Journal: Cell Death and Differentiation
Article Title: Fate decision of satellite cell differentiation and self-renewal by miR-31-IL34 axis
Figure Lengend Snippet: IL34 is required for myogenic lineage progression in cultured SCs. a Fresh SCs were seeded, cultured in growth medium, and then induced to differentiate for various amounts of time. The immunoblots presented here reveal the protein levels of IL34, p-STAT3, STAT3, and unrelated β-tubulin in cultured SCs at different time points after initiation of differentiation. b SCs were isolated from the hindlimbs of WT mice using FACS, cultured in growth medium for 3 days, and treated with shIL34 lentivirus or control shRNA (shScr) for 36 h. The positively infected SCs were then purified by FACS according to the intrinsic enhanced green fluorescent protein (EGFP) fluorescence expressed by the lentivirus. Purified infected SCs were cultured for further analysis. c Western blot analysis of the levels of IL34, p-STAT3, STAT3, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. d Western blot analysis of the levels of MyHC, MyoD, Pax7, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. e Representative merged photomicrographs of SCs stably infected with a lentivirus expressing shIL34 or shScr that were induced to differentiate for one day and stained for Pax7 and DAPI. Scale bar: 30 μm. f Quantification of the percentage of Pax7 + SCs after shScr and shIL34 infection. *** P
Article Snippet: The following primary antibodies were used: mouse anti-Pax7 (1:100, Developmental Studies Hybridoma Bank (DSHB), lowa City, IA), mouse anti-MyoD1 (1:100, DAKO), rabbit anti-MyoD (1:100, Santa Cruz, sc-760), mouse anti-MyoD (1:100, Santa Cruz, sc-32758), mouse anti-embryonic Myosin Heavy Chain BF-45/F1.652 (1:40, DSHB, lowa City, IA), rabbit anti-myogenin (1:200, Santa Cruz, sc-576), rabbit anti-laminin (1:1000, Sigma-Aldrich, L9393), and rabbit anti-Ki67 (1:500, Invitrogen).
Techniques: Cell Culture, Western Blot, Isolation, Mouse Assay, FACS, shRNA, Infection, Purification, Fluorescence, Stable Transfection, Expressing, Staining