Validation of the specificity of Total CDK9 assay by siRNA experiments

HeLa cells were plated in 96-well plates (10,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, or CDK12, as well as with a negative control siRNA. After a 48h incubation, the cells were lyzed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total CDK9 detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #11) was also transferred into the microplate to monitor GAPDH level using the GAPDH Housekeeping Cellular Kit (64GAPDHPET/G/H) . HTRF signals for both kits were recorded after an overnight incubation.

Cell transfection with CDK9 siRNA led to a 68% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed with cells transfected with CDK1, CDK2, CDK4, CDK6, CDK7, and CDK12 siRNAs. The level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrate that the HTRF Total CDK9 is specific for the detection of CDK9 protein and does not cross-react with other CDK family members.

 

 

Assessment of total CDK9 levels in various cell lines

Various cell lines, either adherent HeLa, MCF7, SHSY5Y, NIH-3T3 (mouse) cells or suspension, such as K562 cells, were seeded at 50,000 cells / well in a 96-well microplate. After a 24H incubation, the cells were lyzed with supplemented lysis buffer, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total CDK9 detection reagents. The HTRF signal was recorded after an overnight incubation.

 

Total CDK9 degradation using CDK9 Protac

HeLa cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were then treated with Vehicle (DMSO), CDK9 Protac (Thal SNS-032), CDK9 Binder (SNS-032), CRBN Binder (Thalidomide), or an Irrelevant Protac at 1 µM for 4H at 37°C, 5% C02. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: 16 µL for Total CDK9 and an additional 4 µL (supplemented with 12 µL diluent #11) to check GAPDH levels using the HTRF GAPDH Housekeeping kit (64GAPDHPET/G/H). The corresponding kit detection reagents were added under 4 µL, and HTRF signals were recorded after an overnight incubation. The CDK9 Protac (Thal-SNS-032) induced CDK9 protein degradation with a 84% signal decrease compared to the cells treated with Vehicle (DMSO). No signal decrease was observed for cells treated with CDK9 Binder, CRBN Binder, or an Irrelevant Protac. The level of GAPDH measured remained unchanged under all the conditions tested (no cytotoxicity).

 

 

Total CDK9 degradation on HeLa and MCF7 cells

HeLa and MCF7 cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2.The cells were then treated with increasing concentrations of CDK9 Protac (Thal-SNS-032) for 4H at 37°C-5%CO2. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: 16 µL for Total CDK9 and an additional 4 µL (supplemented with 12 µL diluent #11) to check GAPDH levels using the HTRF GAPDH Housekeeping kit (64GAPDHPET/G/H). The corresponding kit detection reagents were added under 4 µL, and the HTRF signals were recorded after an overnight incubation.

As expected, the results obtained show a dose-response degradation of Total CDK9 in HeLa and MCF7 cells after treatment with CDK9 Protac (half maximal degradation of 166 nM and 60 nM respectively), while the level of GAPDH remained constant.

 

 

HTRF total CDK9 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total CDK9 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total CDK9 assay, 300 cells/well were enough to detect a significant signal, while 10,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF total CDK9 assay is 32 times more sensitive than the Western Blot technique.