Fig 2: SENP1 mediated the deSUMOylation of SMAD4 in response to TGF-ß1-induced ROS generation. A549 cells were either transfected with His-SENP1 or treated with BLM, GA, TGF-ß1, or them in combination. (a) The fluorescence intensity of 2',7'-dichlorofluorescein (DCF) in cells of each group was detected using a reactive oxygen species (ROS) assay kit to show ROS levels in cells of each treatment group. (b) WB analysis of the key oxidases that triggered the ROS outbreak to clarify the role of SMAD4 SUMOylation and SENP1 in TGF-ß1-induced ROS generation. (c) A549 cells transfected with siRNA-SENP1 or treated with BLM, GA, TGF-ß1, NAC, or them in combination were subjected to WB analysis to detect the levels of key protein involved in EMT. Data are presented as the mean ± SD. n = 6, *P < 0.05.

Fig 3: The effect of cryptotanshinone (CTS) on the airway remodeling through transforming growth factor beta 1 (TGF-ß1)/STAT3 in TGF-ß1-induced airway smooth muscle cells (ASMCs). (A) Western blot of TGF-ß1, p-Smad2/3, Smad4, p-STAT3 (Tyr705), and STAT3 in response to the treatment with SB431542 or different doses of CTS in ASMCs. (C) Western blot of TGF-ß1, p-Smad2/3, Smad4, p-STAT3 (Tyr705), and STAT3 under the treatment with SB431542 or 10 µM CTS at different time in ASMCs. (B) and (D) Densitometric analysis was presented as the relative ratio of each molecule to Smad2/3, ß-actin, and STAT3. Data were presented as mean ± SEM (n = 3). #P < 0.05, ##P < 0.01 vs. untreated group; *P <0.05, **P < 0.01 vs. TGF-ß1-induced group. (E) The immunofluorescence analysis of p-STAT3 (Tyr705) in ASMCs showed that this protein was mainly expressed in the cytoplasm of ASMCs. Magnification: 200×. As shown in the pictures, p-STAT3 (Tyr705) was stained green and nuclei was stained with 4',6-diamidino-2-phenylindole showing a blue color.

Fig 4: The effect of cryptotanshinone (CTS) on the airway remodeling was through TWEAK/STAT3 and transforming growth factor beta 1 (TGF-ß1)/STAT3. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) or TGF-ß1-induced airway smooth muscle cells (ASMCs) were treated with the indicated concentrations of CTS for 24 h. (A) and (C) The protein expressions of Fn14, p-Smad2/3, Smad4, p-STAT3 (Tyr705), and STAT3 were analyzed by Western blot. (B) and (D) The band intensities of these proteins were quantified in relative to Smad2/3, ß-actin, and STAT3. (E) Immunofluorescence staining of TWEAK, TGF-ß1, and STAT3 in ASMCs. The magnification was 200×. Fn14 staining (red), p-Smad2/3 staining (red), Smad4 staining (red), p-STAT3 staining (green), and nuclei with 4',6-diamidino-2-phenylindole (blue) is shown. Data were presented as mean ± SEM (n = 3). ##P < 0.01 vs. untreated group; *P <0.05, **P < 0.01 vs. TWEAK-induced or TGF-ß1-induced group.

Fig 5: The increase of cell migration induced by UBE2D1 was reversed by SMAD4. A The mRNA and protein levels of SMAD4 were detected after transduction with SMAD4 overexpression lentivirus using real-time PCR and western blot. ***, P < 0.001 versus vector. B–C Transwell (B) and wound healing C assays were performed to analyze cell migration after transduction with SMAD4 and/or UBE2D1 overexpression lentiviruses in MGC-803 cells. ***, P < 0.001 versus vector; #, P < 0.05, ###, P < 0.001 versus SAMD4 + UBE2D1. D A western blot assay was performed to measure the protein levels of UBE2D1, SMAD4, MMP2, and MMP9