Jasplakinolide

Reagents were obtained from the following manufacturers: Adenosine 5’-diphosphate sodium ADP (A2754), P2X7 antagonist, BBG (B0770), AC5 inhibitor, NKY80 (N2165) and PP1/PP2A phosphatases inhibitor calyculin A (C5552) were obtained from Sigma-Aldrich. P2Y Antagonist II (BPTU; 504187) was obtained from EMD Millipore. 2-Methylthioadenosine diphosphate trisodium salt (2-MeSADP; 1624), (N)-methanocarba-2MeSADP (MRS 2365; 2157), the calpain inhibitor MDL 28170 and jasplakinolide were obtained from Tocris. P2Y1 shRNA and scrambled shRNA plasmids have been previously published and validated (del Puerto et al., 2012 (link)).

Fetal BSA (GIBCO) and 1-Oleoyl lysophosphatidic acid (LPA, Tocris Bioscience) were used at the indicated concentrations. Fibroblast growth factor mFGF-8b and rhFGF-3 (R&D Bioscience), platelet-derived growth factor PDGF-AA (PeproTech), and epidermal growth factor hEGF (Sigma) were used at a final concentration of 100 ng/μl. Pharmacological inhibitors were used at the following concentrations: 10 μM active (−) or inactive Blebbistatin (+) (Tocris Bioscience), 100 nM Latrunculin-A (Sigma), 100 μM Y-27632 (Tocris Bioscience), 500 nM Jasplakinolide (Tocris Bioscience), 20 μM ML-141 (Sigma), and 30 μM L-294002 (Sigma). The percentage of polarized cells was measured after 15 min (Blebbistatin, LatrunculinA, and Jasplakinolide) and 30–60 min (Y-27632, ML-141, Y-294002) of exposure.

For EdU incorporation in cultured MuSCs, EdU was mixed in media to a final concentration of 10 μM for 24 hr, followed by the Click-reaction (Thermo Fisher Scientific) for detection. FACS-isolated MuSCs were immediately treated with DMSO (mock-treatment), Jasplakinolide (100 nM; Tocris Bioscience) or Narciclasine (100 nM; Tocris Bioscience) at for 2 hr, cytospun to coverslip, and processed for IF. For activated MuSCs, they were cultured for 48 h after FACS-isolation, and treated by DMSO, Blebbinstatin (10 μM; Tocris Bioscience), Cytochalasin B (10 μM; Tocris Bioscience), and Y-27632 (10 μM) for 2 hr and processed for IF.

K562 cells and erythroblasts were treated with 5 µM Cytochalasin D, 1 µM Latrunculin A (Sigma-Aldrich), or 1 µM Jasplakinolide (Tocris) for 30 minutes at 37 °C. Erythroblasts were incubated with nocodazole (10 µM; Sigma) or taxol (paclitaxel, 10 µM; Sigma) at 37 °C for 3 hours. As indicated in the figure legends, cells were incubated with 30 µM Myristyl trimethyl ammonium bromide (MiTMAB; Calbiochem), 80 µM Dynasore (Sigma), 30 µM Pitstop-2 (Abcam) or 15 mM Sucrose (Sigma), for 15–45 minutes at 37 °C. All reagents were solubilised in DMSO (Sigma-Aldrich).

The Aspergillus fumigatus wild type H237 and mutant strains were maintained on Aspergillus Glucose Minimal Medium (GMM) [28 (link)]. For analysis of colony morphology and for quantification of radial growth rates, 1 X 10⁴ conidia were inoculated in triplicate onto GMM or YPD (10% w./v. yeast extract, 20% w./v. peptone, 20% w./v. dextrose) solid agar and incubated at 30°C or 37°C, for the indicated times. Colony diameter was measured every 24 hours and results reported as the average diameter in millimeters ± standard deviation. For the HsactB heterokaryon, radial growth analysis was performed in a similar manner, with the exception that GMM solid agar plates were inoculated with agar plugs containing hyphae from the colony periphery of previous GMM culture plates. For germination studies, strains were inoculated into triplicate GMM broth cultures, with or without jasplakinolide (Tocris Cat. No. 2792), at a density of 5 X 10⁵ conidia / ml and incubated at 37°C. Germ tube production was scored microscopically at the indicated times. DMSO was utilized as a control. Data are presented as average percent (%) germination ± standard deviation for each strain.