Fig 2: Knockdown of PPARγ altered epithelial‐mesenchymal transition (EMT) and AKT/GSK3β signalling pathway proteins in T24 cells. A, Transwell invasion and migration assay for T24 cells with or without siRNA. The scale bar is 200 μm. B, Statistical analysis of the transwell assay suggested significantly decreased migration and invasion of the cells after siRNA treatment. C, Western blot results for proteins related to EMT regulation revealed a strong reduction of N‐cadherin, vimentin, slug, MMP2 and MMP9, while E‐cadherin was up‐regulated after siRNA treatment in T24 cells. Proteins associated with the AKT/GSK3β signalling pathway were also detected. GAPDH was used as an internal reference. D, Immunofluorescence staining of N‐cadherin and E‐cadherin, which are mesenchymal and epithelial markers, respectively, confirmed the alteration of EMT. *P < 0.05, **P < 0.01. The scale bar is 200 μm

Fig 3: NT1721 induced tumor suppressor genes and decreased the invasive potential of pancreatic cancer cell lines. a NT1721-mediated induction of tumor suppressor genes (BIM, p21) and CDK2 was assessed by Western blot after 24 h using GAPDH as loading control. b CDK2 mRNA expression was determined after 24 h treatment of Panc1 and Capan1 cells with NT1721; GAPDH was used as reference gene. The graphs represent the mean ± SD from 4 independent experiments. c PDAC cell lines were treated with NT1721 for 16 h. Their invasive potential was quantified using the CytoSelect™ 96-Well Cell Invasion Assay (Cell Biolabs). d qPCR to determine ZEB1 and ZEB2 mRNA expression levels in PDAC cells and non-tumorigenic HPDEC cells. GAPDH was used as reference gene. The graphs represent the mean ± SD from 4 experiments. e Protein expression of EMT markers (ZEB1, ZEB2, CDH2, MMP2) was determined by Western blot in Panc1 and Capan1 cells after 48 h treatment with NT1721. f The mRNA expression of EMT markers (MMP2, CDH2) was assessed in Panc1 and Capan1 cells after 24 h treatment with NT1721

Fig 4: Naltriben upregulated the expression of MMP-2, and increased the phosphorylation of ERK1/2 in U87 cells(A) Representative images of Western immunoblotting. Cells were treated with naltriben (50 μM) for 24 h. Protein expression was then determined by Western immunoblot. Comparison between naltriben-treated cells and the control group was summarized in: (B) ratio of MMP-2/β-actin; (C) ratio of p-ERK1|2/β-actin; (D) ratio of t-ERK1|2/β-actin; (E) ratio of p-ERK1|2/t-ERK1|2; (F) ratio of p-Akt/β-actin; (G) ratio of t-Akt/β-actin; and (H) ratio of p-Akt/t-Akt. * represents p < 0.05 (Student's t-test, n = 3/group).

Fig 5: VBMECs enhanced NSCLC cell invasion via CX3CL1 signaling-mediated activation of the PI3K/AKT pathway. (A) Western blotting analysis of CX3CL1 protein levels in control (sicon) and CX3CL1-KD (siCX3CL1) VBMECs (Vs), and CX3CR1 protein levels in control (shcon) and CX3CR1-KD (shCX3CR1) A549 cells (A549). (B, C) Quantitative analysis of cell invasion in control (shcon) and CX3CR1-KD (shCX3CR1) A549 cells incubated with or without conditioned culture media of indicated VBMECs (VBMEC CM). Data represent the mean ± SEM (n = 3). **P < 0.01 and ***P < 0.001. (D) Expression levels of EMT markers including Vimentin, E-cadherin, MMP-2, and MMP-9 in control (shcon) and CX3CR1-KD (shCX3CR1) A549 cells co-cultured with or without indicated VBMEC conditioned media measured by western blotting. (E) PI3K and AKT phosphorylation in A549 cells co-cultured with or without VBMEC conditioned media measured by western blotting. (F, G) Quantitative analysis of invasion of A549 cells incubated with or without conditioned culture media of VBMECs treated with the PI3K inhibitor LY294002 or AKT inhibitor MK-2206 2HCI. Data represent the mean ± SEM (n = 3). *P < 0.05 and ***P < 0.001. (H) Expression levels of PI3K, p-PI3K (Ser249), AKT, p-AKT (Ser473), Vimentin, E-cadherin, MMP-2, and MMP-9 in A549 cells co-cultured with indicated VBMECs and treated with LY294002 or MK-2206 2HCI measured by western blotting.