Absence of miR‐21 in macrophages promotes foam cell formation Absence of miR‐21 in macrophages promotes foam cell formation Representative images of WT or miR‐21−/− peritoneal macrophages incubated with or without Ac‐LDL (120 μg cholesterol/ml) for 24 h and stained with ORO. Quantification of the ORO‐positive area and integrated optical density (IOD) per cell is shown in the right panels. Data represent the mean ± SEM of the quantification of three images from different fields (n = 3 per group; *P = 0.0126 for ORO and *P = 0.0148 for IOD). Scale bars, 100 μm. Level of significance was determined using one‐way ANOVA with Bonferroni's post‐test.Quantification of total cholesterol (TC) (left panel), free cholesterol (FC) (middle panel), and cholesterol esters (CE) (right panel) corrected by cell protein concentration of WT and miR‐21−/− peritoneal macrophages incubated as in (A). Data represent the mean ± SEM of duplicates (n = 3 per group; *P = 0.022 for TC, *P = 0.042 for FC and *P = 0.025 for CE). Level of significance was determined using one‐way ANOVA with Bonferroni's post‐test.Flow cytometry analysis of DiI‐Ox‐LDL uptake in peritoneal macrophages of WT and miR‐21−/− mice incubated with DiI‐Ox‐LDL (30 μg cholesterol/ml) for 2 h at 37°C. The results are expressed in terms of geometric mean fluorescence intensity (M.F.I.) after subtracting the autofluorescence of cells incubated in the absence of DiI‐Ox‐LDL. Data represent the mean ± SEM of duplicates (n = 3 per group)Cholesterol efflux to apolipoprotein A1 (ApoA1) and HDL in peritoneal macrophages isolated from WT and miR‐21−/− mice stimulated with or without T0901317 (T090). Data represent the mean ± SEM of duplicate samples (n = 3 per group; *P = 0.0417). Level of significance was determined using t‐test. Representative Western blot analysis of ABCG1 and ABCA1 in WT and miR‐21−/− peritoneal macrophages treated with Ac‐LDL (120 μg/ml) for 12 and 24 h. Samples from three different mice per group are shown. Bottom panels show the quantification of band densitometry values of ABCG1 and ABCA1 protein levels expressed in arbitrary units after correction for HSP90 (loading control). Data represent the mean ± SD from blots above (n = 3 per group as indicated; *P = 0.0002 for 12 h and *P = 0.0003 for 24 h). Level of significance was determined using one?way ANOVA with Bonferroni's post?test. Representative Western blot analysis of ABCG1 and ABCA1 of WT and miR‐21−/− peritoneal macrophages treated with Ac‐LDL (120 μg/ml) in the presence of p38 inhibitor (SB202190) for 12 and 24 h. Representative experiments out of three with similar results. Relative protein levels were determined by band densitometry and are expressed in arbitrary units after correction for HSP90 (loading control). n.d., not detectable.Representative Western blot analysis of ABCG1 in WT and miR‐21−/− peritoneal macrophages treated with Ac‐LDL (120 μg/ml) in presence of the proteasome inhibitor MG132 (10 μM) for 12 h. Source data are available online for this figure. Alberto Canfrán‐Duque et al. EMBO Mol Med. 2017;9:1244-1262 © as stated in the article, figure or figure legend