Element software

Forster resonance energy transfer (FRET) analysis of cAMP production was performed in HEK293 cells transiently transfected with plasmids encoding PTH1R-WT or mutant, and the Epac1^CFP/YFP FRET-based biosensor of intracellular cAMP^{68 (link)}. Cells plated on poly-D-lysine–coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies); maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl, and 1 mM CaCl2, as well as 0.1% bovine serum albumin (BSA) (pH 7.4); and transferred on an in-verted Nikon Ti-E equipped with an oil immersion 40× numerical aperture (NA) 1.30 Plan Apo objective and a moving stage (Nikon Corporation). Recordings were performed at room temperature in single cells upon perfusion of agonist ligand or buffer for ~2 min (indicated by horizontal bar in graph) and during a subsequent washout period. The CFP and YFP fluorescent groups were excited with 440- and 514-nm lasers (Melles Griot), respectively. Fluorescence data were extracted using Nikon Element Software (Nikon Corpo- ration), and calculated as corrected CFP/YFP fluorescent ratios^{68 (link)}. Data were normalized to the FRET response induced by forskolin (10 μM) added at the end of test period.

RPE1 cells stably expressing Histone H2B-RFP or MDA-MB-231 cells stably expressing Histone H2B-mCherry were plated on 4-chaamber 35 mm glass bottom dish at least one day prior to do imaging (#1.5 glass, Cellvis). Cells were treated with DMSO (control), PTX (10 nM), or ERI (10nM) 1 hour prior to imaging. High-temporal live-cell imaging was performed using a Nikon Ti2 inverted microscope equipped with a Hamamatsu Fusion camera, spectra-X LED light source (Lumencor), Shiraito PureBox (TokaiHit) and a Plan Apo 20x objective (NA = 0.75) controlled by Nikon Element software and Metamorph (Molecular Devices). Cells were recorded at 37°C with 5% CO2 in a stage-top incubator using the feedback control to maintain the growing media’s temperature (Tokai Hit, STX model). Image analysis was performed using Nikon Element software. Mitotic stages were determined by nuclear staining. The mitotic duration was measured from nuclear envelope breakdown (NEBD) to anaphase onset. Incidences of multi-nuclei, mitotic slippages and unaligned chromosome were analyzed. The experiments were independently repeated 2–3 times for mitotic duration measurements (total of n = 100), and P-values between variants were calculated by One-Way Anova and two-tailed t-test. P-values < 0.05 were considered significant.

The mice were fasted for 24 h (with as libitum access to water) in metabolic cages and then were administered aspirin (200 mg/kg body weight), 6G + ASA (6G was given to mice in the same molar dose of aspirin), and GAS (GAS was given to mice in the same molar dose of aspirin) suspensions by oral gavage. At 4 h after the administration of test compounds, the mice were sacrificed by euthanization with isofluorane and then blood was collected from the heart. After sacrifice, mouse stomachs were removed and cut open along the greater curvature. The gastric contents were emptied, and the stomachs were then washed with PBS. Gastric mucosal lesions were observed under microscope at 10 × magnification and the size of the all lesions were measured and estimated manually. The inner surface was photographed by Nikon stereoscopic zoom microscope (SMZ1500) with DXM 1200 C digital camera system (0.7 × Relay lens) under Nikon Element Software (Nikon Corporation, Kanagawa, Japan). Next the gastric mucosal tissues were removed, frozen in liquid nitrogen and stored at −80 °C.