Microplate reader

Cell counting kit-8 (Thermo, USA), colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) experiment were established to determine the viability and proliferative ability of cells. For CCK-8 assay, cells were seeded in 96-well plates, followed by culture for 24, 48, or 72 hours. CCK-8 reagent (15 μL) was then added into each well to incubate for 1 hour, the absorbance at optical density (OD) 450 nm were evaluated by a microplate reader (BD Biosciences, USA).
To establish colony formation assay, A549 and H1975 cells (500 cells per well) were planted in 12-well plates and placed in 37°C incubator for two weeks. Medium were changed every three days. The colonies were fixed with methanol and dyed by 0.2% crystal violet for 15 minutes. Images of whole well were taken by a camera.
For EdU evaluation of DNA replication, cells were fixed in 4% PFA and permeabilized with Triton X-100, then incubated with EdU reagent (50 μM, RiboBio) for 3 hours. Nuclei were labelled with Hoechst 33342. Five random areas were captured by a confocal microscope.
Cell apoptosis was evaluated by flow cytometry by using FITC-Annexin V/PI detection kit (Beyotime, China) following the manufacturer’s protocol. In short, cells were collected and stained with FITC-Annexin V (5 μL) and PI (5 μL) in dark for 15 minutes, followed by detection on a Calibur C6 system.

Wild-type SIRT6 protein was purified as previously described methods³¹ (link). The full-length human SIRT6 was inserted into the pET28a-LIC vector, and the plasmids were transformed into Escherichia coli BL21 (DE3) cells. Protein was purified with a nickel column and gel filtration. Subsequently, the purified SIRT6 protein was dialyzed into the assay buffer (50 mmol/L Tris-HCl [pH 8.0], 137 mmol/L NaCl, 2.7 mmol/L KCl, and 1 mmol/L MgCl₂) and utilized in FDL assays. Wild-type SIRT6 protein (5 μmol/L) was incubated in a 50 μL of reaction mixture (DMSO/MDL-800/MDL-811, 2.5 mmol/L NAD⁺, 75 μmol/L RHKK-Ac-AMC, and assay buffer) at 37 °C for 2 h. Nicotinamide (40 mmol/L) was used as stop solution and the reaction system was continuously incubated with 6 mg/mL of trypsin at 25 °C for 30 min. Fluorescence intensity was assessed with a microplate reader (BD, Franklin Lakes, NJ, USA) at excitation and emission wavelengths of 360 and 460 nm respectively. EC₅₀ values were calculated by fitting the data points with the dose–response variable slope (four parameters) function in GraphPad Prism 5.

The SMC1A-KD and control cells were seeded into 96-well plates at 2 × 10³ cells/well and cultured for 24 h or 96 h at 37℃ in a humidified atmosphere containing 5% carbon dioxide. They were then incubated with a final concentration of 10 µM 5-bromo-2-deoxyuridine (BrdU; BD Biosciences, San Jose, CA, USA) for 4 h. Cells were then fixed in 1% paraformaldehyde for 15 min and labelled with peroxidase-conjugated antiBrdU antibody (dilution 1 : 1000; Millipore) at 37℃ for 1 h. Next, peroxidase substrate (50 µmol/l tetramethylbenzidine) was added and incubated at room temperature for 15 min. The absorbance values were measured at 450 nm using a microplate reader (BD Biosciences).