Mounting medium with dapi

PLA allows for endogenous detection of protein interaction. We detect the interaction of LDLR and CD3 according to Duolink PLA Fluorescence protocol (Sigma). Cells were fixed with 4% PFA. Block non-specific signal by adding Duolink Blocking Solution and incubate for 60 min at 37 °C. After blocking, add the anti-LDLR and anti-CD3 primary antibodies and incubated for 12 h at 4 °C. Then two PLA probes were diluted and added to the samples and incubated for 60 min at 37 °C. Prepare ligation and amplification buffer to ligate the fluorescence probe and amplify the signal. Mount the samples with in situ Mounting Medium with DAPI (Sigma). The images were captured with Olympus FV1000 or Zess LSM880 confocal microscope, and analyzed with Image J software.
The TIRF-imaging was performed on Nikon N-SIM + N-STORM microscope with a TIRF 100× oil immersion lens. Adjusted the oblique incidence excitation to the appropriate TIRF angle to capture images.

2.5 x 10⁴ MDA-MB-231 and MCF7 cells suspended in 1 ml serum-free medium were loaded onto a upper 8 μm pore size chamber inserted in a 12-well cell culture plate. The lower chamber was filled with DMEM supplemented with 1 % FBS as a chemoattractant. The cells were allowed to adhere before being treated with 10 ng/ml TNF-α and then incubated for 24 h at 37 °C in 5 % CO₂. After this incubation, the inserts were removed and the remaining non-migrating cells on the upper surface of the membrane were removed with a cotton swab. The cells that migrated to the lower surface of the membrane were fixed with 5 % paraformaldehyde (PFA) in PBS for 20 min at room temperature, washed with PBS and then treated with 0.5 % Triton X-100 for 5 min. Next, the cells were stained with DAPI solution (Mounting Medium with DAPI, Sigma-Aldrich) and examined using a Zeiss LSM 510meta microscope at 20× magnification (Plan Apochromat 20×/0,8). Pictures of five randomly chosen fields were taken and migrating cells were counted using ImageJ software. The average number of migrating cells for each condition was calculated and differences in the numbers of cells that migrated through the membrane were calculated.