Ab30394

Integrin α2 and β1 blocking experiment (Fig. 6E-G) was performed by culturing SN organoids in CellCarrier-96 Ultra Microplate. 50 μl of Matrigel containing organoids was seeded in each well. Organoids recovered from cell seeding were treated with 10 ng/ml recombinant rabbit integrin α2 (Abcam, ab181548) and 10 ng/ml mouse IgG1 integrin β1 (Abcam, ab30394) or 10 ng/ml mouse monoclonal 2C11 IgG1 isotype control antibody (Abcam, ab1927) for 6 days with culture medium and antibodies being replenished every 2 days. Organoids were fixed in situ and stained for F-actin (488 ReadyProbes Reagent, ThermoFisher Scientific, R37110) according to the manufacturer's instructions.

Human Foreskin Fibroblasts (HFF) were purchased from ATCC and cultured in DMEM media (Mediatech) supplemented with 10% Fetal Bovine Serum (Corning), 2 mM L-glutamine (Invitrogen) and penicillin-streptomycin (Invitrogen). HFFs were plated on glass coverslips incubated with 10 μg/mL fibronectin (EMD Millipore) for 1 hr at room temperature. Cells were fixed 1 hr after plating by rinsing them in cytoskeleton buffer (10 mM MES, 3 mM MgCl₂, 1.38 M KCl and 20 mM EGTA) and then fixed, blocked and permeabilized in 4% PFA (Electron Microscopy Sciences), 1.5% BSA (Fisher Scientific), and 0.5% Triton X-100 (Fisher Scientific) in cytoskeleton buffer at 37° for 10 minutes. Coverslips were subsequently rinsed three times in PBS and incubated with either a β₁ antibody (1:100; Abcam product #:ab30394) or β₃ antibody (1:100; Abcam product #:ab7166) followed by AlexaFluor 488 phalloidin (1:1000; Invitrogen) and a AlexaFluor647 donkey anti-mouse secondary antibody (1:200; Invitrogen).
Cells were imaged using a 1.2 NA 60X Plan Apo water immersion lens on an inverted Nikon Ti-Eclipse microscope using an Andor Dragonfly spinning disk confocal system and a Zyla 4.2 sCMOS camera. The microscope was controlled using Andor’s Fusion software.

hTERT-RPE1 cells stably expressing zyxin-GFP were incubated with 9 mM RO-3306 (Enzo Life Sciences ALX-270-463) to inhibit CDK1 activity for 15–20 h. Inhibition was released by replacing drug containing media by fresh media at the microscope immediately before imaging. Cells were imaged using a Nikon Eclipse Ti microscope (Nikon) equipped with a Neo-Zyla sCMOS camera (Andor), LED illumination (CoolLED) and a ×60 objective (Plan Apo 60 × /1.4 Oil, Nikon). Images were acquired every 3 min until enough cells had undergone mitotic rounding. At that point, determined by visual inspection, 16% warmed PFA (from a Lego syringe pump placed inside the incubation chamber) was added to cells in media to a final concentration of 4%, and incubated at room temperature for 20 min. They were then washed three times and 0.2% Triton was added for 5 min. 5% BSA in 1X PBS was used to block for 30 min at room temperature, before activated β1 Integrin (Abcam ab30394) primary antibody was added. After incubation and washing, Phalloidin-TRITC (Sigma-Aldrich) and anti-mouse AF647 antibody (Invitrogen) were added. All of these steps were performed automatically using the NanoJ-Fluidics platform, except the identification of the time point when to fix the cells.