Flat 96 well plates

Cells were harvested and seeded in 96-flat well plates (Corning) at a density of 5 x 10⁴ cells/mL overnight. Media was removed and 100 μL media with GW6471 or GW9662 at indicated concentrations was added to each well. Cell viability and toxicity were measured at 48 h. Cells were incubated with 50 μL of a 2 mg/mL solution of (3-(4,5-dimethyl-thiazole-2-yl)-2,5-biphenyl tetrazolium (MTT, Sigma-Aldrich) in PBS for 4 h and then exposed to 100 μL dimethyl sulfoxide (DMSO; Sigma-Aldrich). Cell viability was measured by absorption at 570 nm in a microplate spectrophotometer (Spectromax 250 plate reader). Results are shown as relative cell viability.

Cells were harvested and seeded in 96-flat well plates (Corning) at a density of 5 x 10⁴ cells/mL overnight. Media was removed and 100 μL media with geldanamycin at indicated concentrations was added to each well. Cell viability and toxicity were measured at 48 h. Cells were incubated with 50 μL of a 2 mg/mL solution of (3-(4,5-dimethyl-thiazole-2-yl)-2,5-biphenyl tetrazolium (MTT, Sigma-Aldrich) in PBS for 4 h and then exposed to 100 μL dimethyl sulfoxide (DMSO; Sigma-Aldrich). Cell viability was measured by absorption at 570 nm in a microplate spectrophotometer (Spectromax 250 plate reader). Results are shown as relative cell viability. MTT obtained values were normalized and concentrations transformed to logarithm, the nonlinear regression (curve fit) and IC₅₀ were calculated using a log(geldanamycin) versus response algorithm.

For assays in 2D culture, cells were seeded at predetermined optimal densities in 96-flat-well plates (Corning). For assays in 3D cultures, 1000 cells per well were seeded in Poly-HEMA-coated (0.6 mg per well) 96 round-well plates (Corning). In addition, cells were pelleted at 300 g for 3 min to facilitate more homogenous spheroid growth. Twenty-four hours post seed, compounds for treatments were added and incubated for another 96 h. XTT assay reagent (neoFroxx) was added, and after development, absorbance was read at 463 vs. 670 nm on a TECAN Infinite M1000 Pro reader. Data shown are expressed as percentage of metabolic activity compared to untreated or vehicle-treated cells, corrected by subtraction of blank values. Titration curves were fitted with a “log(inhibitor) vs. response—variable slope (four parameters)” fit (GraphPad Prism). Data were weighted by 1/Y² and the Hill slopes were constrained to >“−3”. Antiproliferative effects were compared by calculating the area under the curve with a 95% confidence interval. Nonoverlapping areas were considered different. Single concentration points were compared by the two-sided Student’s t test or two-sided ANOVA where applicable and corrected for multiple testing where due; see individual figure captions for detailed description of applied statistical tests.