Bf9212

NIH-3T3 cells (2 × 10⁵ cells per well) were cultured in a 6-well plate overnight and were then subjected to TGF-β1 (100.0 ng/ mL) treatment or not. After that, these cells were harvested and protein concentration was measured by BCA protein assay kit. The primary antibodies including α-SMA (1:1000; BF9212; Affinity), FAP (1:500; AF5344; Affinity) and β-actin (1:1000; AF2811; Beyotime), as well as secondary antibodies were incubated in accordance to standard protocols. ImageJ software was used to perform semi-quantitative analysis of the protein band.
CAFs and 4T1 cells were implanted in confocal culture dishes at a density of 5 × 10⁵ cells per dish overnight. Thereafter, the cells were treated with curcumin, mPDA/Cur@M, mPDA/Cur@M/CM, curcumin+laser, mPDA/Cur@M+laser, and mPDA/Cur@M/CM+laser (50 μg/mL curcumin), respectively. Thereafter, proteins were extracted from these cells, and then incubated with the primary antibody HSP90 (1:1000, AF5368; Affinity) as well as secondary antibody, respectively. At the end, ECL western blotting detection kit was used to visualize the different proteins.

The paraffin‐embedded tissue slides were dewaxing and antigen retrieval utilizing citric acid buffer (10 mM, pH 6.0; Servicebio, G1202). Following this, block the glass slides with BSA and incubate overnight at 4°C with the specified primary antibody. The primary antibody employed was as follow: anti‐CCL20 antibody (1:200; Affinity, DF2238); anti‐CD4 antibody (1:500; Elabscience, E‐AB‐22098); anti‐CD116 antibody (1:500; Affinity, DF4820); anti‐alpha SMA (α‐SMA) antibody (1:500; Affinity, BF9212); anti‐vimatin antibody (1:500; Affinity, BF8006). Subsequently, the slides were left at room temperature for 2 h with the appropriate secondary antibodies, which were as follows: Cy3‐conjugated Goat Anti‐Rabbit IgG (1:500; Servicebio, GB21303); FITC‐conjugated Goat Anti‐Mouse IgG (1:200; Servicebio, GB22301). The marked slides were always protected from light and were dyed using DAPI (Servicebio; G1012). The co‐localization of CCL20 with other biomarkers was recorded using confocal laser scanning microscopy (Olympus; FV3000). The fluorescence signal was quantified using ImageJ.