In brief, MCF7, MDA-MB231, and A431 cells seeded onto glass coverslips at 70% confluence were washed once in PBS and were then fixed in 4% (vol/vol) PFA in PBS for 20 min at room temperature (RT). Coverslips were washed three times in excess PBS and were permeabilized in 0.1% (vol/vol) Triton X-100 in PBS for 7 min and blocked in 1% (vol/vol) bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 30 min at RT. The primary antibodies were diluted in 1% (vol/vol) BSA in PBS and incubated for 1 hr at RT. Secondary antibodies (Molecular Probes) were diluted in 1% (vol/vol) BSA in PBS and incubated for 1 hr at RT. Washes were performed in PBS. Coverslips were rinsed in distilled water and mounted in Mowiol (Mowiol 488, Hoechst AG) in 0.2 M Tris-HCl, pH 8.5. The images were taken on a laser-scanning microscope (LSM 510 META, Carl Zeiss, Inc) using a 63× oil lens, NA 1.4. Adjustments of brightness and contrast were applied using ImageJ software (NIH). The LUT of images for PLA were inverted for better visualization of PLA dots in cells.

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