Figure 4

CHSE cells accumulate significant amounts of CpGs in endolysosomes but fail to relocate transgenic SsTLR9 into these compartments; similarly, TO cells, a salmon head kidney-derived cell line do not translocate CpGs into the CpG-containing compartments. A, Live CHSE cells at different time points after addition of Cy5-CpGs (red). Late endosomes and lysosomes are stained with LysoSensor Green (LSG). Endocytosis of the CpG ODNs is visible within 15 min in small vesicles without clear colocalization with the LysoSensor-stained endolysosomes. Colocalization with LysoSensor Green is detectable within 60 min indicating transition of the endocytosed CpGs from early to late endosomes and lysosomes. Within 24 hours, significant amounts of CpGs are detected both in acidic vesicles and in tubular structures, the later of which most likely represent tubular endosomes/lysosomes. B. Unlike the endogenous SsTLR9 detected in primary cells (Figure 2), SsTLR9 overexpressed in CHSE cells does not colocalize with CpGs. To label transfected cells as an indicator of the specificity of the staining, the plasmid expressing TLR9 was mixed with a plasmid expressing EGFP (green). Cy5-CpGs are shown in blue pseudocolor. C, same is observed in salmon TO cells, a poorly-transfectable cell line derived from salmon head kidney. BF stands for bright field. The magnified areas are indicated with dashed boxes.