Myod Is a DNA Damage Signaling-Target with Essential Function in DNA Repair of Muscle Satellite
Supplementary Fig 1: Confocal immunofluorescence analysis of C2C12 cells treated or not with the indicated drugs for 12 hours and then fixed with 4% PFA. Staining with anti -H2AX or P-Nbs1 was performed. To-Pro-3 was used to mark nuclei.
Supplementary Fig 2: Confocal immunofluorescence analysisof C2C12 cells pretreated with imatinib (ABLi) or vehicle alone (NT) and then treated or not with DOX 3μM. 1hour after treatment cells were washed and fixed with 4% PFA at different times, as indicated in the panel and then immunostained with γH2AX. DAPI was used to mark nuclei.
Supplementary Fig 3: ChIP of the myogenin promoter and MCK enhancer was performed in C2C12 myoblasts treated with DOX 0,4 M for 15 hours and then collected, or shifted in DM for 24 hours and then collected. ChIP on nuclear extracts was performed with antibodies against MyoD, serine 5 phosphorylated Polymerase II (S5-PolII), histone lysine4 tri-methylated (H3K4me3), histone lysine 27 tri-methylated (H3K27me3) and control IgG. Graph shows real-time PCR values normalized against the input DNA. Values are mean±SEM. p values showing statistical significance by the Student’s t test between each treatment and the untreated (NT) sample are indicated (*p < 0.05, ** p< 0,005).
Supplementary Fig 4: Nuclear staining of MyoD, γΗ2AX and Pax7 in quiescent versus activated SCs (A-C) Immunofluorescence staining for Pax7, MyoD and -H2AX of tibialis anterior transverse sections of 2 months aged wild type mice, exposed (injury) or not (non injury) to cardiotoxin injection. (D) Graph representing counts of MyoD/H2AX (number of H2AX+cells within the MyoD+ cells), Pax7/H2AX (number of H2AX+cells within the Pax7+ cells) and MyoD/Pax7 (number of Pax7+cells within the MyoD+ cells) positive cells.
Supplementary Fig 5: (A)Immunofluorescence of SCs from single fibers isolated from muscles of ABL fl/fl mouse. After delamination, SCs were infected with adenovirus-GFP-control or with adenovirus-GFP-CRE. 24 hours after infection cells were untreated (untr) or treated with DOX 0,4 uM or MMS 75 uM for 15 hours and then shifted in differentiation medium for 48 hours. After fixation, cells were stained for MyHC and nuclei were stained with Dapi. The bottom panel shows the gal staining that revealed successful CRE-Lox recombination and gene excision in SCs after infection. The graph shows the average myogenic index (numbers of nuclei contained in differentiated cells) in the different time points, calculated in three different experiments.
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