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‐1β), tumor necrosis factor‐alpha (TNF‐α), 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‐1β (IL‐1β), tumor necrosis factor‐alpha (TNF‐α), 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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