Journal: Science signaling
Article Title: MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection
doi: 10.1126/scisignal.aao2387
Figure Lengend Snippet: (A) Primary alveolar macrophages (AMs) and AECII were infected with PAO1 at a multiplicity of infection (MOI) of 20:1 for 1 hour, polymyxin B (100 μg/ml) was added, and cells were cultured for another 1 hour to kill bacteria outside of the cell membrane. Samples were collected at multiple time points over 48 hours, and the expression of MEG3–4 in AMs and AECII cells is time-dependent, as detected by qRT-PCR. (B) Nuclear and cytosolic expression of MEG3–4 in primary AMs and AECII cells was detected by qRT-PCR. lncRNA Xist and H19 were used as nuclear and cytoplasmic controls, respectively. (C) TLR2 and TLR4 expression in AMs from wild-type (WT), Tlr2−/−, and Tlr4−/− mice was measured by immunoblotting. (D) WT, Tlr2−/−, and Tlr4−/− mice (n = 3) were infected with 5 × 106 CFU of PAO1 per mouse for 24 hours. AMs were collected to assess MEG3–4 expression. (E and F) MH-S cells were pretreated with indicated signaling pathway activators (a) or inhibitors (i) for 4 hours and then infected for 2 hours with PAO1 at an MOI of 20:1. MEG3–4 expression before and after infection was analyzed by qRT-PCR. (G and H) MH-S cells were transfected with control siRNA [scrambled siRNA (siNC)] and NF-κB p65 siRNA (si-p65) for 48 hours, respectively, and then infected with PAO1 at 20:1 MOI for 2 hours. Expression and phosphorylation of NF-κB p65 were measured by immunoblotting, and MEG3–4 transcripts were detected by qRT-PCR. Data in (C) and (G) are representative of three independent mice or cell samples. Data in (A), (B), (D) to (F), and (H) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). NS, no significant change.
Article Snippet: Mouse monoclonal antibodies against β-actin (sc-47778), TLR4 (sc-293072), p-ERK (sc-7383), ERK1/2 (sc-514302), p-JNK (sc-6254), JNK (sc-7345), p-NF-κB p50 (sc-33022), NF-κB p50 (sc-166588), NF-κB p65 (sc-8008), p-Stat3 (sc-8059), pro-caspase I (sc-56036), MAPK p38 (sc-7972), and p53 (sc-377567); goat polyclonal antibodies against cleaved IL-1β (sc-23460), IL-6 (sc-1265), TNF-α (sc-1349), and NLRC4 (sc-49395); rabbit polyclonal antibodies against TLR2 (sc-10739), Stat3 (sc-482), p-NF-κB p65 (sc-33020), pro-IL-1β (sc-7884), cleaved caspase I (sc-514), ASC (sc-22514), and Akt1 (sc-8312); and chicken polyclonal antibodies against IL-18 (sc-7954-Y) were obtained from Santa Cruz Biotechnology.
Techniques: Infection, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Transfection
Santa Cruz Biotechnology is a verified supplier 
![(A) Primary alveolar macrophages (AMs) and AECII were infected with PAO1 at a multiplicity of infection (MOI) of 20:1 for 1 hour, polymyxin B (100 μg/ml) was added, and cells were cultured for another 1 hour to kill bacteria outside of the cell membrane. Samples were collected at multiple time points over 48 hours, and the expression of MEG3–4 in AMs and AECII cells is time-dependent, as detected by qRT-PCR. (B) Nuclear and cytosolic expression of MEG3–4 in primary AMs and AECII cells was detected by qRT-PCR. lncRNA Xist and H19 were used as nuclear and cytoplasmic controls, respectively. (C) <t>TLR2</t> and TLR4 expression in AMs from wild-type (WT), Tlr2−/−, and Tlr4−/− mice was measured by immunoblotting. (D) WT, Tlr2−/−, and Tlr4−/− mice (n = 3) were infected with 5 × 106 CFU of PAO1 per mouse for 24 hours. AMs were collected to assess MEG3–4 expression. (E and F) MH-S cells were pretreated with indicated signaling pathway activators (a) or inhibitors (i) for 4 hours and then infected for 2 hours with PAO1 at an MOI of 20:1. MEG3–4 expression before and after infection was analyzed by qRT-PCR. (G and H) MH-S cells were transfected with control siRNA [scrambled siRNA (siNC)] and NF-κB p65 siRNA (si-p65) for 48 hours, respectively, and then infected with PAO1 at 20:1 MOI for 2 hours. Expression and phosphorylation of NF-κB p65 were measured by immunoblotting, and MEG3–4 transcripts were detected by qRT-PCR. Data in (C) and (G) are representative of three independent mice or cell samples. Data in (A), (B), (D) to (F), and (H) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). NS, no significant change.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7737/pmc06637737/pmc06637737__nihms-1039710-f0002.jpg)
