miR-155/KD upregulates natural miR-155 targets and reduces AKT activation. (A-B) Deregulation of natural miR-155 targets in RAW264.7 cells upon stable transduction with the indicated LVs is shown. (A) Western blot for PU.1 was performed on unstimulated and LPS-treated (1 μg/mL for 48 hours) cells. p84/Thoc1 and tubulin were used as loading controls; densitometric quantification of the PU.1 band after normalization to p84 and calibration to the scrambled control is shown below the blots, together with vector copy number in the tested cells. Blots are representative of 2 independent experiments. (B) qPCR for Pu.1 (left) and Ship1 mRNA (right) was performed. Data shown are the fold change to control, normalized to β2m (2^−δδCt); n = 6 to 19 independent experiments. Statistical analysis of the data was performed on 2^−ΔΔCt values by unpaired Student t test. (C) Shown are the results of qPCR for Ship1 mRNA in GFP⁺ T cells and macrophages sorted from the spleens of wild-type mice reconstituted with Ctrl (white) or 155/KD (gray) LV-transduced BMs 24 hours after treatment with 50 µg LPS intraperitoneally. n = 4 to 6 mice. (D) Shown is a simplified scheme of the PI3K/AKT signal transduction pathway. SHIP1, a direct miR-155 target, is a major negative regulator of the PI3K/AKT axis. (E) Phosphorylation of PI3K downstream targets in macrophage RAW264.7 cells transduced with ScrT Ctrl or miR-155/KD vectors (red); shown are western blots for pAKT (Ser473), pGSK3β (Ser9), and pS6 (Ser235/236). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control. (F) The panels show GM-CSF induced activation of AKT in RAW264.7 cells upon miR-155/KD (GFP⁺ cells) or miR-155/OE (OFP⁺ cells). The pAKT (S473) signal in vector-positive cells (middle histograms) and vector-negative cells (right histograms) is shown (FACS plots are representative). The colors represent the following: red, miR-155/KD vector; blue, 155/OE vector; black, Ctrl vector-transduced RAW264.7 cells; and gray, miR-155/KD cells pretreated with the PI3K inhibitor Wortmannin (W) to set the background signal. The MFI of each gated population is indicated. The column bar graph (right) shows pAKT Ser473 MFI in Raw264.7 cells transduced with the indicated vectors. Statistical analysis was performed on n = 4 (155/KD) or n = 3 (155/OE) replicates using a paired Student t test against the corresponding Ctrl vector-transduced samples. *P < .05; **P < .01. bT, bulged targets; MFI, mean flourescent intensity; pT, perfectly complementary targets; RTK, receptor tyrosine kinase; W, Wortmannin.