Journal: PLoS Biology
Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy
Figure Lengend Snippet: HF treatment promotes IL-1β mRNA degradation by targeting it to SGs through recruitment of TIA-1/TIAR. (A) Immunoblot analysis of RBPs, TIA-1/TIAR, in the lysates of J774A.1 macrophages, left untreated or treated with varying concentrations of HF for 3-h, 6-h, and 12-h time points. β-actin was used as loading control. (B) J774A.1 macrophages were transfected with either control siRNA or TIA-1/TIAR siRNA. After 24 h, cells were either left untreated or stimulated with LPS (500 ng/ml) or LPS (500 ng/ml) plus HF (20 nM) for 6 h, ATP (5 mM) was added to the macrophage cultures for 30 min at the end of time point and were assayed for IL-1β levels in culture supernatants by ELISA ( S1 Data ). (C) IL-1β expression in the whole-cell lysates of HEK293T cells transiently expressing various combinations of plasmids (pcDNA3, pcDNA3-IL-1β, and pEYFP-TIA-1/TIAR) as indicated (top). β-actin was used as loading control. (D) RT-PCR analysis of IL-1β expression in RIP material (pull-down using TIA-1/TIAR or IgG) of lysates from J774A.1 macrophages left untreated or treated with LPS or LPS plus HF. (E) Immunoblot analysis of pro–IL-1β or pro–caspase-1 expression in macrophages treated with LPS or LPS plus HF; β–actin was used as a loading control. (F) qRT-PCR analysis of IL-1β mRNA in the RIP material from LPS-primed J774A.1 macrophages treated with the indicated concentrations of HF ( S1 Data ). (G) qRT-PCR analysis of IL-1β mRNA in HEK293T cells transfected with constructs pCMV6-IL1β or pCMV6 IL-1β Δ3'UTR ARE, or cotransfected along with pEYFP-TIA-1/TIAR ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. Data are representative of 1 of 3 independent experiments. HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IgG, immunoglobulin G; IL-1β, interleukin 1β; LPS, lipopolysaccharide; qRT-PCR, quantitative reverse transcription PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; siRNA, small interfering RNA; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.
Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).
Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Construct, Polymerase Chain Reaction, RNA Binding Assay, Immunoprecipitation, Small Interfering RNA