96 well plate

Stress granules experiments were performed as previously described (38 , 59 (link)). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in the presence of penicillin and streptomycin (100 μg/ml) (GIBCO Life Technologies). Cell cultures were maintained at 37 °C and 5% of CO₂ in an incubator. HeLa Cells were plated in 96-well plate (PerkinElmer). The plasmids were designed to express, in mammalian cells, FUS and HUR proteins with a GFP tag and TDP-43 WT or mutants, FUS, HUR, G3BP1, and YB1 bearing an HA tag peptide on N terminus. For transfection, cells were incubated in the presence of 0.3 μg of the plasmid followed by the addition of lipofectamine 2000 reagent (0.2 μl/sample).

C2C12 myoblasts were seeded on a 96 well plate (Perkin Elmer). After differentiation, they were preincubated with 20 μM U73122 for 1 h. Ca²⁺ release was measured using the fluorescent calcium indicator Fluo-4AM (Molecular Probes) according to the manufacturer's suggestions. Ca²⁺ increases are reported as ΔF/F ((F-Fbasal)/Fbasal), where F indicates fluorescence.

Ca²⁺ measurements were taken by co-transfecting H1299 or HeLa cells in a six-well plate with 1 μg cytosolic (cytAEQ) aequorin along with 5 nM of either scramble siRNA or siRNA against Sorcin, using Lipofectamine 3000. Twenty-four hours post-transfection, cells were re-plated into a 96-well plate (PerkinElmer). Forty-eight hours post-transfection, CytAEQ was reconstituted by incubating cells for 1.5 h with 5 µM coelenterazine wt (Santa Cruz Biotech) in modified Krebs Ringer Buffer (KRB: 125 mM NaCl, 5 mM KCl, 400 mM KH₂PO₄, 1 mM MgSO₄, 20 mM Hepes, pH 7.4) supplemented with 0.1% glucose at 37 °C. Luminescence measurements were taken using a PerkinElmer EnVision plate reader equipped with two injector units. After reconstitution, cells were washed with KRB (either with 1 mM Ca²⁺ or 0.5 mM Ca²⁺, depending on the experiment) and luminescence from each well was measured for 1/1.5 min. According to the experiment, 100 μM Histamine (Sigma) or (Carbachol 500 μM + His 100 μM + ATP 100 μM + Bradykinin 100 nM) or (100 ng/mL EGF + CPA 20 μM + EGTA 2 mM) (final concentrations in the wells) was injected to generate Ca²⁺ transients, and then a hypotonic, Ca²⁺-rich, digitonin-containing solution was added to discharge the remaining aequorin pool. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.

siRNA transient transfection was performed using jetPRIME transfection reagent (Polyplus) following the manufacturer’s protocol. For siRNA treatment, B38 cells were seeded in a 96-well plate (PerkinElmer) at 2 × 10⁴ cells/well 1 d before transfection. CFTR Human siRNA Oligo Duplexes (Origene) variants in 10 and 50 nM concentrations were used for CFTR knockdown. Rab5 (RAB5A) and RAB11 Human siRNA Oligo Duplex (Origene) were used in 10 and 50 nM concentration for RAB5/11 knockdown. Transfection with a Universal Scrambled Negative Control siRNA (Origene) was used as a control. Cells were transfected for 48 or 72 h, followed by lytic and extracellular assay detection of CFTR levels. To exclude siRNA cell cytotoxicity an MTS assay was performed with a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer’s protocol. The relative cell viability (%) was calculated as (A_sample − A_background)/(A_control − A_background) × 100%. The absorbance from the corresponding cell-free conditions was used as background.

The fibril formation of Aβ(1–42) and its inhibition by the IDPs and the polymers were monitored by fluorescence enhancement upon ThT binding to amyloid. The fluorescence was recorded with an EnSpire Multimode Plate Reader (PerkinElmer, Boston, MA USA) with excitation and emission wavelengths set at 440 and 484 nm, respectively. The lyophilized Aβ(1–42) was dissolved in dimethyl sulfoxide (DMSO), immediately diluted in 0.5 × PBS, and then added to the IDP solution (in 0.5 × PBS) containing 1 mM ThT. The samples were added to a 96 well plate (PerkinElmer) and incubated at 37℃ without rotation. The ThT and protein concentrations were 20 μM and 25 μM, respectively. The fluorescence intensity was measured from 0 to 72 h.

We adjusted the cell density to 5 × 10⁴ cells/mL per well and added 150 μL per well to a 96-well plate (PerkinElmer, Waltham, MA, USA) and then placed them in an incubator for 24 h. We set seven groups of concentration gradients for the target compound, which were 160, 80, 40, 20, 10, 5, and 0 μM, and set five replicates for each concentration. After 48 h of cell treatment at different working concentrations, 90 uL of culture medium and 10 uL of MTT solution (Solabio, Beijing, China) were added to each well and incubated for 4 h in the dark. After aspirating the supernatant, 110 microliters of dimethyl sulfoxide solution were added to each well. The solution was dissolved by shaking on a shaker at low speed for 10 min, and the absorbance (OD) of each hole was measured at 490 nm. The absorbance (OD) of each well was measured at 490 nm, and the cell viability was calculated according to the formula.