Fig 2: Knockdown of rela enhances myogenic differentiation and impairs activation of the canonical pathway by LTßR. (A) Experimental scheme of myoblast differentiation in the presence of the LTßR agonist or antagonist and transfection with siRNA targeting rela or a non-targeting control siRNA. (B) Incubation at the beginning of differentiation or incubation at day 3 of differentiation markedly decreases myotube diameter compared to the control. Knockdown of rela results in increased myotube diameters independent of incubation with the LTßR agonist or antagonist. n = 3. (C) Representative images of immunofluorescent images from myotubes quantified in (B), myosin heavy chain (MHC) in green and myogenin in red. Nuclei are counterstained with DAPI (blue). Scale bar = 50 µm. (D) Incubation of myotubes with the LTßR agonist at the beginning of differentiation or addition at day 2 of differentiation leads to the cleavage of p100 to active p52 at day 3 of differentiation (E) and increased phosphorylation of RelA (E). *p < 0.05. Error bars represent SEM.

Fig 3: NF-?B induces the expression of Arid5a by binding to its promoter in response to lipopolysaccharide (LPS). (A and B) Expression of Arid5a mRNA and protein in mouse peritoneal macrophages stimulated for 0–24 h with LPS (1 µg/ml). (C) Expression of Arid5a mRNA macrophages that were pretreated with the indicated inhibitors (10 µM) or DMSO (control) for 30 min and then stimulated with LPS for 2 h. (D) Arid5a mRNA expression in the control and IKKß-deficient MEFs after stimulation with LPS (10 µg/ml). (E and F) HEK293T cells were transfected with a pGL3-luciferase vector encoding an Arid5a promoter (-2000 bp upstream of the TSS) or a pGL3 empty vector. Both were transfected with an empty vector (control) or with expression vectors of p65 or c-Rel (E) or p65 mutants (Mu1 and Mu2) (F). p65 Mu1 and p65 Mu2 refer to the K310R/K314R/K315R and S536A mutants, respectively. Luciferase activity was determined 24 h after transfection and normalized to the activity induced by the pGL3-empty vector. The values are shown relative to those after transfection with the empty vector. (G and H) Peritoneal macrophages were stimulated using LPS for 2 h, and ChIP assays were then performed using anti-p65, anti-c-Rel, and anti-IgG (negative control) antibodies. (I) Immunoblot assays of p65 expression in MEF cells transfected with 20 nM control or p65 siRNA. (J) Arid5a mRNA expression in the control and p65 knockdown MEFs after stimulation with LPS (10 µg/ml). (K) Immunoblot assays of p52 expression in MEF cells transfected with 20 nM control or p52 siRNA. (L) Arid5a mRNA expression in the control and p52 knockdown MEFs after stimulation with LPS (10 µg/ml). All data show the mean ± SD of three independent experiments. Error bars indicate the mean ± SD. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Western blot of different proteins related to cancer cell proliferation after treatment with PS fractions. Western blot quantification was normalized against the ß-actin signal. Data were obtained from three independent experiments performed in duplicate and are expressed as mean ± SD from three independent experiments performed in triplicate (**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). (a) membrane that contain proteins of NFkB p100, c-Myc, Cyt C; (b) membrane with CD-44, Caspases 3 and 8, and Bax, (c) membrane with beta-catenin.

Fig 5: CYPA and S100A4 induce p52 via STAT3 signaling. a Immunoblot (IB) using WI-38 cells incubated in regular media (RM) or conditioned media (CM) for 24 h, probed with the indicated antibodies. P-STAT3 (phospho-STAT3). b IB using lysate from WI-38 cells harvested on Day 0 and 7 following prolonged culture, probed with indicated antibodies. c IB using lysate from WI-38 cells at D0 and D7 treated with or without WP1066 (WP, 5 µM). d Quantification of ß-gal-positive WI-38 cells on Day 7 following prolonged culture ± WP1066 (5 µM). Data represent mean value of three biological samples ± SEM. *P < 0.05 (two-tailed t test). e IB using lysate from WI-38 cells transfected with si-STAT3 or si-control (CTL) and incubated with either RM or CM for 24 h. Inset: IB of same cells with indicated antibody following transfection with siRNA. f IB using lysate from WI-38 cells incubated in recombinant CYPA or S100A4 for 24 h with or without WP1066 (5 µM). Blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated