Fig 1: Inhibitor of growth 4 (ING4) alleviated the lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory cytokine expression in RAW 264.7 macrophages. RAW 264.7 cells were transfected with 2 µg pcDNA3.1-ING4, pLVX-shING4 or negative control (NC) plasmid with or without LPS treatment. (a, b) ING4 messenger RNA (mRNA) and protein levels were evaluated 48 h after transfection. (c, d) Inflammatory cytokine transcripts, including those encoding interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-a), IL-6 and monocyte chemoattractant protein 1 (MCP-1), were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) 48 h after transfection with or without LPS treatment and normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript level. The data are presented as the mean ± s.e.m. and represent at least three independent experiments. **P < 0.01 compared with the NC. Statistical significance was determined by a Student's t-test.
Fig 2: Inhibitor of growth 4 (ING4)-overexpressing mice were hyposensitive to lipopolysaccharide (LPS) challenge and displayed reduced organ injury. Each BALB/C mouse was transfected in vivo with 60 µg pcDNA3.1-ING4 or pcDNA3.1-EGFP-C2 [negative control (NC)] through caudal vein injection. Three days after injection, the mice were injected intraperitoneally with 5 or 15 mg kg-1 LPS to establish a mouse in vivo sepsis model. (a) The survival rates of the mice transfected with the NC or ING4 plasmid were observed within 72 h of stimulation with either 5 mg kg-1 (solid lines) or 15 mg kg-1 (dashed lines) LPS (n = 12 in each group). At both doses, there was a significant survival difference between the ING4-overexpressing mice and NC mice (P < 0.005). (b) Enzyme-linked immunosorbent assay analyses were performed to measure interleukin-1beta (IL-1ß), IL-6 and tumor necrosis factor-alpha (TNF-a) levels in the serum from mice transfected with pcDNA3.1-ING4 (n = 8) or NC (n = 8) at 6 h after LPS injection. (c) Hematoxylin–eosin staining was performed with mouse heart, lung, liver and kidney tissue samples from the ING4-overexpressing and NC mice (n = 8; scale bar = 50 µm). ***P < 0.005 and ****P < 0.0001 compared with the negative control. The significance of survival differences was analyzed by the log-rank (Mantel–Cox) test, and other significant differences were determined by a Student's t-test.
Fig 3: Inhibitor of growth 4 (ING4) levels were associated with the lipopolysaccharide (LPS)-induced inflammatory response. (a, c) RAW 264.7 macrophages and peritoneal macrophages were treated with LPS (1 µg mL-1) for various periods. The messenger RNA (mRNA) levels of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-a), IL-6 and monocyte chemoattractant protein 1 (MCP-1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). (b, d) The mRNA level of ING4 during LPS challenge was analyzed by qRT-PCR, and cell lysate aliquots were immunoblotted with the indicated antibodies to reveal protein expression. The data are presented as the mean ± s.e.m. and represent at least three independent experiments. *P < 0.05 and **P < 0.01 compared with LPS treatment at 0 h. Statistical significance was determined by a Student's t-test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SIRT1, sirtuin 1.
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