JCS006619 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. SRF activity and MyoD expression are regulated by actin dynamics during cell cycle activation of synchronized myoblasts. C2C12 myoblasts were transfected with the 3DAluc SRF reporter gene (gift of R. Treisman) and a stable clone expressing the SRF reporter gene isolated, and designated C2luc cells. C2luc cells were synchronized in G0 using suspension culture in methyl cellulose medium (Milasincic et al., 1996, Sachidanandan et al., 2003). Briefly, suspension culture in methylcellulose gel prepared in growth medium causes myoblasts to enter an undifferentiated reversible arrest in G0 as they are anchorage-dependent. These arrested cells re-enter the cell cycle in a synchronized fashion when replated on an adhesive substratum in growth medium. (A) Kinetics of S-phase re-entry in synchronized C2luc myoblasts revealed by pulse-labeling with BrdU (mean ± s.e.m., n=3). A, asynchronously growing population; G0, cells arrested by 48 hours in suspension culture; R6-R30, cells reactivated for 6-30 hours by replating in growth medium. (B) Immuno-blot analysis of total protein isolated from a replating time-course of synchronized C2luc cells. A, asynchronously growing population; G0, cells arrested by 48 hours in suspension culture; R15′-R8, cells reactivated for 15 minutes-8 hours by replating in growth medium; MT, differentiated myotubes. Blots were probed with an anti-MyoD antibody (upper panel), or anti-SRF (lower panel); desmin was as a loading control. MyoD expression is downregulated during suspension arrest but is restored within 4 hours of replating. SRF protein levels are largely unchanged during cell cycle arrest and activation. Data are representative of three independent experiments. (C) SRF reporter activity in clone C2Luc during arrest and re-activation. Mb, asynchrononously growing cells; S48, cells arrested in G0 for 48 hours by suspension culture; R2-R24, cells reactivated into the cell cycle for 2-24 hours by replating in growth medium. SRF activity is induced by 4 hours after reactivation of arrested cells, paralleling the induction of MyoD expression. Values represent the mean ± s.e.m. (n=3). (D) Actin-depolymerising drugs differentially affect SRF activity during cell cycle reactivation. G0 synchronized C2Luc myoblasts were plated in 0.5 μM Cytocalasin D or 0.1 μM Latrunculin B for 24 hrs. Values represent normalized luciferase activity relative to the corresponding vehicle control (mean ± s.e.m.; n=4). (E) Actin depolymerising drugs differentially affect activation of MyoD expression during cell cycle reactivation. Immunoblot analysis of MyoD expression in clone C2Luc in CytoD or LatB as described above. The response of MyoD expression to actin disruption mirrors SRF activity. Taken together with the fact that MyoD is a direct SRF target (SRE/CarG box in DRR enhancer), these experiments suggest that MyoD is regulated by SRF in a manner dependent on the dynamics of the actin cytoskeleton. Since blocking SRF activity during cell cycle re-entry using LatB (Fig. S1D) also blocks MyoD expression (Fig. S1E), it appears that induction of SRF during G1 is required for MyoD activation.

• Supplemental Figure S2 -

  Fig. S2. Inhibition of ROCK does not alter SRF activity or MyoD expression. C2C12 myoblasts synchronized as described above were reactivated in the presence of 10 μM Y27632 (specific ROCK inhibitor) or vehicle for 12 hrs. (A) Immuno-fluorescence analysis. Actin staining (with Oregon-Green−phalloidin) reveals the typical stellate morphology of ROCK-inhibitor treated cells, but MyoD expression (anti MyoD antibody and anti-mouse Alexa 594) is unaffected by inhibition of ROCK. (B) Immuno-blot analysis of MyoD protein levels confirm the immunofluorescence analysis. (The two lanes appear merged in the MyoD panel but are distinct in the desmin panel which was done after stripping the MyoD antibody and reprobing with anti desmin). (C) Inhibition of ROCK does not affect SRF activity. SRF reporter assay of C2Luc myoblasts treated with 10 μM Y27632 or vehicle for 24 hours. Values represent the mean ± s.e.m. (n=3).