Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Expression of IFNs in mock-treated or poly(I∶C) pretreated HepG2 cells.

<p>After pretreatment with poly(I∶C) for 9 h, HepG2 cells were infected with DENV2 at an MOI 1. The cells were harvested at 0, 6, 12, and 24 h p.i. for RNA extraction and real-time RT-PCR for IFN-β (A) or IL-28A/B (B). Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, **, p<0.01; ***, p<0.001.</p

IKK inhibitor reversed the IFN-β induction and virus replication suppression mediated by poly(I∶C) pretreatment.

<p>HepG2 cells were incubated in control or IKK inhibitor for 30 min before poly(I∶C) pretreatment and virus infection. (A) Cells were harvested for measurement of IFN-β expression level; (B) Immunofluorescence microscopy. Cells were fixed at 24 h p.i. and incubated with DENV2 prM antibody. Panel a, mock infected; b, DENV2; c, DMSO+poly(I∶C)+DENV2; d, BMS345541+poly(I∶C)+DENV2; (C) Percentages of positive-stained cells determined by PicCnt 100×; (D) Extracellular viral production determined by TCID50. Supernatants were harvested at 24 h p.i. and titered on C6/36 cells. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, *, p<0.05; **, p<0.01; ***, p<0.001.</p

Pretreatment of poly(I∶C) suppresses the DENV2 replication in HepG2 cells.

<p>HepG2 cells were mock- or pretreated with 5 µg poly (I∶C) for 9 h, then infected with DENV2. (A) Real-time PCR to measure viral mRNA levels. Total RNAs were harvested at 2, 6, 12, and 24 h p.i., and used for real-time RT-PCR. (B) Immunofluorescence microscopy. Infected cells were fixed at 24 h p.i. and incubated with DENV2 prM antibody. (C) Percentages of positive-stained cells determined by PicCnt 100×. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, *, p<0.05; ***, p<0.001.</p

DENV2 replication levels in IFN-β neutralizing antibody treated HepG2 cells.

<p>500 unit/ml IFN-β neutralizing antibody was added at 1 h before poly(I∶C) treatment and kept through poly(I∶C) treatment and virus infection. (A) Real-time PCR detecting the expression of viral mRNA; (B) Extracellular viral production determined by TCID50. Supernatants were harvested at 24 h p.i. and titered on C6/36 cells. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, **, p<0.01; ***, p<0.001.</p

miR-155 induces autophagy in macrophages.

<p>(A–D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). The LC3 levels were detected by Western-blot, and the ratios of LC3-II/β-actin were calculated as shown below the representative blot (C and D). (E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.</p

miR-155 expression is induced after mycobacterial infection.

<p>(A) miR-155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB/c mice 6 weeks postinfection. (B) Murine bone marrow-derived macrophages (BMDMs) were infected with BCG at an MOI of 5 for the indicated times, and the expression levels of miR-155 were measured by real-time PCR. (C and D) RAW264.7 cells were infected with BCG at an MOI of 5 for the indicated time points (C) or at indicated MOIs for 24 h (D). The expression levels of miR-155 were examined by real-time PCR. (E and F) RAW264.7 cells were infected with H37Ra at an MOI of 5 for the indicated time (E) or at indicated MOI for 24 h (F). The expression levels of miR-155 were examined by real-time PCR. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.</p

miR-155 post-transcriptionally represses Rheb expression by targeting its 3′UTR.

<p>(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown. (B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant. (C–F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).</p

miR-155-induced autophagy promotes the elimination of intracellular mycobacteria.

<p>(A and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A). Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B). (C and D) RAW 264.7 cells were transiently co-transfected with control or miR-155 mimic together with a control siRNA or Atg7 siRNA. The expression levels of Atg7 and LC3 were detected by Western-blot (C). Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h (D). Values of LC3-II/β-actin ratios are indicated below the representative blot. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.</p

miR-155-induced autophagy promotes the formation of mycobacterial autophagosomes.

<p>(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.</p