HDAC11 target engagement assay development in cells - CETSA

<strong>Methods</strong> <strong>Permeabilized CETSA</strong> 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,<em> </em>Sigma<em>)</em> 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). <strong>Non-permeabilized CETSA</strong> 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).### 实验方法 #### 透化型细胞热位移测定（Permeabilized CETSA） 将HEK293T细胞以8×10⁵个/孔的密度接种于6孔板中，培养基为添加了10%胎牛血清（FBS）、100 U/mL青霉素与100 µg/mL链霉素的达尔伯克改良伊格尔培养基（DMEM）。接种4小时后，按照X-tremeGENE™ HP转染试剂的说明书操作，使用0.2 µg N端带有NanoLuc标签的HDAC11与1.8 µg空pcDNA3.1载体进行细胞转染。次日，使用胰蛋白酶-EDTA消化液收集细胞，重悬于预热的无酚红Opti-MEM培养基中，调整细胞浓度至1.2×10⁵个/mL。分别使用Opti-MEM与二甲基亚砜（DMSO）配制10倍储存液的洋地黄皂苷（digitonin，500 µg/mL，Sigma）与100倍储存液的蛋白酶抑制剂混合物（cOmplete™, Mini, EDTA-free, Sigma）。以该蛋白酶抑制剂/DMSO储存液作为溶剂对照，配制所有待测化合物的100倍储存液。将10倍洋地黄皂苷储存液与100倍待测化合物/溶剂对照储存液分别以10 µL/孔与1 µL/孔的体积预加入96孔PCR反应板中。随后向每孔加入89 µL细胞悬液，用封板胶膜密封反应板，于37℃孵育1小时。 在加热PCR板前即刻，将NanoBRET NanoGlo底物（Promega）以1:100的比例稀释于Opti-MEM培养基中，配制5倍底物储存液。将PCR热循环仪预热至设定温度后，放入反应板进行加热。对于解链温度（Tm）测定实验，设置复孔板，其中包含固定浓度（30 µM）的待测化合物与溶剂对照，采用覆盖37~68.5℃的11点温度梯度进行3分钟加热。热处理结束后，将反应板置于室温冷却3分钟，随后每孔加入25 µL 5倍底物储存液。为检测NanoLuc活性，将100 µL样品从PCR板转移至白色96孔板中，使用CLARIOstar多功能微孔板检测仪（BMG Labtech）在底物添加后即刻检测总发光强度。 #### 非透化型细胞热位移测定（Non-permeabilized CETSA） 将HEK293T细胞以8×10⁵个/孔的密度接种于6孔板中，培养基为添加了10%胎牛血清（FBS）、100 U/mL青霉素与100 µg/mL链霉素的达尔伯克改良伊格尔培养基（DMEM）。接种4小时后，按照X-tremeGENE™ HP转染试剂的说明书操作，使用0.2 µg N端带有NanoLuc标签的HDAC11与1.8 µg空pcDNA3.1载体进行细胞转染。次日，用待测化合物/DMSO处理细胞6小时。使用胰蛋白酶-EDTA消化液收集细胞，重悬于预热的无酚红Opti-MEM培养基中，调整细胞浓度至1×10⁵个/mL。随后将100 µL/孔的细胞悬液加入预加载的PCR反应板中。在加热PCR板前即刻，将NanoBRET NanoGlo底物（Promega）以1:100的比例稀释于Opti-MEM培养基中，配制5倍底物储存液。将PCR热循环仪预热至设定温度后，放入反应板进行加热。对于解链温度（Tm）测定实验，设置复孔板，其中包含固定浓度（30 µM）的待测化合物与溶剂对照，采用覆盖37~72℃的11点温度梯度进行3分钟加热。热处理结束后，将反应板置于室温冷却3分钟，随后每孔加入25 µL 5倍底物储存液。为检测NanoLuc活性，将100 µL样品从PCR板转移至白色96孔板中，使用CLARIOstar多功能微孔板检测仪（BMG Labtech）在底物添加后即刻检测总发光强度。