Fig. 2: Knockdown of STK25 suppresses inflammation and fibrosis in human aortic endothelial and smooth muscle cells.

hASMCs and hAECs were transfected with STK25 siRNA or NTC siRNA. a, g Representative immunofluorescence images of cells stained with anti-NFκB antibody (green); nuclei stained with DAPI (violet); colocalization shown in white. Quantification of the ratio of nuclear to cytoplasmic staining. b, h Nuclear extracts were analyzed by Western blot using antibody specific for NFκB. Protein levels were analyzed by densitometry; representative Western blots are shown with lamin B (nuclear marker) and vinculin (cytoplasmic marker) used as controls. Cytoplasmic fraction was analyzed by Western blot using antibody specific for STK25 to demonstrate the transfection efficacy; representative Western blots are shown with vinculin used as a loading control. c, i Concentration of secreted cytokines and chemokines. d, j Representative images of calcein acetoxymethyl-stained THP-1 monocytes (green). Quantification of the staining. e, k Relative gene expression of selected fibrosis markers 24 h after TGF-β1 stimulation. f, l Concentration of collagens in media 24 h after TGF-β1 stimulation. In (a, g) and (d, j), the scale bars represent 10 and 50 µm, respectively. Data in (a, g) are mean ± SEM from 30 cells per group; data in (b–f, h–l) are mean ± SEM from 4-12 wells per group. OA, oleic acid. *P < 0.05, **P < 0.01, ***P < 0.001 for STK25 siRNA vs. NTC siRNA.