Fig 1: 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 2: 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; ***).
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