Validation of HTRF total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors

Human U-87 MG cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014, an mTOR inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature. As expected, AZD2014, a potent mTOR inhibitor, induced autophagy activation, leading to a dose-dependent increase in ATG16L1 phosphorylation on Serine 278 without any significant effect on the expression level of the total ATG16L1 protein. Validation of HTRF Total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors Human U-87 MG cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of AZD2014, an mTOR inhibitor, and a dose-response of ULK-101, an autophagy kinase ULK1/2 inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature. As expected, ULK-101, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed autophagy activation induced by AZD2014, leading to a dose-dependent decrease in ATG16L1 phosphorylation on Serine 278, without any significant effect on the expression level of the ATG16L1 total protein.

 

Specificity of HTRF Total ATG16 assay using HAP1 ATG16 knock-out cells

The human total ATG16L1 expression level was assessed using the HTRF Total kit in Wild Type (WT) and ATG16 knock-out (KO) HAP1 cells. The cell lines were cultured in flasks and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. In parallel, GAPDH level was monitor using the GAPDH Housekeeping Cellular Kit (# 64GAPDHPET/G/H). 4 µL of cell lysate were prediluted with 180 µL of diluent #11. Then, 16 µL of this diluted material were mixed with 4 µL of the HTRF GAPDH Housekeeping Cellular detection reagents. HTRF signal for both kits was recorded after an overnight incubation at room temperature. The measured level of GAPDH was equivalent under both tested conditions. In contrast, while the HTRF Total ATG16L1 signal was significantly positive in HAP1 WT cells, it was dramatically decreased (equivalent to the negative signal) in HAP1 ATG16 KO cells, demonstrating the specificity of the HTRF Total ATG16L1 assay for the detection of the ATG16L1 protein. HAP1 ATG16 KO (10bp deletion) cell line from Horizon Discovery # HZGHC000296c010 HAP1 WT cell line from Horizon Discovery # C631

 

Versatility of HTRF Total ATG16L1 assay in cell lines from multiple species

Human (PC-3), mouse (C2C12) and rat (H4-II-E) cell lines were seeded at 100,000 cells/well in a 96-well plate. After a 24h incubation, the cells were lysed with 25 µL of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. The HTRF Total ATG16L1 assay efficiently displayed significant positive signals in the cell lines from the 3 species tested, demonstrating its capability to detect human, mouse and rat versions of the protein, and its suitability for translational research.

 

HTRF Total ATG16L1 Detection Kit compared to Western Blot

HCT116 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then, the cells were treated with 100 nM of Calyculin A for 10 min. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side-by-side comparison of WB and HTRF. The HTRF Total ATG16L1 assay is 4 times more sensitive than Western Blot assay.