Varioscan lux device

Primary tumor cells or GH3 cells were plated in 96‐well plates (10⁴cells/well). Cell proliferation/viability was measured using WST‐1 colorimetric assay (Roche) 48 h after treatment or 24 h, 48 h, 72 h after transfection according to the manufacturer’s recommendations.
Apoptosis assay was performed using Caspase‐Glo^® 3/7 Assay kit (Promega) to assess the activity of caspase‐3/7 48 h after treatment or 24 h, 48 h, 72 h after transfection. Absorbance and luminescence was measured using Thermo Scientific Varioscan LUX device.

Förster resonance energy transfer (FRET) was used to analyze the interaction between Hsp70 and HMGB1. For this purpose, purified human recombinant Hsp70 was labeled with Alexa488 fluorescent dye (Thermo Fisher Scientific, USA) and recombinant HMGB1 was labeled with Alexa555 fluorescent dye (Thermo Fisher Scientific, USA). Labeling procedure was produced according to manufacturer’s protocols. Experimental mixtures containing 50 µg/ml of Hsp70-488 and 10, 20 or 50 µg/ml of HMGB1-555 in 200 µL of TBS were incubated for 1 h, then the fluorescence emission spectrum of each sample was determined using a Varioscan LUX device (Thermo Fisher Scientific, USA). The fluorescence emission spectrum was detected from 515 to 620 nm at the excitation wavelength of 490 nm. Then fluorescence emission at 568 nm at the excitation wavelength of 490 nm was separately measured. For all data, the final fluorescent signals were obtained by subtracting the fluorescent signals with the background noise from а blank well (200 µL of TBS containing proteins without fluorescence labels). The experiments were repeated four times and the average value of fluorescence was taken at each specific condition.