Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This study is the first report of a long-term (21-day) triple co-culture of THP-1 macrophages (THP-1m) with Caco-2 intestinal epithelial cells and HT-29-methotrexate (MTX) goblet cells. We demonstrated that high-density seeded THP-1 cells treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h differentiated stably and could be cultured for up to 21 days. THP-1m were identified by their adherent morphology and lysosome expansion. In the triple co-culture immune-responsive model, cytokine secretions during lipopolysaccharide-induced inflammation were confirmed. Tumor necrosis factor-alpha and interleukin 6 levels were elevated in the inflamed state, reaching 824.7 ± 130.0 pg/mL and 609.7 ± 139.5 pg/mL, respectively. Intestinal membrane integrity was maintained with a transepithelial electrical resistance value of 336.4 ± 18.0 Ω·cm². Overall, our findings suggest that THP-1m can be effectively employed in models of long-term immune responses in both normal and chronic inflammatory states of the intestinal epithelium, making them a valuable tool for future research on the association between the immune system and gut health.

Background/Objectives: Disruptions in adipose tissue dynamics contribute to obesity-related metabolic disorders, emphasizing the need for targeted therapies focusing on adipose tissue cells, including progenitor cells and adipocytes. Peroxisome proliferator-activated receptor gamma (PPARG) ligands are potent insulin sensitizers used in type 2 diabetes treatment. This study investigated the effects of rosiglitazone, a PPARG agonist, and betulinic acid, a PPARG antagonist, on adipogenesis and apoptosis in 3T3-L1 pre-adipocytes. Method: 3T3-L1 pre-adipocytes were treated with rosiglitazone or betulinic acid during adipogenic differentiation. Lipid droplet formation was used to evaluate adipogenesis. Cell growth and cell death were assessed using the resazurin-based cell viability assay, trypan blue exclusion assay, LDH assay, and Annexin V/PI staining. Quantitative PCR was conducted to examine the expression of genes associated with adipogenesis and apoptosis. Results: Betulinic acid reduced adipogenesis only when administered daily for eight days. Rosiglitazone did not alter the overall lipid quantity; however, it promoted a shift toward fewer but larger lipid droplets. Both compounds increased Adipoq and Cfd expression, and betulinic acid also elevated Fabp4. Rosiglitazone induced stronger cell aggregation. Despite increased cell death, overall viability was maintained. Apoptotic cell death was enhanced by both compounds and confirmed via Annexin V/PI staining and flow cytometry, accompanied by downregulation of Ccnd1 and Bcl2. Additionally, rosiglitazone markedly increased the expression of Cebpa, a key regulator that can modulate lipid droplet formation and the balance between cell growth and death. Conclusions: Rosiglitazone and betulinic acid differentially modulate adipogenesis and apoptosis in 3T3-L1 cells, revealing a complex interplay between lipid accumulation and programmed cell death. Together, the findings underscore the potential of dual PPARG-targeting approaches for metabolic disease interventions.

Breast cancer (BC) is the most commonly diagnosed malignancy in women, with triple-negative breast cancer (TNBC) representing the most aggressive subtype that is associated with poor clinical outcomes. Identifying novel biomarkers or therapeutic targets is therefore of great importance. Nectin cell adhesion molecule-2 (NECTIN2) is an immunoglobulin-like glycoprotein involved in cell adhesion and immune regulation that is highly expressed in several cancers but its role in the progression of BC is unclear. Using publicly available data sets, we found that NECTIN2 expression is elevated in BC compared to normal tissues and is associated with poorer recurrence-free survival in TNBC patients. Among TNBC cell lines, MDA-MB-231 cells exhibited the highest expression of NECTIN2 at gene and protein levels, consistent with a more aggressive phenotype. To investigate its functional role, we silenced NECTIN2 in MDA-MB-231 and MDA-MB-468 cells. Relative cell viability was modestly reduced in NECTIN2-depleted MDA-MB-231 cells at later time points, but no significant changes were observed in MDA-MB-468 cells. Interestingly, NECTIN2 depletion significantly reduced migration and invasion in MDA-MB-231 cells, but not in MDA-MB-468 cells. Given this cell line–specific effect, subsequent mechanistic investigations focused on the MDA-MB-231 model. Gene expression profiling suggested a reduction in LIMK1, a regulator of cytoskeletal dynamics and cell motility, following NECTIN2 depletion. Furthermore, ectopic expression of LIMK1 in NECTIN2-deficient MDA-MB-231 cells partially restored migratory capacity and was associated with partial epithelial–mesenchymal transition (EMT)-like changes, including a decrease in E-cadherin and ZO-1, and a higher level of fibronectin and Slug. Collectively, these findings suggest that NECTIN2 contributes to migratory and invasive phenotypes in MDA-MB-231 cells, potentially through LIMK1-associated cytoskeletal remodeling and EMT-related marker changes. However, this mechanism requires further investigation because it appears to be context-dependent and may not be universally applicable across TNBC subtypes.