Additional file 1 of Maresin1 ameliorates MSU crystal-induced inflammation by upregulating Prdx5 expression
Creators: name: Jiang, Hui
name: Song, DianZe
name: Zhou, Xiaoqin
name: Chen, Feng
name: Yu, Qingqing
name: Ren, Long
name: Dai, Qian
name: Zeng, Mei
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. The antibodies were used in Western blotting immunofluorescence assays.Table S2. The targeted siRNA sequence for mice Keap1. Table S3. The sequences of primer for PCR amplification.Table S4. The information about the genes encoded by the DEPs. Fig. S1. MaR1 inhibits the secretion of IL-1β, IL-6, TNF-α. ELISA assay was used to detect IL-1β, IL-6, TNF-α, TGF-β and IL-10 levels in cell culture supernatants. * compared with no FAs + MSU crystals treatment, # compared with FAs + MSUc treatment. * and # means P&lt;0.05. Fig. S2. Relative total mtDNA amounts in BMDMs treated with or without MaR1 for 1 h, followed by FAs + MSUc for 12 h. Shown are the ratio of D-loop mtDNA to Tert nuclear (n) DNA, Cox1 mtDNA to 18S nDNA or mtDNA that is not inserted into nuclear DNA (non-NUMT) to B2m nDNA.* compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P&lt;0.05. Fig. S3. Expression analysis of differentially expressed proteins (DEPs) in BMDMs treated with or without MaR1 for 1 h, followed by FAs + MSUc for 12 h. Heat-map was used to show the DEGs. The colors ranging from red to blue indicate the protein levels from high to low. MSU-1, MSU-2 and MSU-3 represent FAs + MSUc treatment groups, MaR-1, MaR-2 and MaR-3 represent MaR1 and FAs + MSUc treatment groups. N=3 each group. Fig. S4. Effect of FAs, MSU crystals, FAs + MSUc stimulation on PRDX1-6 protein levels in BMDMs and targeting Keap1 siRNA to inhibit KEAP1 protein expression. (A)BMDMs were treated with FAs, MSUc  or FAs + MSUc for 12 h. Western blot detection of PRDX1-6 protein levels. * compared with Vehicle (BSA) treatment. (B)Western blot detection of KEAP1 protein level. *compared to BMDMs transfected with negative control siRNAs. * means P&lt;0.05. Fig. S5. PRDX5 protein expression and Compound C reversed the effect of Prdx5 overexpression on AMPK activation and downstream target proteins. (A)BMDMs were transfected with pcDNA3.1 or Prdx5 ORF was cloned into pcDNA3.1 plasmid. Western blot for PRDX5 protein level. *compared with transfection pcDNA3.1 plasmid. (B)Western blot for p-AMPK, TXNIP, TRX1,TRX2 and p-DRP1protein levels.*compared with pcDNA3.1 + FAs + MSU crystals.# compared with Prdx5 OE + FAs + MSUc. (C) Detection of PRDX5 protein level in BMDMs derived from Prdx5+/+ and Prdx5-/- mice.*compared with BMDMs derived from Prdx5+/+. * and # means P&lt;0.05. (D) BMDMs from Prdx5+/+ and Prdx5-/- mice were treated with or without MaR1, then stimulated with FAs + MSUc. Western blot for p-AMPK, TXNIP, TRX1, TRX2 and p-DRP1 protein levels.*compared with Prdx5+/+ + FAs + MSUc. # compared with Prdx5+/+ + FAs + MSU crystals + MaR1. * and # means P&lt;0.05. Fig. S6. Effects of FAs, MSUc and FAs + MSU crystals stimulation on the expression of FAO related genes. (A) BMDMs were treated with FAs, MSUc or FAs + MSUc for 12 h. Western blot detection of FABP4, CPT1A, FABP3, CACT, CPT2 and CD36 protein levels. * compared with no FAs, MSUc and FAs + MSUc treatment. * means P&lt;0.05. (B) Quantitative PCR to detect the mRNA level of ACSL5. * compared with no FAs + MSU crystals treatment,# compared with FAs + MSUc treatment. * and # means P&lt;0.05. Fig. S7. MaR1 affects AMPK activity mainly through upregulation Prdx5 expression. BMDMs from Prdx5+/+ and Prdx5-/- mice were treated with or without MaR1, then stimulated with FAs + MSUc. Western blot for CPT1A, FABP3,CACT, FABP4 and CPT2 protein levels. *compared with Prdx5+/+ + FAs + MSUc. # compared with Prdx5+/+ + FAs + MSUc + MaR1. * and # means P&lt;0.05. Fig. S8. The heat-map showed the effects of FAs + MSUc on metabolites in BMDMs. Fig. S9. The heatmap listed the effects of Prdx5 overexpression on metabolites in BMDMs treated with FAs + MSUc. Fig. S10. Prdx5 overexpression increases Lysine, L-Methionine and ATP production. (A) The levels of Lysine and L-Methionine.  (B) The levels of ATP.*compared with Vehicle (BSA) treatment, # compared with pcDNA3.1 + FAs + MSUc. Fig. S11. MaR1 treatment inhibited the distribution of CD68, Ly6G, MPO and CPT1A proteins induced by MSU crystals in mice footpad tissue section. Immunofluorescence detection of CD68, Ly6G, MPO and CPT1A protein distribution. *compared with Prdx5+/+ + MSU, # compared with Prdx5+/+ + MSU, &amp; compared with Prdx5+/+ + MSU, $ compared with Prdx5+/+ + MSU + MaR1. *, # and &amp; means P&lt;0.05.