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. 2021 Mar 5;12:1460. doi: 10.1038/s41467-021-21617-2

Fig. 2. Arginase-2 regulates dynamics at mitochondria in response to IL-10 in inflammatory macrophages.

Fig. 2

a IVTT assay for mitochondrial import of pCMV-Arg1 and pCMV-Arg2. Gels were exposed to phosphor-scene for detection of imported proteins. 1 μl of complete IVTT protein was loaded as input control (lanes 1 and 6). Time course over 60 min showing bands representing proteins bound to and found inside the mitochondrial membrane. FCCP is used as a depolarising control. Radiograph representative of three experiments ran and processed in parallel. Graph shows densitometry analysis of the pooled experiments by measuring relative expression. b Immunoblot of Arg1 and Arg2 in fractionated Il10–/– BMDM that were unstimulated (CTL), or stimulated with LPS, LPS + IL-10, or IL-10 alone. VDAC and β-tubulin are used as mitochondrial and cytoplasmic controls, respectively. Representative of three independent experiments. c ImageStream showing colocalization levels of Tom20 and Arg2 in BMDM that were unstimulated (CTL), or stimulated with LPS, or LPS + IL-10. (Left) Representative images shown from ~1000 events. Channels shown are bright field (Ch01), Tom20 (green/Ch02), Arg2 (red/Ch11), and a merge. Original magnification, ×60. (Right) Graph showing colocalization index of Tom20 and Arg2 in the different treatments over two independent biological experiments. Each data point represents median index of ~1000 processed images. d–f BMDM were either left unstimulated (CTL), or stimulated with LPS, LPS + IL-10 or IL-10 alone, and stained with MitoTracker Red-CMXRos. Mitochondrial morphology was observed by confocal microscopy. Lower panels show a higher magnification of the image within the white squares. Arrows show mitochondrial fusion and fission. Graphs show results of mitochondrial morphology analysis as a mean of n = 3 independent biological experiments. Scale bars represent 5 µm. d Wild-type BMDM, e BMDM pre-treated with DMSO or nor-NOHA and f Arg2+/+ and Arg2–/– BMDM. a, d–f Data shown with error bars represents ± SEM. d–f Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for multiple comparisons. P-values are indicated on graphs where there is significance between elongated mitochondrial fractions.