Fig 2: Downregulation of SIRT3 decreases the ability of PCSK9i to impede mitochondrial depolarization. (A) Representative images by fluorescence microscopy and (B,C) flow cytometry analysis of mitochondrial membrane potential detection, expressed as fluorescence intensity ± SD of n = 3 experiments, in EC transfected with NT-siRNA (NT) or with SIRT3 siRNA (SIRT3-/-) and then exposed for 24h to 20 ng/mL IL-6 (NT or SIRT3-/-+IL-6), to 100 µg/mL PCSK9i (NT or SIRT3-/-+PCSK9i) or to 100 µg/mL PCSK9i for 8h before exposure to 20 ng/mL IL-6 for 24h (NT or SIRT3-/-+PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. §p<0.001 vs. NT; **p < 0.01 vs. NT+IL-6.

Fig 3: PCSK9i counteract IL-6-induced inflammation. Evaluation of (A) NLRP3 protein expression and (B) content, (C) PCSK9 protein expression, caspase-1 (D) expression and (E) content, IL-1β (F) expression and (G) content, TNF-α (H) expression and (I) content, (J) IFN-γ levels and (K) NF-κB expression levels in EC treated for 24h with 20 ng/mL IL-6 (IL-6), with 100 µg/mL PCSK9i (PCSK9i) or pre-treated for 8h with 100 µg/mL PCSK9i before being exposed to 20 ng/mL IL-6 for 24h (PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Immunoblotting analyses are represented as floating bars with line representing the mean of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). Lane 1 = Ctr; lane 2 = IL-6; lane 3 = PCSK9i; lane 4 = PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). §p < 0.001 vs. Ctr; **p < 0.01 vs. IL-6.

Fig 4: IL-6 neutralizing antibodies blocked the growth and radioresistance induced by CAFs.A Western blotting analysis of STAT3 and p-STAT3 levels in T47D, MCF-7, and MDA-MB-231 cells. CAF conditioned medium alone or in combination with IL-6 neutralizing antibody (500 ng/ml) was used to treat the cells. B, C Colony formation assay showing that treatment with IL-6 neutralizing antibodies blocked the cell growth induced by CAFs. D Clonogenic survival assays showing that treatment with IL-6 neutralizing antibodies abrogated the radioresistance induced by CAFs. Results are displayed as means ± SD, n = 3, *P < 0.05, **P < 0.01.

Fig 5: Modified PD mitigated inflammation in DSS-induced UC in vivo. Colon tissues of negative control group, model group (DSS-treated) and modified PD group (DSS-treated with modified PD treatment) were collected. (A) qRT-PCR results indicating modified PD treatment reduced mRNA level of IL-6, VEGF and TNF-α (Modified PD represent high dose modified PD treatment mice herein). (B) Effect of modified PD on the protein expression of NLRP3, STAT3, p-STAT3, NF-κB, p-NF-κB, p-p65, IL-1β, IL-6 and TNF-α. Protein expression levels were analyzed by Western Blot. (C) The quantitative analysis for western blot. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Data are expressed as mean ± SEM.