Rapamycin

Rapamycin (AG-CN2-0025, AdipoGen, San Diego, CA, USA) was first dissolved in DMSO at a final concentration of 100 nM. This mixture was then added to standard NGM agar plates. Administration of Rapamycin was performed by culturing on Rapamycin NGM plates for 24 h prior to imaging, unless otherwise indicated. Bafilomycin A1 (BA1; BBVT-0252-C100, BioViotica, Dransfeld, Germany) was suspended in 0.2% DMSO and 0.1% phenol red solution at a final concentration of 50 µM and administered via injection into body cavity at tail position. This was done as BA1 is not cuticle permeable. Injections were performed 24 h before imaging. For axotomy experiments involving BA1 treatment, animals were cultured on NGM plates for 2 h post injection prior to axotomy.

Zymosan A (ZyA; Alfa Aesar), DMSO (Tocris Bioscience), Rapamycin (Adipogen), 6-Diazo-5-oxo-L-norleucine (DON; Sigma-Aldrich), Torin1 (Tocris Bioscience), compound 968 (C968; Calbiochem) were purchased. In vitro experiments used the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS; MP Biomedicals), 1% penicillin-streptomycin (Lonza Walkersville), and recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech). For flow cytometry analysis of myeloid cells, we used FITC-conjugated anti-Gr1 (BD PharMingen), FITC-conjugated anti-Ly6G (BD PharMingen), PerCP-conjugated anti-CD11b (BioLegend), APC-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD11c (eBioscience), and PE-conjugated anti-F4/80 (eBioscience). For the flow cytometry analysis of lymphocytes, we used FITC-conjugated anti-CD4 (eBioscience), APC-conjugated anti-Rorγt (eBioscience), PE-conjugated anti-Foxp3 (eBioscience), and 7-AAD Viability Staining Solution (eBioscience). For the Western blotting analysis, anti-phospho-p70 S6 kinase (Thr389) (Cell Signaling, #97596 S), anti-p70 S6 kinase (Cell Signaling, #9202), anti-phospho-Akt (Ser473) (Cell Signaling, #4060), anti-Akt (Cell Signaling, #2920), and anti-β-actin antibodies (Sigma-Aldrich) were purchased.

Two inhibitors, rapamycin (AdipoGen Life Sciences) and axitinib (Selleck Chemicals LLC), were used to inhibit the TOR and Vegf signaling pathways, respectively. For the starfish P. pectinifera, larvae were treated with inhibitors (20 nM for rapamycin and 20 or 50 nM for axitinib) from the onset of feeding (i.e., 2 dpf) until 7 dpf. For the sea urchin H. pulcherrimus, embryos were treated with rapamycin (500 or 2500 nM), or axitinib (500 or 100 nM) from the 2-cell stage. Based on the trials with a series of concentrations referring to previous studies^{21 (link),24 (link)}, we chose the highest concentration that did not result in general abnormal development.