Reagents

Curcumin (HPLC > 94%) was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and kept at – 20 ℃ [14]. The details of the primary antibodies used for Western blot analysis targeting E-cadherin, N-cadherin, Vimentin, and GAPDH are provided below.

Cell culture and treatment

The Central Lab of Changhai Hospital, Navy Medical University provided SKOV3 and OVCAR-3 cells. TAMs were obtained from ascitic fluid of ovarian cancer patients as previously described [15, 16]. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin–streptomycin solution, and incubated at 37 °C in a 5% CO₂ atmosphere. For specific experimental groups, cells were treated with curcumin at varying concentrations for subsequent analysis. Short tandem repeat (STR) profiling was performed on SKOV3 and OVCAR-3 cell lines to confirm their identity and data has been provided in the supplementary file S1.

Cell viability assay

Cells were seeded in a 96-well plate at a density of 1.0 × 105 cells per well and incubated overnight. Subsequently, the cells were exposed to the indicated treatments. Cell viability was assessed using a CCK-8 assay (Dojindo Molecular Technologies, Japan) [17].

Flow cytometry

Cells were washed three times with cold PBS, and surface staining was performed at room temperature for 30 min using anti-human CD86 antibodies and/or anti-human CD206 antibodies (eBioscience, San Diego, CA, USA) [18]. We typically use 1 × 106 cells in a test. Results were analyzed using a FACSCalibur flow cytometer by FlowJo software (Tree Star Inc., Ashland, OR, USA).

Western blot

Cells were harvested and lysed in a lysis buffer. The protein concentration was determined and separated using SDS-PAGE and electro-transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) [19]. Immunoblotting was conducted using the primary antibodies targeting E-cadherin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), N-cadherin (1:1000, Abcam, Cambridge, MA, USA), vimentin (1:1000, Abcam, Cambridge, MA, USA) and GAPDH (1:5000, Beyotime Biotechnology, Shanghai, China). Membranes were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland; Gilbertsville, PA, USA). Images were obtained by the Odyssey infrared imaging system (LI-COR Bioscience, Lincolin, NE, USA).

Realtime PCR (RT-PCR)

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed with PrimeScript RT Master Mix (Takara, Otsu, Shiga, Japan). The relative gene expression was quantified using the 2ΔΔCT method, with β-Actin serving as the internal reference. RT-PCR was conducted using a Real-Time PCR System and the Fast Start Universal SYBR Green Master (Roche, Basel, Switzerland) [20]. The primers used are listed in Table 1.

Table 1 Sequences of RT-PCR primersEnzyme-linked immunosorbent assay (ELISA)

After stimulation, the cell culture supernatant was collected, and the levels of proinflammatory cytokines, including IL-10 and IL-12, were measured using commercially available ELISA kits (R&D Systems, New York, NY, USA) according to the manufacturer’s instructions [21].

Immunofluorescence analysis

Following stimulation, cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min. After fixation, cells were incubated with 1% bovine serum albumin in PBS and stained with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies) for 2 min. Colocalization was assessed using a confocal laser scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan [22].

Cell scratch test

A cell scratch assay was performed to assess the migratory ability of cells [23]. Cells were seeded into 6-well plates and uniformly scraped using a 200 μL pipette tip to create wounds before transfection (NC or si-TINCR; pcDNA3.1 or TINCR). Each well was washed three times with PBS to remove any floating cells. The initial wound distance (0 h) and the distances traveled by cells at 24 and 48 h post-scratching were recorded microscopically at 100 × magnification for each group. ImageJ software was used to quantify the scratch area.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Prism, San Diego, CA, USA). All results were described as means ± standard error of the mean (SEM). as p < 0.05.

Comparisons between multiple sample groups were performed using a one-way analysis of variance (ANOVA). The LSD test was used for pairwise comparisons when the assumption of homogeneity of variance was met, while Dunnett’s T3 test was applied when this assumption was not met. An independent samples t-test was used to assess the homogeneity of variance between the two groups, and a Welch’s t-test was applied when homogeneity of variance was not assumed. Statistical significance was defined as P < 0.05.