Figure 7.

SRF activities and MAL expression levels are dependent on ALP expression. C2C12 cells were cotransfected with a mouse smALP expression vector (pQE/smALP) or an empty vector (pQE), and the SRF reporter plasmid pSRE3-Luc associated with a Renilla standardization reporter plasmid, and were subsequently placed in differentiation medium for 2 d. SRF activity was determined in (A). In a similar experiment, expression levels of SRF (B), MAL (C), myosin (D), and tubulin (E) were analyzed by Western immunoblotting, while phase contrast images show the aspect of the C2C12 cells transfected with the vector alone (F) or the smALP construct (G) after 2 d in differentiation medium. (H) SRF activity was also determined in ALP-antisense C2C12 cells (Anti 14I) or control C2C12 cells (Mock 13G) that were cotransfected with the SRF reporter plasmid pSRE3-Luc and a Renilla standardization reporter plasmid, and subsequently grown for 2 d in differentiation-promoting conditions. Expression levels of SRF (I) and MAL (J) were detected by Western immunoblotting in ALP-antisense C2C12 cells (Anti 14I) and control C2C12 cells (Mock 13G) grown in proliferation medium (Day 0) or placed in differentiation-promoting conditions for 2 d (Day 2) and 5 d (Day 5). As a control, tubulin was detected under the same conditions (K). Luciferase activities are expressed after correction for Renilla reporter activities. Results are the mean ± SEM of three independent experiments. Asterisks indicate statistical significance at p < 0.003 according to the unpaired Student's t test.