Figure 1. BRAF mediates muscle precursor cell migration independent of MEK/ERK signaling.
(A) Immunofluorescence staining of migrating muscle cells (C2C12) after knock down of the hepatocyte growth factor (HGF) receptor (siMet), Braf (siBraf) and Pax3 (siPax3) or after transfection of cultures with Craf, Braf, a dominant negative form of Braf (DN Braf) and CA Braf (V600E) in the presence or absence of HGF. Con indicates control vector and siCon represents a scrambled siRNA control. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical assessment is shown in the upper part of the panel (n = 15; Mann-Whitney-U test, p*<0.05). siRNA knock-down efficiencies for Met (80%), Braf (70%) and Pax3 (85%) were determined by Western blot analysis. (B) Phosphorylation of ERK1/2 after transfection of C2C12 cells with control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of UO126 (5 µM) or cytochalasin D (5 µM; CytoD), noco (5 µM; nocodazole), Dynasore (80 µM; dyna), colchicine (0.01%; colch), methyl-β-cyclodextrin (3 mM; MβCD), and paclitaxel (5 µg/ml; paclit). Addition of DMSO served as an additional control. (n = 2). Cytochalasin D disrupts actin filaments. Nocodazole, colchicine, and paclitaxel interfere with microtubuli assembly or disassembly. U0126 inhibits the MEK/ERK pathway. Dynasore blocks dynamin-dependent endocytosis. Methyl-β-cyclodextrin removes cholesterol from cultured cells and disrupts lipid rafts. (C) Statistical assessment of the experiments shown in (D) (n = 15; Mann-Whitney-U test, p**<0.01). (D) Microscopic imaging of migrating C2C12 cells after transfection with a control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of DMSO, UO126 and Dynasore. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation.
Figure 1—figure supplement 1. BRAF and PAX3 stimulate limb muscle precursor cell migration.
