Specificity of HTRF Total SMARCA2 assay using relevant cell lines

SMARCA2 expression levels were assessed with the HTRF total SMARCA2 kit in A549 (lung carcinoma) and in HAP1 cells (WT). In parallel, the expression of SMARCA4 was assessed with HTRF total SMARCA4 kit. Cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The different cell lines were plated in 96-well culture plates at a density of 200,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking. Then 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

Low expression of SMARCA2 was detected in A549 cells, whereas SMARCA4 expression was equivalent to the non-specific signal (dotted line), indicating a lack of expression of SMARCA4, as described in the literature (Hoffman, G. R. et al. Proc. Natl Acad. Sci. 2014).

Furthermore, the expression levels of SMARCA2 in HAP1 cells were equivalent to the non-specific signal (dotted line), indicating a lack of expression of SMARCA2, whereas SMARCA4 levels were well detected in the HAP1, as expected.

These results demonstrate the specificity of the HTRF SMARCA2 kit over SMARCA4, despite these two proteins sharing more than 70% sequence homology.

Catalog cell line references (Horizon Discovery): HAP1 Wt #C631.

Validation on various Human cell lines

The adherent Human cell lines HeLa (Cervix), HEK293 (Kidney), and A549 (Lung Carcinoma) were plated in 96-well culture plates at a density of 200,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking.

The suspension Human cell lines HEL92.1.7 (erythroleukemia) and MOLT-4 (T lymphoblastic leukemia) were dispensed under 30 µL in a 96-well plate at a density of 200,000 cells/well, incubated for 1h at 37°C, 5% CO2, and lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30 min at RT under gentle shaking.

The SMARCA2 expression level was assessed with the HTRF Total SMARCA2 kit. Briefly, 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed HTRF detection reagents. The HTRF signal was recorded after an overnight incubation at RT. The dotted line corresponds to the non-specific HTRF signal. Note that the cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The HTRF Total SMARCA2 assay efficiently detects endogenous SMARCA2 in various cellular models expressing different levels of the protein.

PROTAC® induced SMARCA2 degradation
 

HEL92.1.7 cells were seeded in a 96-well plate (100,000 cells/30µL well) and treated with increasing concentrations of PROTAC® compounds ACBI1 and AU-15330, as well as a PROTAC ARV-471 (PROTAC® Estrogen Receptor) used as a negative irrelevant control. After overnight incubation, cells were lysed with 10 µl of supplemented lysis buffer #1 (4X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF total SMARCA2 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cell treatment with the two SMARCA2 PROTAC® degraders triggered a dose-dependent decrease in SMARCA2 protein, while the expression levels of SMARCA2 in the ARV-471 control conditions as well as the ATP levels remained stable. These results suggest a PROTAC® induced specific SMARCA2 degradation, with a DC50* in the nM range, as expected (Farnabay, W. et al. Nat Chem Biol. 2019 and Xiao, L. et al. Nature 2022).

* DC50 corresponds to the concentration of the degrader at which 50% of the targeted protein is degraded.

PROTAC® SMARCA2 versus SMARCA4 profiling

HEL92.1.7 cells were seeded in a 96-well plate (100,000 cells/ 30µL well) and treated with increasing concentrations of  PROTAC® compounds ACBI1 and AU-15330. After overnight incubation, cells were lysed with 10 µl of supplemented lysis buffer #1 (4X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF detection antibodies were added, using Total SMARCA2 or Total SMARCA4 assay reagents. The HTRF signal was recorded after an overnight incubation

The results indicate an ACBI1 and AU-15330 induced dose-dependent SMARCA2 and SMARCA4 decrease. ATP levels remained stable in the same conditions (data not shown). These results are consistent with studies from Farnabay, W. et al. (Nat Chem Biol. 2019) and Xiao, L. et al. (Nature 2022).

HTRF total SMARCA2 assay compared to Western Blot

HEL92.1.7 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 24h incubation followed by cell medium removal, 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#1, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total SMARCA2 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

 

In these conditions, the HTRF total SMARCA2 assay was as sensitive as the Western Blot technique.