Latrunculin b

For latrunculin B, CK666, and VU0285655-1 experiments, cells were preincubated for 10–20 min with either solutions of 10 nM fMLP with 50 nM latrunculin B (Sigma), 100 nM fMLP with 100 μM CK666 (Sigma), or 100 nM fMLP with 4.5 μM VU0285655-1 (Avanti lipids).

For all pharmacological experiments, drugs were added to pre-warmed and equilibrated media before adding to hiCMs. For sarcomere assembly assays, 25 μM SMIFH2 (Sigma-Aldrich, S4826), 25 μM CK666 (Sigma-Aldrich, SML0006), and 100 μM Blebbistatin (Sigma-Aldrich, B0560) (separately) were added 1.5 hr after plating to allow hiCMs to attach to the substrate. hiCMs were subsequently fixed (see below) at 24 hr post plating. For live-cell experiments (as in Figure 5D), media in imaging container was aspirated using a vacuum system, and media containing drug was added to hiCMs on the microscope stage. This allowed the same hiCM to be imaged pre and post-drug application. For the LatrunculinB experiment (Figure 9J), cells were allowed to spread for 18 hr in normal media, and media containing 5 μM LatrunculinB (Sigma-Aldrich, L5288) was added for 6 hr and hiCMs were fixed (for a total spreading time of 24 hr).

Cortisol, EGTA (ethylene glycol bis (β-amino ethyl ether)-N,N,N′,N′-tetraacetic acid), trypan blue, cholesterol, methyl-beta-cyclodextrin (MβCD), latrunculin B, and Cpd5J-4 were purchased from Sigma, St. Louis, MO, USA. Pluoronic^®-F 127 (20% solution in DMSO), Fura 2-AM, and Laurdan were purchased from Thermofisher, Waltham, MA, USA. Cortisol-conjugated BSA was purchased from EastCoast Bio, North Berwick, ME, USA. All other chemicals were of analytical grade and were purchased from local suppliers.