Caspase activity assay
Coral fragments (size ~0.5 – 1.5cm) were dry-frozen and
kept at -80°C. Prior the assay, host and algal cells were separated as
follows. Frozen fragments were placed on ice in 1.5ml tubes with 250μl
of ice cold EB buffer (100mM Tris, 20mM EDTA, pH 8.0). Glass beads
(Sigma Aldrich, G1152) were added to the corals, each fragment was
vortexed in short pulses for 20s and then placed immediately back on
ice. Samples were centrifugated at 500g for 5min at 4°C. 50μl of host
cell containing supernatant was transferred to a new tube and lysed with
Cell Lysis Buffer according to instructions in
EnzChekTM Caspase-3 Assay Kit #1, Z-DEVD-AMC
substrate (ThermoFisher Scientific). Protein concentration was measured
with Qubit Protein Assay Kit (Life Technologies). 50μg of protein in
volume of 50μl or lower was used to measure caspase 3 activity following
the kit instructions. Fluorescence was measured with SpectraMax2
(Molecular devices) using SoftMax Pro v4.8 software. Caspase activity
was analyzed in 6 colonies for heat stress experiment, and in 4 colonies
for the venetoclax and colchicine experiments.