Fig 2: Silencing OSM inhibits the ox-LDL-induced p65-NLRP3 pathway. THP-1 macrophages transfected with si-NC or OSM siRNAs (si-OSM-1 and si-OSM-2) were treated with 40 µg/mL ox-LDL for 48 hours. Protein expression of p65 and NLRP3 was analyzed by Western blotting (A). NLRP3 expression was analyzed by immunofluorescence (magnification, ×400) (B). Protein expression of cleaved caspase-1, ASC, and GSDMD-N was analyzed by Western blotting (C). ASC, apoptosis-associated, speck-like protein containing a caspase-1 recruitment domain; GSDMD-N, gasdermin-D-N; NC, negative control; NLRP3, NLR family pyrin domain containing 3; OSM, oncostatin M; ox-LDL, oxidized low-density lipoprotein.

Fig 3: Silencing OSM inhibits inflammation via the inhibition of NLRP3. THP-1 macrophages transfected with OSM siRNA (si-OSM-2) alone or in combination with pcNLRP3 were treated with 40 µg/mL ox-LDL for 48 hours. TNF-a (A), IL-1ß (B), IL-6 (C), and IL-18 (D) levels in the cell supernatant were measured by ELISA kits. Protein expression of TNF-a, IL-1ß, IL-6, and IL-18 was analyzed by Western blotting (E). n=3. Data presented as mean ± SD. Statistical analysis was performed by ANOVA. *, P<0.05; **, P<0.01 vs. the indicated groups. ANOVA, one-way analysis of variance; ELISA, enzyme-linked immunosorbent assay; NC, negative control; NLRP3, NLR family pyrin domain containing 3; OSM, oncostatin M; ox-LDL, oxidized low-density lipoprotein; SD, standard deviation.

Fig 4: OSM is highly expressed in ox-LDL-treated THP-1 macrophages. THP-1 cells were treated with PMA to cause differentiation into macrophages. ox-LDL in concentrations of 10, 20, and 40 µg/mL was used to treat cells for 12, 24, and 48 hours. Untreated cells were used as controls. The mRNA (A,B), protein (C,D), and cell supernatant content (E,F) of OSM were measured by qRT-PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA), respectively. n=3. Data presented as mean ± SD. Statistical analysis was performed by ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001 vs. non-treated controls. ANOVA, one-way analysis of variance; ns, no significant; OSM, oncostatin M; ox-LDL, oxidized low-density lipoprotein; PMA, phorbol-12-myristate-13-acetate; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SD, standard deviation.

Fig 5: Silencing OSM inhibits foam cell formation via the inhibition of NLRP3. THP-1 macrophages transfected with OSM siRNA (si-OSM-2) alone or in combination with pcNLRP3 were treated with 40 µg/mL ox-LDL for 48 hours. The mRNA (A) and protein (B) levels of NLRP3 were measured by qRT-PCR and Western blotting, respectively. Lipid accumulation (C) and total cholesterol content (D) were measured by Oil Red O staining and a commercial kit, respectively. Scale bars =20 µm. n=3. Data presented as mean ± SD. Statistical analysis was performed by ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001 vs. the indicated groups. ANOVA, one-way analysis of variance; NC, negative control; NLRP3, NLR family pyrin domain containing 3; OSM, oncostatin M; ox-LDL, oxidized low-density lipoprotein; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SD, standard deviation.