Volume 68, Issue 1, Pages 35-46 (July 2005) Lipoxin A4 inhibits TNF-α-induced production of interleukins and proliferation of rat mesangial cells  Sheng-Hua Wu, Chao Lu, Ling Dong, Guo-Ping Zhou, Zha-Guang He, Zi-Qing Chen  Kidney International  Volume 68, Issue 1, Pages 35-46 (July 2005) DOI: 10.1111/j.1523-1755.2005.00379.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Proliferation of mesangial cells assessed by incorporation of [3H]-thymidine (TdR) and expressed as cpm/μg protein. Cultured and serum-starved glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. TNF-α-stimulated proliferation of mesangial cells was inhibited by LXA4 in a dose-dependant manner. Data were mean ± SEM of six independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Real-time polymerase chain reaction (PCR) analysis of interleukin (IL)-1β and IL-6 mRNA expression. Cultured and quiescent mesangial cells were exposed to tumor necrosis factor-α (TNF-α) (10 ng/mL) for 12 hours with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Expression data of TaqMan-PCR analysis of IL-1β and IL-6 mRNA are shown as ratio of copies to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Controls consisting of ddH2O were negative in all runs. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Proteins of interleukin (IL)-1β and IL-6 assessed by enzyme-linked immunosorbent assay (ELISA) in supernatant of cultured mesangial cells. Cultured and serum-deprived glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. TNF-α-stimulated release of IL-1β and IL-6 of mesangial cells were inhibited by LXA4 in a dose-dependant manner. Nuclear factor-κB (NF-κB) inhibitor pyrrolidine dithio-carbamate (PDTC) completely abolished secretions of IL-1β and IL-6 induced by TNF-α. Data were mean ± SEM of six independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4 or PDTC. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Semiquantitative analysis using UVP-gel densitometry (A) of electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR) products from cyclin E mRNA (B) in rat mesangial cells. Cultured and serum-restricted glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 12 hours, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. (B) is representative of four independent experiments, the lower panel shows RT-PCR products from β-actin served as an internal control. In (A), arbitrary unit (AU) = (Acyclin E/Aβ-actin) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Semiquantitative analysis using UVP-gel densitometry (A) of electrophoresis of Western blotting for cyclin E protein (B) in rat mesangial cells. Cultured and growth-arrested glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. (B) is representative of four independent experiments, the lower panel shows Western blotting of β-actin protein serving as a loading control. In (A), arbitrary unit (AU) = (Acyclin E/Aβ-actin) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Semiquantitative analysis using UVP-gel densitometry (A) of electrophoresis of Western blotting for phosphorylated Akt1 (P-Akt1) protein (B) in rat mesangial cells. Cultured and serum-starved glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 15 minutes, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. (B) is representative of four independent experiments, the lower panel shows Western blotting of β-actin protein serving as a loading control. In (A), arbitrary unit (AU) = (AAkt1/Aβ-actin) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Semiquantitative analysis using UVP-gel densitometry (A) of electrophoresis of Western blotting for p27kipl protein (B) in rat mesangial cells. Cultured and quiescent glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. (B) is representative of four independent experiments, the lower panel shows Western blotting of β-actin protein serving as a loading control. In (A), arbitrary unit (AU) = (Ap27kip1/Aβ-actin) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated signal tranducers and activators of transcription (STAT3) (B) in rat mesangial cells. Cultured and serum-deprived glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 1 hour, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Thirty micrograms of nuclear protein extracts were prepared for detection of STAT3 activity by EMSA with γ-[32P]-labeled double-stranded oligonucleotide probe of STAT3. In (B),the upper arrow denotes the specific STAT3-DNA complexes. In (A), arbitrary unit (AU) = (ATNF-α or ALXA4 or ATNF-α+ LXA4/A0.5% FCS) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Semiquantitative analysis using UVP-gel densitometry (A) of immunoprecipiation and immunoblotting analysis for phosphorylated Src homology 2 containing protein-tyrosine phosphatase (SHP-2) (B) in mesangial cells. Cultured and serum-starved glomerular mesangial cells were treated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 30 minutes, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Whole-cell extracts (500 μg) were immunoprecipitated with 1 μg of antiphosphotyrosine antibody PY20, and immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with anti-SHP-2 antibody. In (B), the lower panel shows immunoblotting analysis of β-actin protein serving as a loading control. In (A), arbitrary unit (AU) = (ASHP-2/Aβ-actin) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated nuclear factor-κB (NF-κB) (B) in rat mesangial cells. Gel supershift assay (C) demonstrated the specialty of activation of NF-κB by tumor necrosis factor-α (TNF-α). Cultured and growth-arrested glomerular mesangial cells were treated with TNF-α (10 ng/mL) for 1 hour, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Thirty micrograms of nuclear protein extracts were prepared for detection of NF-κB activity by EMSA with γ-[32P]-labeled double-stranded oligonucleotide probe of NF-κB. In (B), the upper arrow denotes the specific NF-κB-DNA complexes. In (A), arbitrary unit (AU) = (ATNF-α or ALXA4 or ATNF-α+ LXA4 or ATNF-α+PDTC/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4 and pyrrolidine dithio-carbamate (PDTC). #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated nuclear factor-κB (NF-κB) (B) in rat mesangial cells. Gel supershift assay (C) demonstrated the specialty of activation of NF-κB by tumor necrosis factor-α (TNF-α). Cultured and growth-arrested glomerular mesangial cells were treated with TNF-α (10 ng/mL) for 1 hour, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Thirty micrograms of nuclear protein extracts were prepared for detection of NF-κB activity by EMSA with γ-[32P]-labeled double-stranded oligonucleotide probe of NF-κB. In (B), the upper arrow denotes the specific NF-κB-DNA complexes. In (A), arbitrary unit (AU) = (ATNF-α or ALXA4 or ATNF-α+ LXA4 or ATNF-α+PDTC/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. *P < 0.05 compared to the cells treated with TNF-α and 0.5% fetal calf serum (FCS) without LXA4 and pyrrolidine dithio-carbamate (PDTC). #P < 0.05 compared to the cells treated with 0.5% FCS alone. Kidney International 2005 68, 35-46DOI: (10.1111/j.1523-1755.2005.00379.x) Copyright © 2005 International Society of Nephrology Terms and Conditions