Additional file 1 of Ackr3-Venus knock-in mouse lights up brain vasculature

Additional file 1: Figure S1. G protein activation profile. A–C HEK-293 cells were co-transfected with the indicated receptor (hDOR, mACKR3 or mACKR3-Venus), Gβ1, hGγ3-RlucII (BRET2 sensor donor), hGRK2-GFP10 (BRET2 sensor acceptor) and the indicated human (h) or mouse (m) Gα subunits. Mock condition is with cells not co-transfected with a Gα subunit encoding cDNA, thus detecting receptor-mediated activation of endogenous G proteins. A G protein activation profile of the human Delta opioid receptor (hDOR) following a 10 min stimulation with Met-Enkephalin (30 µM). B, C G protein activation profile of the untagged (ACKR3) or Venus-tagged (ACKR3-Venus) mouse ACKR3 receptor following a 10 min stimulation with CXCL12 (1 µM). Data are expressed as the mean ± SEM of 3 independent experiments. D HEK-293 cells were transiently transfected with ACKR3 or ACKR3-Venus and stimulated with 50 nM CXCL11-12AF647 for 20 min at 37 °C and scavenging was assessed by confocal microscopy. Nuclei were stained with DAPI. Cells not expressing ACKR3 or ACKR3-Venus (on the right, DAPI positive) did not take up CXCL11-12. Figure S2. H&E staining of ACKR3-Venus expression in peripheral mouse organs. Representative images of amplified ACKR3-Venus expression from eight peripheral tissues prepared from adult (P56 to P72) female and male mice. Organs from Ackr3Venus/Venus mice were sectioned transversely (4µm), and the expression of Venus was revealed with a mouse Venus (GFP) antibody (brown) and counterstained with hematoxylin labeling of cell nuclei (blue) and eosin labelling of cytoplasm (pink). Shown are representative images of organs where ACKR3-Venus is observed A heart (male) B stomach (female) C intestine (male) D kidney (female) E Thymus (female) F colon (female) G lung (male) H Fat (male). Arrowheads are located at regions with ACKR3-Venus expression. A yellow box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) and 20× (inset) objectives. Scale bar is 25µm. Figure S3. ACKR3-Venus expression in spleen. Representative images of amplified ACKR3-Venus expression from adult (P56 to P72) female and male mice. Organs from Ackr3Venus/Venus mice were sectioned transversely (4 µm) and the expression of Venus was revealed with a mouse Venus (GFP) antibody. The consecutive slices were incubated with hematoxylin and eosin (H&E) staining to reveal the histological structure of the spleen. Shown are representative images where ACKR3-Venus is observed, A Red pulp B Peripheral region of white pulp C Central region of white pulp D Marginal zone E Red pulp (H&E) F Peripheral region of white pulp (H&E) G Central region of white pulp (H&E) H Marginal zone (H&E). Arrowheads are located at regions with ACKR3-Venus expression. A red or yellow box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) and 20× (inset) objectives. Scale bar is 25µm. Figure S4. ACKR3-Venus expression in female peripheral organs. Representative images of amplified ACKR3-Venus expression from twelve peripheral tissues prepared from adult (P56 to P72) female mice. In addition to the eight organs which show considerable ACKR3-Venus expression (see Figure 5), shown here are all 12 organs side-by-side with the comparable control Ackr3WT/WT samples. Organs from Ackr3Venus/Venus mice were sectioned transversely (4 µm), and the expression of Venus was revealed with a mouse Venus (GFP) antibody (brown) and counterstained with hematoxylin labeling of cell nuclei (blue). Shown are representative images of the following organs A fat B stomach C intestine D colon E liver F lung G pancreas H heart I thymus J kidney K uterine horns L–N spleen L red pulp M marginal zone N white pulp. Expression in Ackr3Venus/Venus (n = 3) were compared to any background staining observed in Ackr3WT/WT (n = 3). A red box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) 20× (inset) objectives. Scale bar is 25 µm. Figure S5. H&E staining with ACKR3-Venus expression in female peripheral organs. Representative images of amplified ACKR3-Venus expression from twelve peripheral tissues prepared from adult (P56 to P72) female mice. In addition to the eight organs which show considerable ACKR3-Venus expression (see Figure 4 and S3), shown here are all 12 organs side-by-side with the comparable control Ackr3WT/WT samples. Organs from Ackr3Venus/Venus mice were sectioned transversely (4 µm), and the expression of Venus was revealed with a mouse Venus (GFP) antibody (brown) and counterstained with hematoxylin and eosin (H&E) staining to reveal the histological structure. Shown are representative images of the following organs A fat B stomach C intestine D colon E liver F lung G pancreas H heart I thymus J kidney K uterine horns L–N spleen L red pulp M marginal zone N white pulp. Expression in Ackr3Venus/Venus (n = 3) were compared to any background staining observed in Ackr3WT/WT (n = 3). A yellow box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) 20× (inset) objectives. Scale bar is 25 µm. Figure S6. ACKR3-Venus expression in male peripheral organs. Representative images of amplified ACKR3-Venus expression from thirteen peripheral tissues prepared from adult (P56 to P72) male mice. In addition to the eight tissues which show considerable ACKR3-Venus expression (see Figure 4 and S3), shown here are all 13 organs side-by-side with the comparable control Ackr3WT/WT samples. Organs from Ackr3Venus/Venus mice were sectioned transversely (4 µm), and the expression of Venus was detected with a mouse Venus (GFP) antibody (brown) and counterstained with hematoxylin labeling of cell nuclei (blue). Shown are representative images of the following organs A fat B stomach C intestine D colon E liver F lung G pancreas H heart I thymus J kidney K testis L seminal vesicles M–O spleen M red pulp N marginal zone O white pulp. Expression in Ackr3Venus/Venus (n = 3) were compared to any background staining observed in Ackr3WT/WT (n = 3). A red box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) 20× (inset) objective. Scale bar is 100µm. Figure S7. H&E staining with ACKR3-Venus expression in male peripheral organs. Representative images of amplified ACKR3-Venus expression from twelve peripheral tissues prepared from adult (P56 to P72) male mice. In addition to the eight organs which show considerable ACKR3-Venus expression (see Figure 4 and S3), shown here are all 13 organs side-by-side with the comparable control Ackr3WT/WT samples. Organs from Ackr3Venus/Venus mice were sectioned transversely (4µm), and the expression of Venus was revealed with a mouse Venus (GFP) antibody (brown) and counterstained with hematoxylin and eosin (H&E) staining to reveal the histological structure. Shown are representative images of the following organs A fat B stomach C intestine D colon E liver F lung G pancreas H heart I thymus J kidney K testis L seminal vesicles M–O spleen M red pulp N marginal zone O white pulp. Expression in Ackr3Venus/Venus (n = 3) were compared to any background staining observed in Ackr3WT/WT (n = 3). A yellow box on the inset indicates where the higher magnification image was taken. Organ sections were imaged on the Hamamatsu’s NanoZoomer® Digital Pathology system 2HT with 80× (large image) 20× (inset) objectives. Scale bar is 25 µm. Figure S8. Intrinsic and amplified cellular expression of ACKR3-Venus in the adult brain of Ackr3WT/WT mice. To check for non-specific tissue signals generated by naturally occurring autofluorescence or non-specific interactions between antibodies, we immunostained Ackr3WT/WT brain sections (n = 4) and observed negligible background signals. Images were taken on Olympus IX73 epifluorescent microscope, 10× objective. Scale bar is 200 µm. Figure S9. Oligodendrocyte progenitor cell staining in Ackr3-Venus adult mouse brain. To examine oligodendrocyte progenitor cells, brain sections from Ackr3Venus/Venus mice were stained with anti-NG2 (cyan), a marker of oligodendrocytes progenitor cells and anti-Venus (red). Shown regions include A the olfactory bulb (OB), B orbital frontal cortex (OFC) and C Hippocampus (HPF). Amplified Venus and NG2 expression in brain sections were imaged on an Olympus FV1200 laser scanning confocal microscope, 20× objective. White boxes (left column) correspond to the 5× magnified images. Insets contain mouse brain reference atlas images from Allen Brain Atlas, with the region locations outlined by black boxes. Scale bar of 20× image is 100 µm (1st column) and 20 µm (2nd–3rd columns).

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