Fig. 4
PI3K-p110β mRNA, a direct mRNA target of QKI, was dynamically regulated by QKI in the cytoplasmic P-bodies. A PI3K-p110β mRNA were analyzed after RAW264.7 cells incubated with MRSA for indicated time. B RAW264.7 cells transfected with plasmids encoding Flag-tagged QKI were fixed and doubly immuno-stained using anti-flag and anti-EDC4 antibody. Immunofluorescence showed the expression of flag-tagged QKI and EDC4, and DAPI. DAPI: blue, QKI: pink, EDC4: green (Scale Bar: 10 μm). The co-localization of QKI and EDC4 was showed on the top. C RNA-FISH detection of PI3K-p110β mRNA and immuno-stained with Flag-tagged QKI expressed RAW264.7 cells. Representative fluorescence images of protein expression of flag-QKI and localization of PI3K-p110β mRNA. DAPI:blue, PI3K-p110β mRNA:green, flag-QKI:pink (Scale Bar:10 μm). The co-localization of QKI and PI3K-p110β mRNA was showed on the top. D RNA-FISH detection of PI3K-p110β mRNA and immunostained with EDC4 in various cells. Representative fluorescence images of protein expression of EDC4 and localization of PI3K-p110β mRNA. DAPI:blue, PI3K-p110β mRNA:green, EDC4:red (Scale Bar:10 μm).The co-localization of EDC4 and PI3K-p110β mRNA was showed on the top. E RNA immunoprecipitation (RIP) was used in overexpress QKI RAW264.7 cells and the results indicated QKI protein interacts with PI3K-p110β mRNA. F Construction of luciferase reporter vectors (PI3K-p110β full-length 3’UTR), and PI3K-p110β 3’UTR QRE motif site mutant. Luciferase activity assays was used to examine the functional QRE motif site in PI3K-p110β 3’UTR affected by QKI. G The mRNA stability in macrophage was measured by incubating cells with actinomycin D to block transcription at the indicated time points. Real-time PCR was performed for the analysis of PI3K-p110β mRNA expression. All the bars represented the mean of measurements from three independent experiments, and the error bars indicated ± SD. (B–D) are representative of three experiments, **p < 0.01. (student’s t test). (F) are representative of three experiments. ***p < 0.001 one-way ANOVA with Tukey’s multiple comparisons test. (G) are representative of one experiment (n = 3 wells/group), *p < 0.05, **p < 0.01, not significant (ns). (student’s t test)