Fig 2: TBHQ, an ERK activator, rescued the inhibitory phenotype of GC cells by RECK overexpression. A, B Colony formation assay (A) and EdU assay (B) for GC cell proliferation evaluation in RECK-overexpressed AGS and HGC-27 cells treated with TBHQ. C, D Cell cycle (C) and cell apoptosis (D) were detected by flow cytometry in RECK-overexpressed AGS and HGC-27 cells treated with TBHQ. E, F The migration and invasion abilities of GC cells were measured using wound-healing and Matrigel invasion assays in RECK-overexpressed AGS and HGC-27 cells treated with TBHQ. G Western blot representations of cell proliferation markers, EMT markers, and MEK/ERK signaling markers levels in RECK-overexpressed AGS and HGC-27 cells treated with TBHQ (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. DM: DMSO; TB: TBHQ)

Fig 3: Ectopic expression of RECK blunts IL-18-induced inhibition in the expression of SMC markers and the induction of proinflammatory phenotype markers. (A–F) Forced expression of RECK restores IL-18-induced suppression of SMC markers and inhibits the induction of proinflammatory phenotype markers. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi10 for 24 h), made quiescent, and then treated with IL-18 at 10 ng/mL for 48 h (experimental design in (A)). The expression levels of SMC markers ACTA2 (B,C) and MYH11 (D,E) were analyzed by RT-qPCR (B,D) and Western blotting (C,E). The proinflammatory phenotype markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α were analyzed by RT-qPCR using TaqMan™ probes (F). (C,E) While a representative immunoblot is shown, the intensities of immunoreactive bands from three independent experiments were semiquantified by densitometry and are summarized on the right. (B,D,F) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 5). (C,E) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Fig 4: EMMPRIN is a prognostic marker in renal cell carcinoma. Kaplan-Meier analyses show RECK-positive (0) and RECK-negative (1–3) staining intensities (A) or low EMMPRIN (0/1) and high EMMPRIN (2/3) staining intensities (B) connected with overall survival of the patients after nephrectomy. Oncocytomas were excluded. Curves of all 387 cases (386 for EMMPRIN) are shown at the left, curves of the selected 181 pN0/M0 cases only (180 for EMMPRIN) are depicted at the right. High EMMPRIN expression was significantly associated with a decrease in overall survival, even in pN0/M0 cases only, while RECK did not discriminate subgroups. Censored cases are marked (+).

Fig 5: Empagliflozin inhibits OxLDL and CT-1-induced SMC proliferation and migration. (a) Empagliflozin inhibits OxLDL-induced miR-30b expression. Quiescent SMCs were treated with empagliflozin for 15 min followed by the addition of OxLDL or nLDL for 2 hr and analyzed for miR-30b expression by RT-qPCR using a TaqMan® probe. (b, c) Empagliflozin reverses OxLDL-induced RECK suppression. Quiescent SMCs were treated with empagliflozin as in (a), were exposed OxLDL or nLDL for 6 hr and analyzed for RECK expression by western blotting. Semiquantification of the intensity of immunoreactive bands by densitometry is shown in panel (c). (d, e) Empagliflozin inhibits OxLDL- or CT-1-induced MMP2 (d) and MMP9 (e) activity. SMCs made quiescent in medium supplemented with ITS-G 1x and no FBS were exposed to empagliflozin as in (a), followed by OxLDL (black) or CT-1 (red) for 18 hr and analyzed for MMP2 and MMP9 activity using SensoLyte® 520 fluorimetric assay. (f, g) Empagliflozin inhibits OxLDL- or CT-1-induced SMC proliferation and migration. Quiescent SMCs exposed to empagliflozin were treated with OxLDL (black) or CT-1 (red) for 48 hr (f) or 18 hr (g) and then analyzed for proliferation. (h) Empagliflozin reverses OxLDL-induced suppression of the mRNA expression of SMC markers αSMA and MYH11 and the proinflammatory markers ICAM1, Galectin 3, and Olr1 as analyzed by RT-qPCR. (a) ∗P < 0.01 versus nLDL, †P < 0.05 versus OxLDL (n = 5); (c) ∗P < 0.05 versus nLDL, †P < 0.05 versus OxLDL (n = 3); (d–g) P < 0.05 versus nLDL, †P < 0.05 versus OxLDL or CT-1 (n = 4 or 5), (h) ∗P < 0.05 versus nLDL, †P < 0.05 versus OxLDL (n = 5).