiation in N2-based medium (A′) . Scale bars, 100 µm. (B–F) hiPSC-derived myoblasts were differentiated according to the protocol shown in A-A′ , fixed at different timepoints, and immunostained for myogenic markers. Skeletal muscle cells were derived from control and SOD1 D90A mutant hiPSC lines. (B) Representative confocal images of samples immunostained for myogenin (MyoG) and myosin heavy chain (MYH1) at timepoints as indicated. Scale bars, 100 µm. (C,D) Quantification of the percentage of MyoG positive nuclei (C) and of percentage of nuclei within MYH1 positive cells (differentiation index; D) as a function of differentiation time. Graphs depict mean ± SD. (E,F) Quantification of MyoG (E) and MYH1 (F) mean fluorescence intensity of positive cells. At least three biological replicates were analyzed per condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies and dye concentrations were as follows: anti-Myogenin (MyoG; LSBio; cat. no: LS-C334865; dilution: 1/400), anti-Myosin Heavy Chain 1 (MYH1; DSHB; cat. no: MF20-c; dilution: 1/800), anti-α-actinin (Invitrogen; cat. no: MA1-22863; dilution: 1/400), anti-vesicular acetylcholine transporter (vAChT; Synaptic Systems; cat. no: 139 103; dilution: 1/400), 4’,6-Diamidino-2-Phenylindole (DAPI; Roche; cat. no: 10236276001; 1 mg/mL; dilution 1:1000); αBGT-AF488 (Invitrogen; cat. no: B13422; dilution: 1/500), αBGT-AF647 (Invitrogen; cat. no: B35450; dilution: 1/500), Donkey α-mouse AF-488 (Invitrogen; cat. no: A21202; dilution 1:1000), Donkey α-rabbit AF-555 (Invitrogen; cat. no: A32794; dilution 1:1000), Donkey α-mouse AF-647 (Invitrogen; cat. no: A31571; dilution 1:1000).

Techniques: Marker, Expressing, Derivative Assay, Control, Mutagenesis, Fluorescence