Anti mouse minus pla probes

After treatment with DOX, fixation, permeabilization, blocking, and primary antibody incubation steps were performed as described in the immunofluorescence section. Once cells were washed three times with 1% BSA and 2% FCS in PBS, incubation with PLA probes (i.e., anti-rabbit PLUS, anti-mouse MINUS PLA probes), ligation, amplification, and mounting of the coverslips were all performed according to the manufacturer's instruction (Sigma-Aldrich). Slides were imaged using a Leica TCS SP5 confocal laser-scanning microscope and analyzed using ImageJ software. The number of PLA foci was manually counted in a blind fashion (100 cells per each condition in the three independent experiments). Cyto-3 plasmic foci were defined as fluorescent spots that did not colocalize with the nuclei stained with DAPI. Statistical significance in the number of PLA foci was tested by using a two-tailed t-test.

In situ proximity ligation assay (PLA) was performed using Duolink Anti-Rabbit PLUS and Anti-Mouse MINUS PLA Probes (Sigma), according to the manufacturer’s instructions. Briefly, prepare samples on slides, then treat for fixation, retrieval, and permeabilization. Block slides with Duolink® Blocking Solution at 37 °C for 60 min. Apply diluted antibodies CDH1 (Abcam, ab227784) and PIN1 (Santa Cruz, sc-46660) or CDK4 (Santa Cruz, sc-23896), followed by diluted PLUS and MINUS PLA probes, each with specific washes in between. Mix and incubate with a ligation solution, and then with an amplification buffer-polymerase mix, ensuring protection from light during amplification. After final washes, mount slides and analyze using fluorescence microscope.