Figure 2.

SpCas9 plasmid transfection of IMhu cells results in Cas9 expression but does not induce cytokine responses. (A) Experimental Streptococcus pyogenes (Sp) Cas9 expression plasmid (SpCas9 plasmid) and non-coding control plasmid (NC plasmid) used to assess the effect of Cas9 on IMhu cells. In the NC plasmid the Cas9 encoding sequence is removed. (B) Immunostaining of Cas9 in transfected IMhu cells. Cells were separately transfected with either the SpCas9 plasmid or the NC plasmid. At 24 h post-transfection, cells were stained with Cas9 antibodies (red) and DAPI (nuclei, blue). SpCas9 plasmid-transfected IMhu cells expressed Cas9 after SpCas9 plasmid transfection, whereas NC plasmid-transfected cells did not express Cas9. Data are representative of three independent experiments. Scale bar 100 µm. NLS nuclear localization sequence, ori origin of replication, AmpR ampicillin resistance gene, FLAG protein tag. (C) Heatmap of cytokine levels in IMhu supernatant 24 h after transfection with the SpCas9 plasmid. Cytokine levels were determined using Proteome Profiler Antibody Arrays. Colors represent cytokine ratios of SpCas9 plasmid-transfected versus L3000 treated cells (left column) or NC plasmid-transfected versus L3000 treated cells (right column) respectively.