Additional file 1 of Pangenome analysis of Shewanella xiamenensis revealed important genetic traits concerning genetic diversity, pathogenicity and antibiotic resistance

Supplementary Material 1

Additional file 1 of Molecular characterization of extensively drug-resistant hypervirulent Pseudomonas aeruginosa isolates in China

Supplementary Material 1: Figure S1. Heatmap of XDR-hvPA carrying resistance genes; Figure S2. Heatmap of XDR-hvPA carrying virulence gene

Enhanced expression and phosphorylation of Sirt7 activates smad2 and ERK signaling and promotes the cardiac fibrosis differentiation upon angiotensin-II stimulation

<div><p>Cardiac fibroblasts (CFs) phenotypic conversion to myofibroblasts (MFs) represents a crucial event in cardiac fibrosis that leads to impaired cardiac function. However, regulation of this phenotypic transformation remains unclear. Here, we showed that sirtuin-7 (Sirt7) plays an important role in the regulation of MFs differentiation. Sirt7 expression and phosphorylation were upregulated in CFs upon angiotensin-II (Ang-II) stimulation. Sirt7 depletion by siRNA in CFs resulted in decreased cell proliferation and extracellular matrix (ECM) deposition. Further, examination of Sirt7-depleted CFs demonstrated significantly lower expression of α-smooth muscle actin (α-SMA), the classical marker of MFs differentiation, and decreased formation of focal adhesions. Moreover, overexpression of Sirt7 increased α-SMA expression in Ang-II treated CFs and exacerbated Ang-II-induced MFs differentiation. Moreover, Sirt7 depletion could largely reverse Ang-II induced increase of nuclear translocalization and activity of smad2 and extracellular regulated kinases (ERK) in CFs. Importantly, the increased differentiation of CFs to MFs was also abolished by smad2 siRNA or U0126. Our findings reveal a novel role of Sirt7 and its phosphorylation in the phenotypic conversion of CFs to MFs and might lead to the development of new therapeutic and prognostic tools for cardiac fibrosis.</p></div

Smad2 partially mediates the effects of Sirt7 on fibrosis of CFs.

<p>(A) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II. The p-smad2 was analyzed by western blot. (B) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II. The nuclear localization of smad2 was analyzed by nuclear proteins western blot. (C) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II. The transcriptional activity of smad2 was measured through luciferase assay. (D) CFs were transfected with NC or si-samd2 and empty vector or Sirt7 plasmid. The expressions of α-SMA, (E) Paxillin and (F) Vinculin were analyzed by western blot. All experiments were repeated at least three times. (*<i>P</i><0.05)</p

Sirt7 affects the function of CFs.

<p>(A) CFs were transfected with NC or si-Sirt7, and the expression of PAI-1, FN, Collagen I and Sirt7 were analyzed by western blot. (B) CFs were transfected with empty vector or Sirt7 plasmid, and treated with or without 100nM Ang-II. The expression of FN was analyzed by western blot. (C) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II for 24h. The hydroxyproline content was determined and analyzed. (D) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II. The cell viability was determined and analyzed. All experiments were repeated at least three times. (*<i>P</i><0.05)</p

The schematic diagram illustrates the mechanism for the effect of the Ang-II/Sirt7/ERK and Ang-II/Sirt7/Smad2 axises on differentiation of CFs to MFs during cardiac fibrosis.

<p>Activation of Sirt7 by Ang-II stimulation promotes phosphorylation of ERK and Smad2. Then ERK and Smad2 regulate the activity of some transcription factors in the nucleus and further mediate expression of some genes about differentiation of CFs to MFs.</p

Increased expression and activity of Sirt7 regulates CFs fibrosis upon Ang-II stimulation.

<p>(A) CFs were transfected with Sirt7 siRNAs pool (si-Sirt7) and negative control (NC) siRNA. At 48 h after transfection, cells were lysed and subjected to western blot analysis for Sirt7 and GAPDH. (B) Cell lysates from Ang-II-treated CFs were immunoprecipitated with Sirt7 followed by western blotting using anti-p-Ser/Thr antibody. (C) CFs were treated with or without 100nM Ang-II for 24h followed by immunostaining with anti-Sirt7 antibody (red). (D) CFs were treated with or without 100nM Ang-II. The nuclear localization of Sirt7 was analyzed by nuclear proteins western blot. (E)CFs were transfected with empty vector or Sirt7 plasmid, and treated with or without 100nM Ang-II. The expression of α-SMA was analyzed by western blot. The expressions of α-SMA, (F) Paxillin and (G) Vinculin were analyzed by western blot. (H) CFs were transfected with negative control siRNA or Sirt7 siRNAs pool, and treated with or without 100nM Ang-II for 24h. All experiments were repeated at least three times. (*<i>P</i><0.05)</p

Sirt7 regulates CFs fibrosis through synergistic activation of smad2 and ERK pathways.

<p>(A) CFs were treated with Ang-II for the indicated time. The expression of p-ERK and ERK were analyzed by western blot. (B) CFs were treated with or without Ang-II followed by immunostaining with anti-p-ERK antibody (green). (C) CFs were transfected with NC or si-Sirt7, and treated with or without 100nM Ang-II. The p-ERK was analyzed by western blot. (D) CFs were treated with or without U0126 and transfected with empty vector or Sirt7 plasmid. The expressions of α-SMA and Vinculin were analyzed by western blot. (E) CFs were transfected with empty vector or Sirt7 wild-type plasmid (WT) or Sirt7 phosphorylation site mutant plasmid (Sirt7-T153A), respectively. The expression of p-ERK, ERK, p-smad2, smad2 and GAPDH were analyzed by western blot. (F) The cell proliferation was measured by CCK-8 assay. CFs were transfected with empty vector or Sirt7 plasmid, and treated with or without 100nM Ang-II. All experiments were repeated at least three times. (*<i>P</i><0.05)</p