Janus workstation pin tool

Cells were plated at 1000 cells/well in 50 µL/well of media in 384 well white culture plates (Thermo). NMC797 cells were allowed to adhere overnight before adding 100 nL of compound in DMSO from compound stock plates using a Janus Workstation pin tool (PerkinElmer). After addition of compound, plates were incubated for 72 hours at 37°C. Cell viability was read out using the ATPlite kit (PerkinElmer). Plates were brought to room temperature prior to reagent addition. Lyophilized powder was resuspended in lysis buffer and diluted 1:2 with DI water. 20 µL of this solution was added to each well and plates were incubated for 15 min at room temperature before signal was read on an Envision 2104 plate reader (Perkin Elmer).

293T and NIH/3T3 cells were plated at 1000 cells per well in 50 µL of appropriate media in 384-well white culture plates (Thermo Fisher Scientific). Cells were allowed to adhere overnight before adding 100 nL of compound in DMSO from compound stock plates using a Janus Workstation pin tool (PerkinElmer). After addition of compound, plates were incubated for 72 hours at 37 ºC, and ATPlite 1 Step Luminescence Assay System (PerkinElmer) was used to determine ATP-dependent luminescence as an approximation of cellular viability. Plates were brought to room temperature prior to reagent addition. Lyophilized powder was resuspended in lysis buffer and diluted 1:3 with deionized water. 20 µL of this solution was added to each well, plates were incubated for 15 minutes at room temperature, and luminescence was measured on an Envision 2104 plate reader (PerkinElmer). Cell viability values were calculated by normalizing to DMSO treated wells for each cell line. Data were analyzed and plotted using GraphPad PRISM v6 and the ‘log(inhibitor) vs. response -- Variable slope (four parameters)’ analysis module.

A clonal 293T line stably transfected with pCMV-IRES-RenillaLUC-IRES-IKZF1-FireflyLUC reporter plasmid^{18 (link)} (a kind gift from W. Kaelin, Dana-Farber Cancer Institute) was used to evaluate compound activity on IKZF1-Fluc fusion protein levels. Cells were plated at 2000 cells per well in 50 µL of appropriate media in 384-well white culture plates (Thermo Fisher Scientific). Cells were allowed to adhere overnight before adding 100 nL of compound in DMSO from compound stock plates using a Janus Workstation pin tool (PerkinElmer). After addition of compound, plates were incubated for 24 hours at 37 ºC, prior to luminescence assessment. Fluc and Rluc detection was performed as described above for the FKBP12^WT and FKBP12^F36V dual luciferase assay (with Rluc activity being measured using Nluc buffer). Fluc values were normalized by dividing by control Rluc values. Fluc/Rluc ratios were then normalized by setting DMSO only wells to 100%. Data were analyzed and plotted using GraphPad PRISM v6 and IC₅₀ values were determined using the ‘log(inhibitor) vs. response -- Variable slope (four parameters)’ analysis module.