Methods Permeabilized CETSA HEK293T cells were seeded in 6-well plates (8 x 105 cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc-tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were harvested using Trypsin-EDTA, and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1.2 x 105 cells/ml. A 10x stock solution of digitonin (500 µg/ml, Sigma) and 100x stock solution of protease inhibitor cocktail (cOmplete™, Mini, EDTA-free, Sigma) was prepared in Opti-MEM and DMSO, respectively. The protease inhibitor / DMSO stock solution was used as vehicle to prepare 100x stock solutions of all test compounds. The 10x digitonin stock solution and 100x test compound / vehicle stock solution were pre-dispensed into 96-well PCR reaction plates at 10 µl/well and 1 µl/well, respectively. Next, 89 µl/well of the cell suspension was added to the pre-loaded PCR plates. Plates were then covered with adhesive plate sealing tape and were incubated at 37°C for 1 h. Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-68.5°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25 µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech). Non-permeabilized CETSA HEK293T cells were seeded in 6 well plates (8 x 105 cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were tretaed with compounds/DMSO for 6 h. Cells were harvested using Trypsin-EDTA and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1 x 105 cells/ml. Next, 100 µl/well of the cell suspension was added to the pre-loaded PCR plates. Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-72°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25 µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech).