Journal: Rheumatology (Oxford, England)

Article Title: A novel estrogen receptor 1: sphingomyelin phosphodiesterase acid-like 3B pathway mediates rituximab response in myositis patients

doi: 10.1093/rheumatology/keac687

Figure Lengend Snippet: The impact of in vitro rituximab treatment on immortalized muscle cells. CCK-8, cell counting kit 8; ESR1, estrogen receptor 1; IL-13, interleukin 13; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor alpha. Raji (B-cells), THP-1 (macrophages) and immortalized human myoblasts and myotubes were treated with PBS or 5, 10 or 20 µg/mL rituximab. (A, B) Cell viability assessed by staining with trypan blue or with CCK-8 reagent. (C, D) Cytokine expression assessed using V-PLEX proinflammatory panel 1 (human) kit. (E) ESR1 expression measured using RTqPCR, fold change calculated by the 2-ΔΔCt method with HPRT1 as reference

Article Snippet: Immortalized muscle cells Immortalized human myoblasts (a kind gift from Dr Vincent Mouly at the Centre for Research in Myology in Paris, France) were grown at 3000 cells/cm 2 with skeletal muscle cell growth media (Promocell, Heidelberg, Germany) with 20% v/v fetal calf serum and 1% penicillin/streptomycin on flasks coated with 0.4% gelatin.

Techniques: In Vitro, CCK-8 Assay, Cell Counting, Saline, Staining, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Cap1 mRNA and protein levels are downregulated during myogenic differentiation. (A,E) Bright-field images (×20) of murine C2C12 (A) and human LHCN-M2 (E) cells upon differentiation for 4 and 6 days (d4 and d6), in comparison to undifferentiated control cells (d0). Cells were stained with crystal violet. (B,F) Relative Cap1 mRNA in differentiating C2C12 (B) and LHCN-M2 (F) cells, quantified by qRT-PCR and normalized to a set of housekeeping mRNAs. (C,G) Immunoblots of lysates from differentiating C2C12 (C) and LHCN-M2 (G) cells, using antibodies against myosin heavy chain polypeptides 1, 2, 4, and 6 (MYH), CAP1 and GAPDH as a control. (D,H) Quantification of CAP1 immunoblots at myogenic differentiation for 4 and 6 days, normalized to undifferentiated control cells. Error bars , SEM ( n = 3); ** p < 0.01, *** p < 0.001 (Student’s t -test). Scale bar, 200 μm.

Article Snippet: Human immortalized LHCN-M2 myoblasts (Evercyte, Vienna, Austria; Cat. no. CkHT-040-231-2) were cultured in MyoUp medium (Evercyte, MHT-040).

Techniques: Staining, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: miRNA (miR-1, miR-133, and miR-206) regulate the expression of Cap1 in murine and human myoblast. (A) The abundance of the indicated miRNAs in total lysates of undifferentiated and differentiated C2C12, determined by RNA-Seq. CPM; counts per million ( n = 3). (B) Schematic of the 3′-UTR of murine Cap1 with the STOP-codon at position 1 and the polyadenylation signal at 1,020 and 1,058 bp. Predicted binding sites for miR-1, miR-133 and miR-206 are indicated by yellow boxes. (C,D) Cap1 mRNA expression in undifferentiated C2C12 (C) and LHCN-M2 (D) cells transfected with the indicated miRNA mimic for 72 h ( n = 3). (E,F) Representative immunoblots of cells transfected with the indicated miRNA. (G,H) Quantification of the CAP1 protein from three independent experiments. Error bars, SEM ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: Human immortalized LHCN-M2 myoblasts (Evercyte, Vienna, Austria; Cat. no. CkHT-040-231-2) were cultured in MyoUp medium (Evercyte, MHT-040).

Techniques: Expressing, RNA Sequencing Assay, Binding Assay, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Satellite cells delivered in their niche efficiently generate functional myotubes in three-dimensional cell culture

doi: 10.1371/journal.pone.0202574

Figure Lengend Snippet: Muscle cell sources and cell culture conditions.

Article Snippet: Immortalized human myoblasts C25Cl48 , 500 000 myoblasts , Promocell, Skeletal Muscle Cell Growth Medium Kit (C-23160) supplemented with 20% FBS and Gentamycin (50 μg/ml), 2 days , Promocell, Skeletal Muscle Cell Differentiation, Medium Kit (C-23161) supplemented with gentamicin (50μg/ml), 6 days.

Techniques: Cell Culture, Cell Differentiation, In Vitro, Isolation

Journal: PLoS ONE

Article Title: Satellite cells delivered in their niche efficiently generate functional myotubes in three-dimensional cell culture

doi: 10.1371/journal.pone.0202574

Figure Lengend Snippet: Immortalized human myoblasts C25Cl48 were embedded in collagen I (500 000 cells / 100 μl: A and D), Fibrin (500 000 cells / 100 μl: B and E) or PEG-Fibrinogen (500 000 cells / 100 μl: C and F), cultured in proliferation medium for 2 days (A-C) and then switched to differentiation medium for 6 days (D-F). After 2 days in proliferation medium, cells were pulsed with the thymidine analogue 5-Ethynyl-2’deoxyuridine (EdU) for 2 hours and then fixed, before EdU incorporation was visualised and myoblasts immunolabelled for β-Tubulin and nuclei counterstained with DAPI. After 6 days in differentiation medium, cells were fixed and immunolabelled for myosin heavy chain (MyHC) to visualise myotubes and counterstained with DAPI. Proliferating EdU positive (arrow-heads) and β-Tubulin myoblasts were aligned in collagen I (A) and Fibrin (B) gels but randomly orientated in PEG-Fibrinogen scaffold (C). After 6 days in differentiation medium many MyHC positive cells were present but only a few small myotubes were detected in collagen I (D) or Fibrin (E) gels. In PEG-Fibrinogen scaffold (C), small myotubes were randomly orientated. Scale bar represents 100 μm. Representative images from 3 or more independent experiments. Expression of the sarcomeric proteins Myosin Heavy chain ( MYH2 , 3 , 8 ) (G), Actinin α3 (ACTN3 ) (H) and Tropomyosin 1 ( TPM1 ) (I) and creatine kinase (CKM ) (J) after 7 days of differentiation were analysed by RT-qPCR. Expression was normalized to the house keeping gene TBP . Data are mean±SEM from 3 independent gels where an asterisk denotes a significant difference ( p <0.05) from the Collagen gel using an unpaired two-tailed Student's t-test.

Article Snippet: Immortalized human myoblasts C25Cl48 , 500 000 myoblasts , Promocell, Skeletal Muscle Cell Growth Medium Kit (C-23160) supplemented with 20% FBS and Gentamycin (50 μg/ml), 2 days , Promocell, Skeletal Muscle Cell Differentiation, Medium Kit (C-23161) supplemented with gentamicin (50μg/ml), 6 days.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test