Extraction of total and cytosolic DNA and mtDNA measurement

Total and cytosolic DNA extraction method was performed (Lai et al., 2018) with some modification. Cells (4 × 10⁶) were divided into two equal aliquots. One aliquot, for normalization control, was used to extract total DNA using a NucleoSpin Tissue kit (Macherey-Nagel, Duren, Germany). The other was resuspended in 400 μl of buffer containing 150 mM NaCl, 50 mM HEPES pH 7.4, and 25 mg/ml digitonin (EMD Chemicals, Gibbstown, NJ, USA). After rotaion end-over-end for 15–20 min at 4°C, the samples were then centrifuged at 980 × g for 3 min three times to remove cellular debris. The supernatants were collected and spun at 17,000 × g for 10 min to remove the remaining cellular residue and to yield cytosolic preparations free of nuclear, mitochondrial, and endoplasmic reticulum contamination. The DNA in the cytosol fraction was then isolated by running the sample through a NucleoSpin Tissue column (Macherey-Nagel) and subsequently eluted with buffer.

To measure the levels of mtDNA, 20 ng of isolated DNA was subjected to qPCR with KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) with 200 nM mtDNA primers (16S and ND5 for THP1 cells; 12S and D-loop for BMDCs) or nuclear DNA (nDNA) primers (B2M and TERT for THP-1 cells; actin and albumin for BMDCs). Two mtDNA primer pairs were used to quantify mtDNA. The levels of mtDNA in total cell lysates were calculated as mtDNA normalized to nDNA. To quantify mtDNA in the cytosolic fraction, 20 ng of a purified plasmid encoding the FLAG gene (PCR3.1-flag) was added to the eluted solution according to the protocol published by Aguirre et al. (Aguirre et al., 2017), except that the FLAG gene was used instead of the enhanced green fluorescent protein (EGFP) gene. The specific primers for both endogenous mtDNA and the FLAG plasmid were used to measure the relative content of cytosolic mtDNA expression normalized to that of FLAG (the primers for PCR are given in Table S1). The relative mtDNA abundance indicates the relative mtDNA content in DENV-infected cells normalized to that in mock-infected cells.