Fig 2: The effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) low expression on the phosphorylation of STAT3 in the TWEAK-induced airway smooth muscle cells (ASMCs). (A) The expressions of TWEAK and Fn14 in the TWEAK-silenced ASMCs were verified by reverse transcription polymerase chain reaction. (B) The protein levels of p-STAT3 (Tyr705) and STAT3 in ASMCs were detected by Western blot after knockdown with TWEAK small interfering RNA. (C) 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 < 0.01 vs. untreated group; *P < 0.05, **P < 0.01 vs. TWEAK-induced group. (D) 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 3: TWEAK participates in neointimal formation after wire injury. A) Relative Tnfrsf12a-/- or Tnfsf12-/- expression levels normalized to 18S rRNA of non-injured (N = 4) or injured (N = 5) femoral artery from wild type mice. Data represent the mean ± SEM (Student´s t test) *p < .05 vs non-injured artery. B) Representative images of non-injured and injured femoral artery cross-sections immunostained with anti-Fn14 or anti-TWEAK antibodies. Negative controls were incubated with non-specific IgG. Scale bars 50 µm. C) Representative images of injured femoral artery cross sections stained with hematoxylin and eosin from wild type, Tnfrsf12a-/- or Tnfsf12-/- mice. Scale bars 50 µm. M = media; I = intima. D) Cell number quantification per mm2, intima and media area, and intima/media ratio of cross-sections of injured femoral arteries from wild type (N = 11), Tnfrsf12a-/- (N = 11) or Tnfsf12-/- (N = 10) mice. Data represent the mean ± SEM (One-way ANOVA with Bonferroni's post-test) *p < .05 vs WT, **p < .01 vs WT and ***p < .001 vs WT.

Fig 4: Endogenous Fn14 is processed by protease ?-secretase Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon ?-secretase inhibition. The cells were treated overnight with ?-secretase inhibitor DAPT (1 µM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 µM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14.Quantification of blots from panel (A). The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1.Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon ?-secretase inhibition. The cells were treated overnight with ?-secretase inhibitor DAPT (1 µM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 µM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14.Quantification of blot from panel (C). The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1.sFn14 is reduced upon ?-secretase inhibition in MDA-MB-231 cells. Conditioned media of the treated cells were collected after overnight DAPT (1 µM) or vehicle treatment. sFn14 concentration was measured by human Fn14 ELISA.sFn14 is reduced upon ?-secretase inhibition in SKOV-3 cells. Conditioned media of the treated cells were collected after 48-h DAPT (1 µM) or vehicle treatment. sFn14 concentration was measured by human Fn14 ELISA. Data Information: All quantification data are shown as mean ± SEM. All the panels have N = 3 biological replicates. For panels (B) and (D), the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. For panels (E) and (F), two-tailed unpaired t-test is used. The P-values that are above 0.05 have not been included into the panels.

Fig 5: Inhibiting ?-secretase in MDA-MB-231 cells enhances Fn14 mediated NF?B signaling MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or nontargeting control (Ctrl) siRNA. A day after transfection, cells were treated with ?-secretase inhibitor DAPT (1 µM) or vehicle overnight. Either TWEAK (100 ng/ml) or positive control TNF (10 ng/ml) were applied for indicated time points. The cell lysate was blotted against pI?B and I?B to evaluate NF?B activation or against Fn14 to verify the effect of the DAPT and siFn14 treatment, or against calnexin as a loading control. Shown are representative blots from N = 4 experiments.MDA-MB-231 cells were treated with ?-secretase inhibitor DAPT (1 µM) or vehicle overnight. TWEAK (100 ng/ml) was applied for 10 min. The cell lysate was blotted against pP65 and P65 to evaluate NF?B activation or against ß-actin as a loading control. Shown are representative blots from N = 3 experiments. The dashed vertical line indicates that sample were run on the same blot but not directly next to each other.