Fig 2: MiR-4521 overexpression regulates malignant properties of HCC cells by the p-FAK/p-AKT pathway. (A) Expression levels of p-FAK and p-AKT, (B) Snail, N-cadherin, E-cadherin and Vimentin, (C) MMP2, MMP9 and TIMP-1, (D) MDM2, P53, BAX and BCL-2, (E) Cytochrome-C, Caspase-3 and Caspase-9 were examined via WB analysis in HCCLM3 and HepG2 miR-4521 mimic transfected cells compared with miR-NC. P < 0.05, 0.01, 0.001 and 0.0001 are denoted by *, **, *** and ****.

Fig 3: TIMP1 protects against BBB injury in an MMP-independent manner. (A) Recombinant rAla-TIMP1 protein expressed in eukaryotic cells (293T) was analyzed by SDS-PAGE and Western blots analysis. (B)–(F) HBMECs treated with 1 µg/mL rTIMP1 or rAla-TIMP1 were subjected to hypoxia plus 20 ng/mL IL-1ß for 24 h. (B) HBMECs from the indicated treatment groups were subject to transwell permeability assay. Data represent mean ± SEM of six independent experiments (ns, not significant; **P < 0.01). (C) HBMECs from the indicated treatment groups were subject to TEER assay. Data represent mean ± SEM of six independent experiments (ns, not significant; **P < 0.01). (D) Western blot analysis and quantification of indicated proteins from total cell lysates (ns, not significant; *P < 0.05; **P < 0.01). (E) Western blot analysis and quantification of indicated proteins from membrane fragments. Data represent mean ± SEM of three independent experiments (ns, not significant; *P < 0.05; **P < 0.01). (F) Cells were also subjected to IF staining of VE-cadherin, claudin-5, occludin and ZO-1. Scale bars, 10 µm.

Fig 4: Effects of conditioned medium from HMC-1 cells on the production of collagens and MMPs by HFL-1 cells. (A) Following culture in normoxic or hypoxic conditioned medium from HMC-1 cells, the mRNA expression of collagen type I and III, MMP-9, MMP-13 and TIMP-1 in HFL-1 cells were assessed by reverse transcription-quantitative polymerase chain reaction analysis, normalized to the expression of ß-tubulin, and expressed as fold change relative to the expression of the control group. (B) Western blot analysis of collagen type I and III, MMP-9, MMP-13 and TIMP-1 protein expression in HFL-1 cells cultured in conditioned medium from HMC-1 cells. There were 4 biological replicates/condition. Error bars depict the standard deviation. *P<0.05, **P<0.01, NS, not significant. COL I, collagen type I; COL III, collagen type III; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of metalloproteinase-1; N-CM, normoxic conditioned medium; H-CM, hypoxic conditioned medium; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.

Fig 5: TIMP1 knockdown induces trans-endothelial barrier leakage and degradation of endothelial JPs. (A) Illustration of the in vitro BBB model to evaluate barrier function. An HBMECs monolayer seeded on top membrane of the cell culture insert followed by measurement of the concentration of 40 kDa FITC-dextran leaking into abluminal to determine the paracellular permeability (left) or the trans-endothelial electrical resistance (TEER) to estimate the TJ integrity (right). (B) HBMECs transfected with control or siRNA against hTIMP1 were subject to transwell permeability assay. Data represent mean ± SEM of six independent experiments (**P < 0.01). (C) HBMECs transfected with control or siRNA against hTIMP1 were subject to TEER assay. Data represent mean ± SEM of six independent experiments (**P < 0.01). (D) Western blot analysis and quantification of the indicated proteins in total cell lysates of HBMECs treated with siRNA. GAPDH was used as loading control. Data represent mean ± SEM of three independent experiments (ns, not significant; *P < 0.05; **P < 0.01). (E) Western blot analysis and quantification of the indicated proteins on membrane fragments of HBMECs treated with siRNA. Na/K ATPase was used as loading control. Data represent mean ± SEM of three independent experiments (**P < 0.01). (F) The distribution of junctional proteins in HBMECs treated with siRNA were analyzed by immunofluorescence (IF) staining using antibodies against VE-cadherin (green), claudin-5 (green), occludin (green) or claudin-5 (green) and counterstained with Hoechst33342 (blue) for nuclear labelling. Scale bars, 10 µm.