Fig. 2

Latent HIV infection disrupts IFN responses downstream of PRR signaling a,b qRT-PCR analysis of Jurkat vs. JLat9.2 cells (a) or A3.01 vs. ACH2 cells (b) after infection with 100 HAU/ml Sendai virus (SeV) for indicated times. c qRT-PCR analysis of Jurkat vs. JLat9.2 cell lines left untreated (NT), or transfected with nonstimulatory xRNA or RIG-I stimulatory PAMP RNA for 24 h. Both RNAs contain 5'-triphosphate. For all qRT-PCR data (panels a-c), fold change (FC) was calculated relative to untreated, uninfected cells (ΔΔCt method), and each symbol represents mean FC of replicates from a single experiment. Data from three independent experiments are shown. Statistical significance relative to similarly treated control cells (Jurkat or A3.01) was calculated by unpaired Student’s t-test; asterisks denote significance (*p < 0.05, **p < 0.01, ***p < 0.001). d, e Immunoblot analyses of total and phosphorylated IRF3 (pIRF3) in Jurkat or JLat9.2 cells infected with 100 HAU/ml SeV for indicated times. One representative image is shown (d) from three independent experiments quantified (e). Target protein abundance relative to actin was quantified with ImageJ software, and statistical significance of JLat9.2 relative to similarly treated Jurkat cells calculated by two-way ANOVA with multiple comparisons (Holm-Sidak) (p < 0.5; ns = not significant). f,g ImageStream quantification of IRF3 nuclear localization in mock-treated or SeV-infected (100 HAU/ml, 24 h) Jurkat or JLat9.2 cells. One ImageStream experiment was performed with 20,000 cells per sample. Representative Imagestream images of brightfield, red (DAPI-stained nuclei), green (IRF3), and red/green merged images. For each cell, IRF3 nuclear translocation status (positive or negative) was determined by IRF3/DAPI similarity over an arbitrary cutoff value of 2.3 (see Imagestream gating in Additional file 1: Figure S2a)