Table 1.
Amino acid sequences of peptides that bind human and mouse CD177.
Table 2.
Quantitative phage peptide binding assay.
Fig 1.
HPLNs displaying Peptide H bind human CD177 on cells.
CHO cells expressing human CD177 were incubated for 3 h at 37°C with 75 μg/ml Peptide H-HPLNs. Cells were fixed and permeabilized, and CD177 was detected using a mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The HPLN particles are fluorescent red. As a negative control, HPLN particles displaying a scrambled peptide were used. The experiment was carried out twice, with at least 100 cells examined in each experiment. A direct correlation between the expression level of CD177 (based on fluorescence intensity) and the Peptide H-HPLN signal could be observed. The cell in the lower panel shows a typical cell distribution of CD177 and Peptide H-HPLNs after 3 h incubation. In contrast, the two cells shown in the upper panel are characteristic of the CD177 staining in the absence of Peptide H-HPLNs and in the presence of Scrambled Peptide H-HPLNs. Scale bar, 50 μm.
Fig 2.
Some Peptide H-HPLN particles co-localize with LAMP2 after prolonged incubation, suggesting delivery into the lysosomes.
CHO cells expressing human CD177 were incubated for 3 h and 17 h at 37°C with 75 μg/ml Peptide H-HPLN particles. Cells were fixed and permeabilized, and the lysosomes were stained with a mouse anti-hamster LAMP2 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. The selected micrographs are representative of more than 100 cells with similar labeling patterns. Scale bar, 50 μm.
Fig 3.
HPLN particles displaying Peptide M become internalized by CHO cells expressing mouse CD177-HA.
CHO cells expressing mouse CD177 containing an HA-tag were incubated with 75 μg/ml Peptide M-HPLN particles for 2 h on ice to allow binding. Cells were then washed and kept on ice or warmed to 37°C for 1 h. Cells were then treated with subtilisin to remove surface-bound Peptide M-HPLN particles, or treated with buffer alone. After fixation and permeabilization, mouse CD177-HA was stained with a mouse anti-HA antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. Most cells out of more than 100 cells visualized on the slides had a similar staining pattern as those seen in these micrographs. Scale bar, 50 μm.
Fig 4.
Peptide H-HPLN particles bind CD177-expressing human neutrophils in whole blood.
Peptide H-HPLN particles were incubated with whole human blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. Scrambled Peptide H-HPLN particles were used as a negative control. The experiment was carried out four times using blood from four different donors. In each case, slides with several hundred cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.
Fig 5.
Peptide M-HPLN particles bind CD177-expressing mouse neutrophils in whole blood.
Peptide M-HPLN particles were incubated with whole mouse blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with rabbit anti-mouse CD177 polyclonal antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody. Peptide H-HPLN particles were used as a negative control. The experiment was carried out twice. The micrographs represent the results from >100 cells. Scale bar, 10 μm.
Fig 6.
Flow cytometry confirms Peptide H-HPLN binding to CD177-positive neutrophils in a pool of purified human leukocytes.
Leukocytes were incubated on ice in the presence or absence of anti-CD177 antibody, washed and incubated on ice in the presence or absence of Peptide H-HPLN and Alexa Fluor 488 goat anti-mouse antibody. A. Forward and side scatter plot of human leukocytes (and remaining red blood cells) with the neutrophil population shown in a circle. B. FL-1 histogram of CD177 expression. Left panel: Total cell population in the absence and presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). Right panel: Gated neutrophil population in the absence or presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). C. FL2 plot showing Peptide H-HPLN binding. Total cell population in the absence or presence of Peptide H-HPLNs (left panel) and gated neutrophil population in the absence and presence of Peptide H-HPLNs (right panel). D. FL1-A and FL2-A plot showing fluorescence of the total cell population (left panel) and gated neutrophil population (right panel). The experiment was carried out twice with similar results. The blood sample shown had a higher percentage of CD177-positive neutrophils than the blood sample from the other donor.
Fig 7.
Time course of CD177 and Peptide H-HPLN redistribution in human neutrophils.
Purified human neutrophils were incubated with Peptide H-HPLNs at 37°C with aliquots taken at 0, 15, 30, 60 and 120 min. Cells were rinsed with PBS, centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once using blood from a donor with a high CD177 expression level. Hundreds of cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.
Fig 8.
Mouse C5aR1 siRNA and human C5aR1 siRNA pool result in receptor knockdown.
CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.
Fig 9.
Mouse C5aR1 ASO results in knockdown of mouse C5aR1-GFP in CHO transfectants.
CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells (p value <0.0001; ***).