Sc 32245

Aortas were embedded in paraffin and then sectioned at a thickness of 5 μm. The sections were dewaxed and rehydrated before immunofluorescence staining. VSMCs were then incubated on round coverslips in 24-well plates. When cells grew to 50% of the total area of the coverslip, they were fixed in 4% paraformaldehyde for 10 min and then rinsed with PBS 3 times before immunofluorescence staining. Aortic sections or VSMCs coverslips were blocked with 5% normal goat serum in PBS for 30 min at room temperature and incubated overnight with primary antibodies at 4 °C. To detect the LKB1 expression level in VSMCs of aortas, fluorescent double labeling of aortic sections was performed with anti-LKB1 antibody (1:100, sc-32245, Santa Cruz) and anti-α-SMA antibody (1:200, ab5694, Abcam). To evaluate the co-localization of LKB1 and SIRT6, fluorescent double labeling of VSMCs coverslips was performed using anti-LKB1 and anti-SIRT6 antibodies (1:100, #12486, Cell Signaling Technology). After washing, sections or coverslips were incubated with secondary antibodies at 37 °C for 30 min and then stained with Mounting Medium with DAPI (ab104139, Abcam).

The adenocarcinoma cell line A549 was purchased from the American Type Culture Collection. Cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium purchased from Hyclone and supplemented with 10% fetal bovine serum; cells were grown at 37 °C in a humidified atmosphere with 5% CO₂. Mouse monoclonal antibodies against LKB1 (sc-32245) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-166545) were purchased from Santa Cruz Biotechnology. A549 cells, a cell line with endogenous LKB1 deficiency, were transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-MCS-3Flag (control) or pLenti-EF1a-mcherry-P2A-Puro-CMV-stk11 stable plasmids. The cells were then subjected to puromycin selection (4 ng/μl) for 2 weeks, after which we collected puromycin-resistant stable clones. The expression of LKB1 in established stable transfected A549 cells was validated by Western blot. We also transfected the LKB1 (K78I) kinase-dead mutant plasmid into the A549 cell line by means of transient transfection and used the A549 cell line transfected with PCDNA3.0 vector plasmid as a control. We verified the expression of LKB1 by western blot.

Serial cross-sections (10 μm thick) from each frozen left GC muscle were transversally cut on a cryostat microtome set at −20°C (HM 525 NX, Thermo Fisher Scientific) and mounted on slides (SuperFrost^® Plus, Menzel Gläser, Thermo Fisher Scientific). 2B4 and SF1 cells were grown on plates at seeding density of 2.5×10⁴ for 11 days. Tissue sections and cells were fixed in paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% TWEEN 20 (Sigma-Aldrich) and 0.1% bovine serum albumin (Sigma-Aldrich) in PBS for 45 min at RT. After three washes with PBS, sections and cells were blocked in saturation buffer (0.1% bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer. After three washes in PBS, sections and cells were incubated with 488/594 Alexa Fluor-conjugated secondary antibodies (1:1000; A21206 and A32744, Thermo Fisher Scientific) for 45 min at RT. Nuclei were stained with PureBlu^TM Hoechst 33342 Nuclear Staining Dye (1:50; Bio-Rad). Images were captured using a dark-field microscope (CL-I Eclipse Nikon) at 20× magnification.