Supplemental Material - Rouger et al. - Vol. 55, pp 607-618

Online Supplemental data for Rouger et al.: Figures and experimental procedures.

Files in this Data Supplement:

• Rouger et al., Procedure SProc 1
• Rouger et al., Figure SF1 - Immunofluorescent labeling of turkey muscle-derived cells. Mice MDC, turkey MDC, and turkey fibroblasts were seeded at 2.5x104 cells/cm2 on several coverlips and grown in proliferation medium. Fibroblasts were used as negative controls for the different antibodies (Ab) directed against myogenic cell-specific proteins. Two time points were used in these experiments: day 4 and day 12 to perform immunolabeling with Pax7, and M-cadherin, desmin, respectively. Indeed, the transcription factor Pax7 had an expression restricted to the first steps of cell proliferation whereas M-cadherin and desmin have an increased expression in differentiated cells. At these times, primary cultures were fixed in 4% cold PFA and some of them were stained with the standard May-Grünwald-Giemsa, to visualize the cell culture morphology. In parallel, MDC-derived cultures and fibroblast-derived ones were tested for embryonic/adult fast myosin heavy chain (MHC) and vimentin expression, respectively, in order to validate the origin of the cell cultured in vitro. All cultures were investigated for their M-cadherin, Pax7, and desmin expression. Finally, they were counterstained with propidium iodide and slides were viewed using confocal laser scanning microscopy. Immunocytochemistry revealed that Pax7 was expressed by a large majority of the turkey mononucleated MDC, as observed for mice MDC. Also, cytoplasmic labeling was observed for M-cadherin and desmin, especially in myotubes that corroborated the results obtained in mice MDC-derived cultures. In contrast, any signal related to these myogenic specific lineage markers was detected in fibroblast-derived cultures. This indicated that the three Ab have cross-reactivity in turkey animal. Moreover, the fibroblast-derived cultures were labeled with the vimentin Ab but not with the desmin one, revealing that it did not exist reactivity between both Ab.
• Rouger et al., Figure SF2 - A representative western blotting analysis for desmin. Immunoblot analysis of desmin was carried out on turkey skeletal muscle, and also on rat skeletal muscle and rat liver corresponding to positive and negative controls, respectively. Indeed, as previously demonstrated by immunocytochemistry and western blotting, the Ab reacts with the desmin-equivalent protein in rat (Debus et al. 1983). After tissue processing and protein determination, 75 g of each muscle protein extracts and 150 g of liver protein extract were loaded onto a 10% SDS-polyacrylamide gel. (A) The amounts of loaded proteins were detected by Ponceau-red staining of the nitrocellulose membrane. (B) After separation and western blotting analysis, the protein was detected with mAb to human desmin. Molecular weight markers (MW) were indicated on the left of the membranes. Desmin immunoreactive band at 53 kD was arrowed on the right of the gels. This band was consistently detected in turkey skeletal muscle, revealing that the mAb well displayed cross-reactivity with turkey desmin. As expected, any protein in liver was recognized by this mAb that was directed against muscle-specific intermediate filament.
• Rouger et al., Figure SF3 - Immunocytochemistry using QH1 antibody. Defined as being specific for cell surface determinants on quail endothelial cells and hematopoietic cells (Pardanaud et al. 1987), QH1 mAb was tested on aorta tissue samples and bone marrow cells. (A) Cryostat transverse sections (8 m) of frozen aorta tissues from quail (positive control), chicken (negative control) and turkey (investigated sample) were collected on Polysin-coated slides and incubated with QH1 mAb. After several washes in PBS, sections were incubated with Alexa Fluor 488-conjugated secondary Ab and then mounted using Vectashield containing prodidium iodide. Serial sections were stained with HES. Immunofluorescent data showed that the turkey endothelial cells located in the condensed layer around aorta were reactive with QH1 mAb, similarly to those observed on quail aorta. In contrast, any labeling was found in chicken tissue, as expected. These results indicated that QH1 mAb cross-reactivity exists with the turkey endothelial cells. (B) 40,000 turkey bone marrow cells were isolated from the tibial and femoral bones and deposited on Polysin microscope slides, using Cytospin. Also, quail and chicken bone marrow cells were used as positive and negative controls, respectively. Part of the bone marrow cells was labeled in both quail and turkey sample, while any chicken cells displayed immunolabeling. This indicated that QH1 mAb also recognizes the turkey hematopoietic cells.
• Rouger et al., Figure SF4 - Western blotting analysis for QH1 antibody. QH1 mAb was tested on lung tissue from quail, chicken and turkey animals. Using western blotting, it has been shown to detect many bands in quail extract but any in chicken one. Freeze lung tissues, that contain many endothelial structures, were disrupted and homogenized with a tissue-lyser system and protein content was determined using the BCA kit. Then, 150 g of protein extracts were added in each lane from a 10% SDS-polyacrylamide gel. (A) The equivalent amount of loaded proteins was detected by Ponceau-red staining of the nitrocellulose filter. (B) The membrane was labeled using QH1 mAb to quail vascular endothelial cells. Molecular weight markers (MW) were indicated on the left of the membranes. While any band appeared in chicken lung extract, multiple bands ranging from 35 to 215 kD were observed in lane corresponding to quail lung extract. This profile was similarly obtained with turkey extract, indicating that it exists a cross-reactivity of the QH1 mAb in turkey animal.
• Rouger et al., Figure SF5 - A representative western blotting analysis for M-cadherin. Immunoblot analysis of M-cadherin was carried out on turkey myogenic cells, and also on turkey fibroblasts and mice myogenic cells corresponding to negative and positive (Ab was supplied as cross-reacting with mice M-cadherin) controls, respectively. Indeed, as previously described, M-cadherin is undetectable in fibroblast-derived cultures (Donalies et al. 2001). Primary myogenic cultures were grown in proliferation medium during four days. As M-cadherin is up-regulated following the induction of myotube formation (Eng et al. 1997), proliferation medium was replaced by differentiation medium, in which cells were maintained for four days. After cell culture processing and protein determination, 100 g of each protein extracts were loaded onto a 10% SDS-polyacrylamide gel. (A) The amounts of loaded proteins were detected by Ponceau-red staining of the nitrocellulose membrane. (B) After separation and western blotting analysis, the protein was detected with Ab to human M-cadherin. Molecular weight markers (MW) were indicated on the left of the membranes. M-cadherin immunoreactive band at 130 kD was arrowed on the right of the gels. This band was consistently detected in turkey and mice myogenic cell cultures, revealing that the mAb well displayed cross-reactivity with turkey M-cadherin. As expected, any protein in fibroblasts was recognized by this mAb that was directed against muscle-specific Ca+-dependent cell adhesion molecule.
• Rouger et al., Figure SF6 - A representative western blotting analysis for Pax7. Primary turkey myogenic cultures, turkey fibroblast cultures (negative control), and mice myogenic ones (positive control; Ab was supplied as cross-reacting with mice Pax7) were grown in proliferation medium during 4 days. Then, proliferative cells were processed for western blotting and 30 g of each protein extract were loaded onto a 10% SDS-polyacrylamide gel. (A) The amounts of loaded proteins were detected by Ponceau-red staining of the nitrocellulose membrane. (B) After separation and transfer, the protein was detected with mouse Ab to chicken Pax7. Molecular weight markers (MW) were indicated on the left of the membranes. Western blotting revealed immunoreactive band at 57 kD (indicated by arrow on the right of the membranes) in both lanes corresponding to mice and turkey myogenic cells. This indicated that the Ab also recognizes the Pax7-equivalent protein in turkey. In contrast, any band was detected in fibroblast protein extract with this Ab that was directed against myogenic-specific transcription factor.