Examining PANX1 Role in Myoblast Differentiation

For the differentiation assays, HSMM (70 000 cells/well) were seeded in 24-well dishes containing one collagen coated glass cover slip. Cells were transduced with a lentivector containing either GFP or PANX1 [46 (link)] one day prior to induction of differentiation. Based on GFP, a transduction efficiency of approximately 50% was achieved. Differentiation was initiated by switching cells into differentiation medium which consisted of DMEM supplemented with 5% horse serum and 1% penicillin/streptomycin. Cells were fixed in 3.7% PFA on day 5 of differentiation. Cells were stained for myosin heavy chain (MHC) (1:500, MF20, MAB4470, R&D Biosystems, Minneapolis, MN, USA), and mounted with Fluoromount-DAPI (Southern Biotech, Birmingham, AL, USA). Cells were imaged with the EVOS FL Auto fluorescent microscope (Thermo Fisher Scientific) under 20X magnification. The differentiation index (number of MHC positive cells/ total number of nuclei (%)) and fusion index (number of nuclei in myotubes/total number of nuclei (%)) were analyzed from 3–5 randomly selected fields of view [34 (link)].

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Freeman E., Langlois S., Leyba M.F., Ammar T., Léger Z., McMillan H.J., Renaud J.M., Jasmin B.J, & Cowan K.N. (2024). Pannexin 1 dysregulation in Duchenne muscular dystrophy and its exacerbation of dystrophic features in mdx mice. Skeletal Muscle, 14, 8.

Publication 2024

MAB4470 Fluoromount-DAPI EVOS FL Auto fluorescent microscope

Corresponding Organization :

Other organizations : Children's Hospital of Eastern Ontario, University of Ottawa

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Variable analysis

independent variables

Lentivector containing either GFP or PANX1

dependent variables

Differentiation index (number of MHC positive cells/ total number of nuclei (%))
Fusion index (number of nuclei in myotubes/total number of nuclei (%))

control variables

HSMM (70 000 cells/well) were seeded in 24-well dishes containing one collagen coated glass cover slip
Cells were transduced with a lentivector one day prior to induction of differentiation
Differentiation was initiated by switching cells into differentiation medium which consisted of DMEM supplemented with 5% horse serum and 1% penicillin/streptomycin
Cells were fixed in 3.7% PFA on day 5 of differentiation
Cells were stained for myosin heavy chain (MHC) (1:500, MF20, MAB4470, R&D Biosystems, Minneapolis, MN, USA) and mounted with Fluoromount-DAPI (Southern Biotech, Birmingham, AL, USA)
Cells were imaged with the EVOS FL Auto fluorescent microscope (Thermo Fisher Scientific) under 20X magnification

controls

Positive control: Cells transduced with lentivector containing GFP
Negative control: Not explicitly mentioned

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