Fig. 3.

UPR signaling regulates arsenic trioxide-inhibited bacterial engulfment and clearance of E. coli (K-12 strain) BioParticles® fluorescents conjugated bacteria by murine macrophage. (A) Raw 264.7 cells were treated with ATO (2 μM for 16 h) alone as well as in combination of PBA (1 mM for 24 h) followed by incubation with latex beads coated with fluorescently labeled rabbit-IgG for 45 min at 37 °C. Engulfed fluorescent-beads were observed using a fluorescent microscope. Intensity of green fluorescence indicates the engulfed beads. (B) Mean fluorescence intensity (MFI) of engulfed fluorescence beads were recorded in 96 wells black bottom plate at excitation 485 nm and emission at 535 nm by microplate reader. Saline-treated cells receiving an identical treatment with similar procedure as described above except for the incubation temperature which was 4 °C instead of 37 °C served as negative control. (C) Infection load of opsonized E. coli (K-12 strain) BioParticles® fluorescents conjugated bacteria in Raw 264.7 cells-treated with ATO (2 μM for 24 h) in the presence and absence of PBA and NAC were observed under fluorescent microscopy. At the end of treatment, cells were incubated with fluorescent tagged E. coli bacteria (Bacteria:cells 50:1 ratio) at 37 °C for 2 h and baseline was imaged under the fluorescent microscope. Cells were further incubated at 37 °C to complete the process of bacterial clearance. After 24 h, cells were recaptured for fluorescent imaging. (C-I) Saline-treated control cells effectively cleared the fluorescence tagged E. coli bacteria within 24 h. (C-II) ATO-treated macrophage could not clear this bacterial load and continued to show the presence of fluorescent tagged opsonized bacteria. (C-III & IV) PBA and NAC pretreatment of Raw 264.7 cells restored the ATO-impaired clearance/phagocytosis of engulfed bacteria.