Fig. 3: TFEC induced Gpnmb expression in macrophages during atherosclerosis progression.

A mRNA levels of MITF (left) and TFEC (right) in human carotid artery atheromatous plaques and control intact tissue from hypertensive patients (mean ± SEM, n = 32, based on dataset GDS5083). B The relationships between Gpnmb expression with Mitf expression (left) or Tfec expression (right) in human carotid artery atheromatous plaques (based on dataset GDS5083). C The relationships between Gpnmb expression with Mitf expression (left) or Tfec expression (right) in macrophages from human carotid plaque (based on dataset GSE21545, n = 126). Representative western blot images showing increased expression of GPNMB and TFEC in the aortas of ApoE^−/− mice fed with a WD (D) and WT BMDMs treated with OxLDL (E) (mean ± SEM, n = 3). F Representative images from immunofluorescence of TFEC (magenta) with Hoechst-labeled cell nucleus (cyan) in WT BMDMs treated with or without OxLDL and corresponding models built by Imaris (left). The quantitative analysis of total fluorescence intensity (right) and the ratio of nucleus fluorescence intensity to cytoplasm fluorescence intensity (middle) of TFEC, N = 3 independent experiments, mean ± SEM, n = 26 in NC group, n = 34 in OxLDL group. G, H 293T cells were transfected with pEGFP-TFEC plasmid, pEGFP-MITF plasmid, pEGFP-TFEB plasmid, or pEGFP-TFE3 plasmid, or co-transfected with pEGFP-TFEC plasmid and one plasmid of the other three plasmids. Representative western blot images showing the expression of GPNMB and the overexpression efficacy of these plasmids with an antibody against GFP (G) and the quantitative analysis (H, mean ± SEM, n = 3). I Activation of Gpnmb promoter in HEK293T cells transiently expressing pEGFP-TFEC plasmid, pEGFP-MITF plasmid, pEGFP-TFEB plasmid, pEGFP-TFE3 plasmid, or negative control plasmid was measured using a dual luciferase reporter assay (mean ± SEM, n = 3). J Representative images from immunofluorescence of TFEC (magenta) with Hoechst-labeled cell nucleus (cyan) in WT BMDMs transfected with siRNA against Tfec (siTfec) or nontarget control siRNA (siNC) and the quantitative analysis of total fluorescence intensity of TFEC, N = 3 independent experiments (mean ± SEM, n = 23 in siNC group, n = 20 in siTfec group). K Quantitative PCR analysis of Tfec and Gpnmb expression in WT BMDMs transfected with siTfec or siNC (mean ± SEM, n = 4). L Representative western blot images showing the expression of GPNMB and TFEC in WT BMDMs transfected with siTfec or siNC (left) and the quantitative analysis (right) (mean ± SEM, n = 3). P values were calculated by unpaired Student t-test (A, D, E, F, and J–L) or one-way ANOVA (H–I). Differences are significant for *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001. Spearman rank correlation test, P < 0.05 was regarded as the statistical criterion to set thresholds (B, C).