Fig 2: Roles of MMP9 and SPP1 in non-canonical NF-?B-mediated HCC invasion. (a) The PPI network was constructed to reveal the target genes of the non-canonical NF-?B pathway using the STRING online tool. (b–e) In the TCGA_LIHC cohort, gene expression of MMP9 (b) and SPP1 (d) in various HCC stages was quantified, and their influence on survival was analysed (c, e). (f, g) After treatment of HCC SMMC7721 cells with 0.25 µg mL-1 NETs DNA for 48 h, intracellular MMP9 and SPP1 were tested by Western blot assay (n = 3). (h) In the NETotic and control nude mice, DNASE1L3 knockdown (KD) and wild-type (WT) H22 cells were subcutaneously engrafted, respectively. Three weeks later, the expression of MMP9 and SPP1 was measured by Western blot assay (n = 3). Data are shown as mean ± SD. **P < 0.01 vs. WT in control group. # P < 0.05 vs. WT in NETotic group.

Fig 3: M1-EVs tsRNA-5006c inhibitor suppress osteogenic differentiation of AVICs.(A) Representative image of Alizarin red staining of AVICs pretreated with M1-EVs after tsRNA-5006c silencing. Scale bar: 50 µm. (B) The mRNA expression of RUNX2, BMP2, and SPP1 of osteogenesis-related genes in AVICs pretreated with M1-EVs after tsRNA-5006c silencing was detected by RT-qPCR. (C) The protein expression of RUNX2, BMP2, and osteopontin of osteogenesis-related genes in AVICs pretreated with M1-EVs after tsRNA-5006c silencing was detected by and western blot. (D) The protein expression of a-SMA and Collagen I of fibrotic markers in AVICs-pretreated with M1-EVs after tsRNA-5006c silencing was detected by western blot. NC is the negative control of tsRNA-5006c inhibitor. * P < 0.05, ** P < 0.01.

Fig 4: M1-EVs promote osteogenic differentiation of AVICs.(A) Representative image of DiI-labeled M1-EVs was internalized by AVICs. Scale bar: 50 µm. (B) Representative image of Alizarin red staining of AVICs pretreated with M1-EVs or NC- EVs. Scale bar: 50 µm. (C) The mRNA expression of RUNX2, BMP2, and SPP1 of osteogenesis-related genes in AVICs after incubated with M1-EVs was detected by RT-qPCR. (D) The protein expression of RUNX2, BMP2, and osteopontin of osteogenesis-related genes in AVICs after incubated with M1-EVs was detected by western blot. (E) The protein expression of a-SMA and Collagen I of fibrotic markers in AVICs after incubated with M1-EVs was detected by western blot. ns P > 0.05, * P < 0.05, ** P < 0.01.

Fig 5: Expression pattern of OPN in isolated smooth muscle cells of aneurysmal and control tissue. (A) depicts the determination of uncleaved OPN expression in isolated smooth muscle cells (full length 60 kDa bands). In (B) the quantification of the expression of the cleaved 40 kDa band from aneurysmal and control smooth muscle cells is shown. In (C) the quantification of the smooth muscle specific total OPN expression is depicted (60 kDa and 40 kDa). In (D) the corresponding images of Western blot results for the detection of full length and cleaved OPN, as well as GAPDH (37 kDa) as loading control are shown (Control n = 10; TAV n = 10; BAV n = 10) (** < 0.01; *** < 0.001).