Fluoview version 1.4a software

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.

Murine tumor tissue sections were routinely deparaffinized and rehydrated, followed by antigen retrieval using 10 mM sodium citrate buffer (pH 6.0). After the samples were blocked with 1× blocking solution at 37 °C for 1 h, they were incubated with mouse αRat IgG2a (BioLegend) and rabbit αPD-L1 (ABclonal, Wuhan, Hebei, China) overnight at 4 °C. Then, PLA was performed with Duolink In situ reagents (Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Then, the samples were imaged using Olympus FluoView version 1.4a software (Olympus, Tokyo, Japan). Images of cells and sections were acquired, and positively stained areas were analyzed by ImageJ software (NIH, Bethesda, MD, USA).