Duolink in situ reagents

MEF cells endogenously expressing different GR proteins were treated with 1 μM Dex or vehicle control (0.01% EtOH) for 45 min. After treatment cells were fixed using 4% paraformaldehyde in preparation for the PLA using the Duolink In situ reagents from Sigma-Aldrich. The PLA plus and minus oligonucleotides were conjugated to a monoclonal GR antibody purchased from Santa Cruz Biotechnology (GR-F10; sc-376426). Fixed cells were blocked for 60 min with the blocking solution provided and subsequently incubated for 2 h with the conjugated antibodies. This was followed by two wash steps after which the cells were incubated with ligase to facilitate the ligation of the plus and minus oligonucleotides. The annealed oligonucleotides served as a template for the production of numerous DNA circles after incubating the cells with a polymerase solution, which also included a labeled complementary oligonucleotide probe that generates a fluorescent signal. The signal was visualized as a fluorescent dot using the LSM780 confocal microscope with ELRYA PS1 super-resolution platform (Zeiss). To visualize the nucleus, the fixed cells were stained with 10 μg/ml Hoecht33258 stain (Sigma-Aldrich). The PLA signal in the nucleus only was quantified using ImageJ software. Data were expressed as means ± SD, and statistical analysis was performed using a Student’s t test.

Purified naive CD4⁺ T cells were treated by NC siRNA or p62 siRNA for 24 h. Then, CD4⁺ T cells were cultured in 48-well plates with plate-bound anti-CD3 and anti-CD28 for another 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 3% bovine serum for 30 min, the cells were incubated with mouse anti-TGF-β1 and rabbit anti-LC3B antibodies overnight at 4 °C. Then, PLA was performed with Duolink In situ reagents (Sigma–Aldrich) according to the manufacturer’s instructions. Then Samples were imaged using Olympus FluoView version 1.4a software (Olympus Corp). Images of cells and sections were acquired, and positively stained areas were analyzed.

A total of 5 × 10³ OS cells were seeded in Ibidi µ-Slide VI 0.4 during 25 h. The medium was then replaced with DMEM. At 24 h after, TGF-β1 (5 ng/ml) was either added for 45 min or not. The cells were then fixed with 4% PFA for 15 min at room temperature and incubated overnight at 4°C with primary antibody against YAP (1:100; Cell Signaling, 14074) and/or Smad3 (1:1,000, Abcam, ab40854). In situ PLA was performed using DuoLink in Situ Reagents (Sigma-Aldrich, St. Quentin-Fallavier, France) according to the protocol of the manufacturer.

253J-L cells exposed to sHA-F (5-μg/ml) for 24 hours were fixed in paraformaldehyde (4%) and permeabilized using 0.25% Triton X-100. Permeabilized cells were subjected to PLA using the Duolink ®In situ reagents (Sigma Aldrich) as per the manufacturer's protocol, that involved incubation of the permeabilized cells with rabbit anti-PI-3K p85 (1:650) and mouse anti-CD44 (1:650) antibodies at 37°C for 1 hour. The slides were observed under a Zeiss LSM700 Confocal microscope equipped with multi-variant fluorescence filters in two channels (red and blue) under a 63X oil-immersion objective lens, as described before [33 (link)].