D12345

All preparation steps were performed in the dark. Stock solutions of hypericin (PhytoLab GmbH & Co.KG) in DMSO (Molecular Probes™ D12345) with concentrations of 0.1 mM, 0.25 mM, 0.125 mM, 0.05 mM, and 0.005 mM were prepared and frozen at − 20 °C. Aliquots of this stock were freshly thawed for each experiment. After thawing, a 1:100 dilution of hypericin (1 µM, 2.5 µM, 1.25 µM, 0.5 µM, 0.05 µM) in complete cultivation medium and additional 10% FBS was prepared. This procedure was adopted from [15 (link)]. Excess medium was removed from each single well and the hypericin/MEM mixture was added at a final concentration of 1 µM for incubation time experiments. Spheroids were incubated for 5 min, 30 min, 125 min, 10 h + 30 min, and 35 h + 15 min in the hypericin cultivation medium, respectively. Varying hypericin concentrations (2.5 µM, 1.25 µM, 0.5 µM, 0.05 µM) were incubated for a predefined time period of 30 min. Non-hypericin-incubated control spheroids were treated with a 1:100 dilution of DMSO in complete cultivation medium and additional 10% FBS for 30 min. Afterwards, the hypericin and control incubation were disrupted by removing the incubation medium and applying three washing steps with phosphate buffered saline (PBS). Following the washing steps, U-87 MG spheroids were directly preserved by fixation. All experiments were executed in triplicate.

Fluo-4 AM NW dye and probenecid (Molecular Probes, 36206) were prepared according to manufacturer’s instructions. The dye was further supplemented to a final 4 mM CaCl₂ concentration. Yoda1 was prepared as 10 mM stock in 100% DMSO (Molecular Probes, D12345). 24 h after HEK293T^ΔP1 cells were seeded in 96-well plates (Corning, 3603) and transfected with Piezo1-myc, S260R-myc, or S2211L-myc constructs. Media was aspirated to reduce background and adherent cells were loaded with dye/probenecid solution for 40 min at 37 °C and 8% CO₂. On loading, baseline fluorescence was measured for 5 min, and activation by 20 µM Yoda-1 (in 0.2% DMSO) or 0.2% DMSO alone was measured for 18 min. Maximal calcium signal was determined by addition of A23187 ionophore (Millipore Sigma, C7522) and assayed for 25 min. All Relative Fluorescence Unit (RFU) measurements were performed on a BioTek Synergy H1 at 37 °C with excitation 488 nm and emission 518 nm, and 70 s between reads. Normalized RFU = (signal RFU - baseline RFU) / (maximal post-A23187 - baseline RFU). Signal RFU = mean well RFU at t = 24 min, baseline RFU = mean well RFU at t = 5 min, maximal post-A23187 = mean signal at t = 52 min. Data point represents n = 9 to 15 normalized wells. Each well is plated, transfected, treated, and assayed independently, and as such, represents individual experiments.

The stocks were prepared weighing 3–6 mg of crude extract into a micro centrifuge tube. The crude extract was dissolved in DMSO (dimethyl sulfoxide anhydrous from Life technologies cat # D12345) to reach a 60 mg/mL final concentration and stored at −20 °C. For the assay, a different amount of stock material (from 1 to 20 µL) was added to 21 mL of medium to make a working extract. Different concentrations were tested for cell/tissue toxicity and one concentration was chosen for further work (data not shown). Specifically, to obtain “low” (0.007 mg/mL) and “high” (0.015 mg/mL) functional concentrations, 2.45 µL or 5.25 µL of stock extract (60 mg/mL) were added to 21 mL of medium, respectively. The final concentration of DMSO was 0.012% and 0.025% in the “low” and “high” extract concentrations, respectively. The extracts were sterilized using a 0.22 µm filter.