Glass bottom plates

The hBMEC cells were cultured on 6-well Glass Bottom Plates (∅35mm, Cellvis, CA, USA) for 7–9 days until monolayers were confluent. Cells were replenished with DMEM medium, and BifA or BifA variants, at a final concentration of 10 μg/ml, was added to the wells. The plates were cultured in a controlled environmental chamber at 37°C in 5% CO₂. Time-lapse images were acquired at an interval of 30 s for 300 min through an EC Plan-Neofluar 20×/0.50 M27 lens on an Axiom Observer.Z1/7 microscope, using the Applied Precision motorized stage (Carl Zeiss, Japan). ZEN software was used for image processing.

Cells were seeded to reach 40%–60% confluence in 96-well or 24-well glass bottom plates (Cellvis, Mountain View, USA) coated with poly-L-lysine (Sigma, P4707). After washed with PBS, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed with PBS for three times. Next, fixed cells were incubated in blocking solution (containing 5% (v/v) Normal Goat Serum (Bioss Antibodies, C01-03001), 0.3% (v/v) Triton X-100 in PBS) for 2 h at room temperature. After that, the cells were incubated with primary antibody of RXRα (Santa cruz, sc-515929), PPARγ (Cell Signaling, 2435 S) or mCherry (Thermo Fisher scientific, M11217) overnight at 4 °C. Next, cells were washed three times in PBS and then incubated with secondary antibodies conjugated to Alexa Fluor 555 (Cell Signaling, 4409 S), Alexa Fluor 488 (Cell Signaling, 4412 S) or Alexa Fluor Plus 488 (Thermo Fisher scientific, A48262) at 1:1000 dilution for 2 h at room temperature. During this period, 96-well plate was wrapped in foil to keep it in dark environment. Cells were then washed three times in PBS for 10 min. Nuclei staining was performed with DAPI (YEASEN, 40728ES10). Images were acquired at the Nikon Spinning Disk microscope with 100× oil immersion objectives. Fluorescence intensity was obtained using FIJI (National Institutes of Health, Bethesda, USA).

Related to Figure 1. To calculate the fraction of cell area after drug treatments, cells were plated on six-well glass-bottom plates (Cellvis) pre-coated overnight at 4 °C with 10 µg/mL human plasma fibronectin (cat #: FC010). Prior to time-lapse imaging, cells were given 2 h to adhere in a tissue culture incubator. Drugs were pre-diluted in 400 µL of media before adding at “0 min” to wells containing 2 mL of media. Cell area was measured in Fiji (http://fiji.sc/Fiji) using the free-hand circle tool at the time points indicated in each figure. “Fraction of cell area” was calculated in Microsoft Excel (Redmond, WA) as the measured area divided by the area at time 0. For percent protruding measurements, cells were similarly plated on fibronectin-coated glass-bottom plates and adhered for 2 h before imaging. Cells were then imaged at 2 min intervals for 5 h by spinning disk confocal. Cells that extended lamellipodia for at least five successive frames were classified as “protruding.” The fraction of cells protruding was calculated in Microsoft Excel, and all results were plotted using GraphPad Prism (San Diego, CA).