Figure 6: DENV-ADE infection of K562 induces greater autophagy to facilitate viral production. | Scientific Reports

Figure 6: DENV-ADE infection of K562 induces greater autophagy to facilitate viral production.

From: Antibody-dependent enhancement of dengue virus infection inhibits RLR-mediated Type-I IFN-independent signalling through upregulation of cellular autophagy

Figure 6

K562 cells pretreated with different concentrations of rapamycin (a) or 3-MA (b) 1 h before infection with DENV-3 or DENV-ADE. Forty-eight hours post-infection, intracellular DENV RNA was extracted and quantified using qRT-PCR. N = 3; error bars show the means ± SEM. (c) K562 cells were infected with DENV alone, DENV-antibody complex, or mock. At both 0.5 h and 1 h post-inoculation, cellular RNA was extracted and used for gene expression quantification of ATG5 and ATG12 using qRT-PCR. N = 3; error bars show the means ± SEM, the results from each group were compared using Student’s t test. **P < 0.01; *P < 0.05. (d) K562 cells were infected with DENV alone, DENV-antibody complex, or mock. Cell lysates were subjected to detection of LC3 I/II, ATG5 and β-actin at the indicated time points. N > 3. (e) K562 cells transfected with paired sgRNA (1.1 + 1.2, 1.1 + 2.3) targeting the ATG5 locus were assayed by PCR (using the ATG5-Out-Fwd and ATG5-Out-Rev primers). Clonal isolates carrying a homozygous deletion in the ATG5 locus was were using PCR and sequencing. (f) Each isolate was subjected to DENV-ADE/DENV infection. Intracellular dengue genome RNA was quantified using the qRT-PCR method with GAPDH as an internal control, and the fold enhancement in each K562 isolate was expressed as a ratio of DENV/DENV-ADE. N = 3; error bars show the means ± SEM, the results from each group were compared using Student’s t test. **P < 0.01; *P < 0.05. K562 cells were stably transfected with plasmid expressing ATG5 or empty plasmid (control). All cell lines were subjected to DENV and DENV-ADE infection. (g) 4 h post-inoculation, cell lysates were subjected to detection of iκ-B, NF-κB, NOS2 and β-actin. N = 3. (h) At 48 h post-inoculation, cells were harvested for total RNA, and the intracellular viral RNA was detected using qRT-PCR with GAPDH as an internal control. N = 3; error bars show the means ± SEM, the results from each group were compared using Student’s t test. **P < 0.01; *P < 0.05.

Back to article page