Fig. 3: MSR-1 scavenger receptor is involved in H-ferritin uptake by human macrophages.

a Representative confocal microscopy images of colocalization of HFt with AcLDL in human macrophages. hMDM were incubated simultaneously with HFt-AF488 (50 μg/ml) (green) and AcLDL-AF594 (5 μg/ml) (red) for 15 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and AcLDL (yellow foci) for hMDM. Scale bar = 20 μm (10 µm in zoomed region). b Flow cytometry analysis of internalized HFt-AF488 (5 μg/ml) by hMDM in the absence or presence of AcLDL at indicated concentrations within 30 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors. c Representative confocal microscopy images of internalized HFt-AF488 (5 μg/ml) (green) by hMDM in the absence or presence of AcLDL (100 μg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. d Flow cytometry analysis of internalized AcLDL-AF488 (1 μg/ml) by hMDM in the absence or presence of HFt at indicated concentrations within 30 min at 37 °C. Data are presented as mean fluorescence intensity (MFI) of AcLDL-AF488. Data are presented as mean ± SEM from n = 3 different donors. e Representative confocal microscopy images of internalized AcLDL-AF488 (1 μg/ml) (green) by hMDM in the absence or presence of HFt (2,5 mg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. f–h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I), poly(G) and poly(C) (control) (f), fucoidan and mannan (control) (g) and dextran sulfate or chondroitin sulfate (control) (h) Flow cytometry data are presented as % of HFt-AF488 uptake in untreated, control cells. Data are presented as mean ± SEM from n = 3 (f, h) or 6 (g) different donors (hMDM). i–k Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I) and poly(C) (control) (i), fucoidan and mannan (control) (j) and dextran sulfate or chondroitin sulfate (control) (k). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. l Quantitative real-time PCR analysis of MSR1, SCARA5, and MARCO mRNA expression in hMDM. Data are presented as mean ± SEM from n = 3 different donors (hMDM). m Western blot analysis of MSR1 protein expression in monocytes and hMDM. Representative western blot images are shown. n Flow cytometry analysis of MSR1 cell surface staining in hMDM upon stimulation with HFt (200 µg/ml) for indicated time points at 37 °C. For comparison, untreated cells were used (Control). Data are presented as mean ± SEM % of MSR1 expression in control cells, n = 3 independent donors (hMDM). o Western blot analysis of MSR1 protein expression in hMDM that were untreated (Control) or treated with cycloheximide (CHX) (20 µg/ml) for 1 h prior to HFt stimulation (200 µg/ml) (CHX+HFt) for the indicated time points at 37 °C. For comparison, cells treated only with CHX were used. Representative western blot images are shown. p Quantitative analysis of relative MSR1 expression in hMDM shown in (o). Data are presented as mean ± SEM from n = 3 independent replicates. q Western blot analysis showing MSR1 expression in either untransfected hMDM (Control) or cells transfected with one of the following siRNA sequences: scramble siRNA (siScr), no. 1 siRNA targeting MSR1 (siMSR1-1), no. 2 siRNA targeting MSR1 (siMSR1-2) at 72 h after transfection. r Quantitative analysis of western blot for MSR1 expression shown in (q). Data are presented as mean ± SEM % of MSR1 expression in control cells (Control), n = 3 different donors (hMDM). s and (t) Flow cytometry analysis of internalized AcLDL-AF488 (5 µg/ml) (s) or HFt-AF488 (100 μg/ml) (t) by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as mean ± SEM % of ligand uptake in control cells (Control), n = 3 different donors. u and (v) Representative confocal microscopy images of internalized AcLDL-AF488 (5 μg/ml) (green) (u) and HFt-AF488 (50 μg/ml) (green) (v) within 30 min at 37 °C by THP-1 macrophages after MSR1 gene-knockdown. Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. w Western blot analysis of MSR1 protein expression in hMDM macrophages polarized to M1 or M2 phenotypes. Representative western blot images are shown. x Relative expression of MSR1 in M1 and M2 hMDM macrophages quantified based on western blot analysis shown in (w). Data are presented as mean ± SEM from n = 4 different donors. y Flow cytometry analysis of internalized HFt-AF488 by M1 and M2 hMDM macrophages given at 25 µg/ml within 20 or 60 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 different donors. The one-way ANOVA and Dunnett’s post-hoc test were used for statistical analysis in panels b, d, r–t. The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panels f–h. The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis in panels (p, y). The Student’s t-test was used for statistical analysis in panel (x). For all panels, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file.