Deckglaser

Cells were imaged in quartz-top flow cells as described previously^{21 (link),32} . Briefly, flow cells were assembled using a clean quartz piece (Proscitech, Australia) and a bottom cleaned and (3-Aminopropyl)triethoxysilane (Alfa Aesar, A10668">A10668, UK)-treated cover-slip (Marienfeld, Deckglaser, 24 mm × 50 mm, No 1.5, German) using double-sided sticky tape (970XL ½ × 36 yd, 3 M, United States), and sealed with 5-min epoxy (Parfix). Quartz top pieces were designed to be able to insert inlet and outlet tubing (PE-60, Instech labs). Prior to imaging, cells were revived from a −80 °C DMSO stock in 500 μL of EZ-rich defined media (Teknova, CA, US), supplemented with 0.2% (v/v) glucose in 2-mL microcentrifuge tubes at 30 °C. Cultures were set to shake in an Eppendorf Thermomixer C (Eppendorf, Australia) at 1000 rpm. On the following day, cultures were reset by inoculating fresh growth medium 1:200 fold, and continued to shake for ~3 h at 30 °C prior to imaging. Cells expressing plasmid-expressed UvrB were grown in growth medium supplemented with spectinomycin (50 μg per mL) to ensure retention of the plasmid. Cells in early exponential phase were loaded in flow cells at 30 °C, followed by a constant supply of aerated EZ-rich defined media at a rate of 30 µL per min, using a syringe pump (Adelab Scientific, Australia).

Cells were imaged in quartz-top flow cells as described previously^{23 (link)}. Briefly, flow cells were assembled using a clean quartz piece (Proscitech, Australia) and a bottom cleaned and (3-Aminopropyl)triethoxysilane (Alfa Aesar, A10668, UK)-treated coverslip (Marienfeld, Deckglaser, 24 mm × 50 mm, No. 1.5, German) using double-sided sticky tape (970XL ½ × 36 yd, 3 M, United States), and sealed with 5-min epoxy (Parfix). Quartz top pieces were designed to be able to insert inlet and outlet tubing (PE-60, Instech Labs). Prior to imaging, cells were revived from a −80 °C DMSO stock in 500 μL of EZ-rich defined media (Teknova, CA, US), supplemented with 0.2% (v/v) glucose in 2-mL microcentrifuge tubes at 30 °C. Cultures were set to shake in an Eppendorf Thermomixer C (Eppendorf, Australia) at 1000 rpm. On the following day, cultures were reset by inoculating fresh growth medium 1:200 fold, and continued to shake for ~3 h at 30 °C prior to imaging. For experiments involving plasmid-expressed UvrA-YPet or UvrA(Δ131–250)-YPet, spectinomycin (50 μg per mL) was added to the growth media. Cells in early exponential phase were loaded in flow cells at 30 °C, followed by a constant supply of aerated EZ-rich defined media at a rate of 30 µL per min, using a syringe pump (Adelab Scientific, Australia).

The A549 cells were seeded into 6-well plates containing cover glasses (22 mm × 22 mm, Deckglaser, Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and incubated at 37 °C for 24 h. The cells were infected by DENV2 for 2 h and then were replaced with 10% FBS DMEM [31 (link)]. The cells were fixed in 3.7% formaldehyde for 30 min. After washing with phosphate-buffered saline (PBS), the cells were incubated for 30 min in 0.1% Triton X-100 in PBS. The primary antibodies were added to the plate and incubated at 4 °C overnight. The primary antibodies LC3B (MBL), capsid (GeneTex), envelope (GeneTex), NS1 (Sigma), NS3 (GeneTex) and NS4B (GeneTex) were used to detect specific proteins. Hoechst (5 mg/mL; Sigma) was used at a dilution of 1:500 in PBS to stain the nucleus. The secondary antibodies: Anti-Mouse IgG Antibody (FITC or PE conjugate) and anti-Rabbit IgG Antibody (FITC or PE conjugate) were used and the mounting media is glycerol gelatin aqueous slide mounting medium (Sigma-Aldrich). The fluorescent change of the cells was detected under a multi-photon confocal microscope (Olympus, FV1000MPE, Tokyo, Japan).