ASCs were seeded in flasks or multiwell plates at a density of 7.5 × 103 cells/cm2 (70%–80% confluence) and allowed to adhere for 24 h. Cells were washed twice in PBS and serum starved in FBS‐free standard culture medium (SFM) for a further 24 h. On the day of stimulation, medium was aspirated, cells washed twice in PBS and stimulated with 100 ng/mL of GDF6 (Peprotech) in SFM as previously described.
3 (link),
6 (link),
7 (link) Stimulation was performed for a variety of durations which are described for each experiment and forms the temporal‐nature of these studies.
To assess the importance of intracellular signaling mechanisms on the early response genes following GDF6 stimulation, Smad1/5/8 phosphorylation was inhibited by pre‐incubation with 10 μM dorsomorphin (Merck, cat no. 171261) and ERK1/2 phosphorylation was inhibited by pre‐incubation with 10 μM U0126 (Merck, cat no. 662009).
7 (link) Furthermore, protein translation was inhibited by pre‐incubation with 10 μg/mL cyclohexamide (Sigma, cat no. C4859) to help elucidate whether gene expression was directly downstream of GDF6‐mediated signaling or reliant upon de novo protein synthesis. In all experiments, following stimulation, cells were washed twice with ice‐cold PBS before either protein or RNA extraction.
3 (link),
6 (link),
7 (link) Stimulation was performed for a variety of durations which are described for each experiment and forms the temporal‐nature of these studies.
To assess the importance of intracellular signaling mechanisms on the early response genes following GDF6 stimulation, Smad1/5/8 phosphorylation was inhibited by pre‐incubation with 10 μM dorsomorphin (Merck, cat no. 171261) and ERK1/2 phosphorylation was inhibited by pre‐incubation with 10 μM U0126 (Merck, cat no. 662009).
7 (link) Furthermore, protein translation was inhibited by pre‐incubation with 10 μg/mL cyclohexamide (Sigma, cat no. C4859) to help elucidate whether gene expression was directly downstream of GDF6‐mediated signaling or reliant upon de novo protein synthesis. In all experiments, following stimulation, cells were washed twice with ice‐cold PBS before either protein or RNA extraction.
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