Clear bottom 96 well plate

E. coli cells were grown on a clear-bottom 96-well plate (Nunc) with a final volume of 150 μl of solution in each well. The plates were loaded into an Infinite 200Pro fluorescence plate reader (Tecan, Männedorf, Switzerland) and incubated at 30 °C in the presence of various concentrations of A22 drug (0.4–12 µg/ml). Agitated samples were shaken with orbital agitation (2.5 mm amplitude) for a period of ~5–15 h. Cell density was measured at 600 nm at 15 min intervals, measured in biological triplicates.

All samples were repeated at least in biological duplicates.
Bacterial growth experiments in bulk. E.coli cells were grown on a clear-bottom 96-well plate (Nunc) with a final volume of 150 μl of the solution in each well. The plates were loaded into an Infinite 200Pro fluorescence plate reader (Tecan, Männedorf, Switzerland) and incubated at 30 °C in the presence of various concentrations of Novobiocin drug (25, 50, and 100 µg/ml). Samples were shaken with orbital agitation (2.5 mm amplitude) for a period of ~12 h. The cell density was measured at 600 nm with 15 min intervals, measured in biological triplicates.

Recombinant MoPrP(23–230) was generated as described previously for recombinant Syrian hamster PrP (34 (link)). ScCAD5 cells were homogenized as described above, and then protein levels in homogenates quantified using the BCA assay. RT-QuIC was carried out as described previously, with conditions optimized for detection using MoPrP as the substrate (89 ). Briefly, RT-QuIC substrate mixture was prepared as follows: 0.1 mg/ml (4 μM) recombinant MoPrP(23–230), PBS (130 mM NaCl, 10 mM sodium phosphate pH 7.3), 10 μM ThT, and 1 mM EDTA. Prior to addition to the reaction mixture, recombinant MoPrP was centrifuged at 21,000g for 15 min to remove any preexisting aggregates. A tenfold molar excess (40 μM) of either G418 or puromycin was added where indicated. Ninety eight microliters of this reaction mixture was added to each well of a black, clear bottom 96-well plate (Nunc). Then, 2 μl of the seed mixture [PBS containing 0.05% (w/v) SDS, 1× N-2 Supplement (ThermoFisher), and 10 ng of ScCAD5 homogenate] was added to each well. The plate was sealed and then incubated at 42 °C in a BMG CLARIOstar microplate reader. The plate was subjected to alternating cycles of 1 min shaking (700 rpm, double orbital) and 1 min rest. ThT fluorescence (excitation: 444 ± 5 nm; emission: 485 ± 5 nm) was measured every cycle (every 2 min) for up to 38 h total using a gain setting of 1600.

HTLA cells
were a gift from the laboratory of G. Barnea and were maintained in
DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin
and 100 μg/mL streptomycin, 2 μg/mL puromycin, 100 μg/mL
hygromycin B, and 100 μg/mL G418, in a humidified atmosphere
at 37 °C in 5% CO². On day 1, cells were plated at
a density of 1x10⁵ cells/cm² in a black wall,
clear bottom 96 well plate (Nunc). On the following day (day 2), cells
were transfected with a 10× solution of 3:1 mixture of HTR2C-TANGO
(Addgene #66411):Optifect Transfection Reagent (Thermo) in un-supplemented
DMEM. On day 3, 1× drug stimulation solutions were prepared in
filter-sterilized unupplemented DMEM. The transfection media was shaken
or aspirated from the wells, and drug stimulation solutions were gently
added. On day 4, drug solutions were removed from one well every 10
s (to maintain consistency of incubation time) and 50 μL per
well of Bright-Glo solution (Promega) diluted 20-fold in HBSS was
added. After incubation for 2 min at room temperature, luminescence
was counted with an integration time of 10 s in a Spectramax i3×
plate reader (Molecular Devices). Drug concentrations were experimentally
measured in triplicate. Statistical analysis was performed using GraphPad
Prism 9.

Purified 6xHisLip-tagged VIN3_VEL (residues 500–603) was diluted to a final concentration of 15 mg/mL in a 100 μL reaction volume in a clear bottom 96-well plate (Nunc). TEV protease and DTT were mixed bubble-free in reaction buffer (200 mM NaCl, 25 mM Tris pH7.4, 0.06% NaN₃, 5 mM DTT and TEV protease in a 1:50 ratio, TEV:protein). Measurements were done in a Pherastar (BMG Labtech) at 30°C and 330 nm (20 flashes per cycle) in 2 min intervals with 20 s shaking (250 rpm, 3 mm double orbital) prior to measurement. Values were subtracted against a blank well and analyzed in Prism8 (Graphpad). Experiments were performed four times from which SEM values were calculated.

For live cell membrane protein labelling, Piezo1^−/− HEK293T cells were plated on 96 well clear bottom plate (ThermoFisher) coated with 0.1 mg/ml of Poly-L-Lysine (Sigma). Cells were transfected with pIRES-GFP constructs carrying WT or mutant Piezo1 (125 ng cDNA per well) with PEI. Sixty to 72 h after transfection, live labelling was performed by incubating the cells with anti-HA (Sigma, 1:100) antibody for 20 min at 37°C. The cells were then washed with DMEM six times before being incubated with Alexa Fluor 555 anti-mouse secondary antibody (1:200) for 15 min at room temperature (22°C). Cells were washed again 5 times with DMEM and twice with phosphate buffered saline (PBS), then fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. PFA was washed off and replaced with PBS before confocal analysis.
For co-labelling of Piezo1 and the endoplasmic reticulum (ER), HeLa cells were transfected with WT or mutant Piezo1 fused with GFP. After transfection for 60–72 h, cells were washed with PBS twice then incubated with 1 μM of ER-Tracker Red dye (Invitrogen) for 20 min at 37°C. The cells were washed again with PBS three times and fixed with 4% PFA for 20 min at room temperature. PFA was then replaced with PBS, and the ER or Piezo1-GFP signals were visualized using confocal microscopy (Zeiss LSM 700 inverted).