A1 confocal microscopy

For cell proliferation detection, HEK293T and HeLa cells were plated in 24‐well, and were transfected by the designated plasmids using Lipofectamine® 3000. The cells transfected successfully were marked by GFP. The images taken by Nikon A1 confocal microscopy at 488 nm channel was used to acquire the images at the same position of the 24‐well plate at different time points. ImageJ (https://imagej.nih.gov/ij/download.html) was used to measure the total area of GFP positive cells. The cell proliferation rates were derived by the changes of the area of GFP positive cells over time. The formula is as follows:
Relative Proliferation Rate [fluorescence intensity] = A₀/AA: Total area of cells containing fluorescence at definite time after transfection.
A₀: Total area of cells containing fluorescence at time 0 was defined for different experiments.
We defined cells post transfection 20 h as time 0.
For MTT assay, cells were incubated in MTT solution at a final concentration of 0.5 mg/ml in DMEM for 4 h at 37°C. DMSO was added to dissolve the formazan crystal. The value of optimal density was measured at 490 nm using a microplate reader (GENios, TECAN).

SMMC-7721 cells were plated into 35 mm culture dishes. Fluorescent images for FRET samples, donor samples, and acceptor samples were captured 16 h after transfection using an A1 confocal microscopy (Nikon, Tokyo, Japan) with 3-FRET filter cubes for EGFP/DsRed: EGFP (488/515 nm), DsRed (543/585 nm), and FRET (488/585 nm) [18] (link). Regions of interest were selected for all non-saturated fluorescent cells in any given field. The background was subtracted from all images before analysis. Donor (pEGFP-CD147) and accepter (DsRed1-N1-vinculin) images were utilized to obtain CoA and CoB data for the next FRET calculation with NIS-Elements software (Nikon, Tokyo, Japan). FRET calibration and net FRET were calculated with analysis software (Nikon, Tokyo, Japan). FRET analysis was performed using a three-filter setup system based on the sensitized emission method. Each experiment was repeated at least three times, and similar results were obtained for each [19] (link).

Cells grown on coverslips were fixed in 4% paraformaldehyde PFA for 20 min and permeablized with 0.5% Triton-X100. After blocking with 2% BSA in PBS for 30min, coverslips were incubated with indicated primary antibodies for 1 h at room temperature in 2% BSA in PBS. Following three times washing with PBS, cells were incubated with Alexa-488-, Alexa-594- or Alex-633-conjugated secondary antibodies for 1 h. Cells were then washed three times with PBS and mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were acquired by Olympus Epifluorescence microscopy, Perkin Elmer Ultraview VoX spinning disc confocal or Nikon A1 confocal microscopy.