Fig. 8

Downregulation of NDUFS2 leads to pro-inflammatory phenotype and disrupted phagocytosis in macrophages. The protein levels of NDUFS2 (red) and HSP60 (green) were measured by immunofluorescence staining in TP cells (a) and RAW264.7 cells (b). Scale bars = 20 μm for representative images at low magnification in the white solid box. Scale bars = 10 μm for enlarged image at higher magnification. The protein levels of NDUFS2 (red) in AMs (marked by HSP60, green) was quantified as red fluorescence intensity. TP cells (c) and RAW264.7 cells (d) were transfected with or without 3 different pieces of siRNAs against NDUFS2/Ndufs2 for 48 h, when the mRNA and protein levels of NDUFS2/Ndufs2 were measured by qPCR (left) and western blotting (right). siNDUFS2-2 and siNdufs2-3 were selected for further experiments in TP cells and RAW264.7 cells, respectively. *P < 0.01, **P < 0.01 vs. siNC (Student’s t-test). e, f TP cells (e) and RAW264.7 cells (f) were transfected with siNDUFS2-2 and siNdufs2-3 for 48 h respectively y, when the mRNA levels of IL1B, IL6, TLR4, TGFB1, NLRP3, IFNG, IL23, STAT3, TNF, CD80, CXCL8, CXCL10, CXCL11, CXCL12, CCL2, CD209, MACRO, SIRPA were measured by qPCR. *P < 0.01, **P < 0.01 vs. siNC (Student’s t-test). g, h TP cells (g) and RAW264.7 cells (h) were transfected with siNDUFS2-2 and siNdufs2-3 for 48 h respectively, when the phagocytosis was assessed by Cell Meter™ Fluorimetric Phagocytosis Assay Kit. The images were taken under fluorescence microscopy (Scale bars = 20 μm). The red fluorescence intensity of phagocytosed beads reflects the digestion extent of the ingested particles in macrophages, while the average number of engulfed beads within every cell was calculated to indicate the engulfing ability of macrophages. **P < 0.01, ***P < 0.001 or vs. siNC. (Student’s t-test)