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• Research Paper Volume 13, Issue 17 pp 21142-21154

  Serglycin promotes proliferation, migration, and invasion via the JAK/STAT signaling pathway in osteosarcoma

  Relevance score: 15.457615

  Bin Lv, Guangyu Gao, Yuhong Guo, Zhiping Zhang, Renfeng Liu, Zhengzai Dai, Cheng Ju, Yiping Liang, Xiaofeng Tang, Min Tang, Xiao-Bin Lv

  Keywords: SRGN, osteosarcoma, GEO, bioinformatics analysis, JAK/STAT

  Published in Aging on September 7, 2021

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  Background: Osteosarcoma (OS) is a common disease in the world, and its pathogenesis is still unclear. This study aims to identify the key genes that promote the proliferation, invasion, and metastasis of osteosarcoma cells.

  Method: GSE124768 and GSE126209 were downloaded from the Gene Expression Omnibus (GEO) database. The gene ontology and enrichment pathway were analyzed by FunRich software. qPCR and Western blot were used to detect the gene expression. After gene knockdown, Transwell and wound healing assays were conducted on osteosarcoma cells to detect whether the genes were defined before enhancing the invasion of osteosarcoma.

  Results: Totally, 341 mRNAs were found to be regulated differentially in osteosarcoma cells compared to osteoblasts. In addition, the expression level of Serglycin (SRGN) in osteosarcoma cells was higher than that in human osteoblasts. The invasion and proliferation ability of osteosarcoma cells with upregulated Serglycin was significantly increased, and on the contrary, decreased after Serglycin knockdown. Moreover, we preliminarily found that Serglycin may associate with the JAK/STAT signaling pathway.

  Conclusions: By using microarray and bioinformatics analyses, differently expressed mRNAs were identified and a complete gene network was constructed. To our knowledge, we describe for the first time Serglycin as a potential biomarker.

• Research Paper Volume 13, Issue 4 pp 5185-5196

  A short deletion in the DNA-binding domain of STAT3 suppresses growth and progression of colon cancer cells

  Relevance score: 12.610607

  Yi-Jia Xiong, Dong-Yang Liu, Rong-Rong Shen, Yong Xiong

  Keywords: STAT3, gain of function, JAK/STAT pathway, colon cancer

  Published in Aging on February 1, 2021

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  In this study, we investigated the effect of a short deletion in the DNA-binding domain of STAT3 (STAT3^del) on the transcriptional activation of STAT3 target genes and its relationship with colon carcinogenesis. We used the CRISPR-CAS9 gene editing system to delete a short sequence encoding amino acids 400-411 in the DNA-binding domain (amino acid sequence: 317-567) from STAT3 gene in SW480, SW620 and HCT116 colon cancer cells. ChIP sequencing analysis showed that STAT3^del occupancy was significantly reduced in 1029 genes and significantly increased in 475 genes compared to wild-type STAT3. The mutation altered the DNA motifs recognized by STAT3^del as compared to the wild-type STAT3. We observed a strong correlation between expression of the STAT3 target genes and the loss or gain of STAT3^del binding to their promoters. CCK-8, wound healing, and TUNEL assays showed reduced proliferation, migration, and survival of SW480, SW620 and HCT-116 cells expressing STAT3^del as compared to the corresponding controls. These findings demonstrate that a short deletion in the DNA-binding domain of STAT3 alters its genome-wide DNA-binding and transcriptional profile of STAT3-target proteins, and suppresses the growth, progression and survival of colon cancer cells.

• Research Paper Volume 12, Issue 11 pp 10896-10911

  SPTBN1 suppresses the progression of epithelial ovarian cancer via SOCS3-mediated blockade of the JAK/STAT3 signaling pathway

  Relevance score: 13.416022

  Mo Chen, Jia Zeng, Shuyi Chen, Jiajia Li, Huijie Wu, Xuhui Dong, Yuan Lei, Xiuling Zhi, Liangqing Yao

  Keywords: SPTBN1, epithelial ovarian cancer, SOCS3, JAK/STAT pathway, EMT

  Published in Aging on June 8, 2020

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  SPTBN1 plays an anticancer role in many kinds of tumors and participates in the chemotherapeutic resistance of epithelial ovarian cancer (EOC). Here, we reported that lower SPTBN1 expression was significantly related to advanced EOC stage and shorter progression-free survival. SPTBN1 expression was also higher in less invasive EOC cell lines. Moreover, SPTBN1 decreased the migration ability of the EOC cells A2780 and HO8910 and inhibited the growth of EOC cells in vitro and tumor xenografts in vivo. SPTBN1 suppression increased the epithelial mesenchymal transformation marker Vimentin while decreasing E-cadherin expression. By analyzing TCGA data and immunohistochemistry staining of tumor tissue, we found that SPTBN1 and SOCS3 were positively coexpressed in EOC patients. SOCS3 overexpression or JAK2 inhibition decreased the proliferation and migration of EOC cells as well as the expression of p-JAK2, p-STAT3 and Vimentin, which were enhanced by the downregulation of SPTBN1, while E-cadherin expression was also reversed. It was also verified in mouse embryonic fibroblasts (MEFs) that loss of SPTBN1 activated the JAK/STAT3 signaling pathway with suppression of SOCS3. Our results suggest that SPTBN1 suppresses the progression of epithelial ovarian cancer via SOCS3-mediated blockade of the JAK/STAT3 signaling pathway.

  

  SPTBN1 is closely related to the progression of epithelial ovarian cancer. (A) Analysis of TCGA data. The expression of SPTBN1 was significantly decreased as EOC staging increased. NS: stage 1 vs stage 2, 3, and 4; *P<0.05 vs stage 3 and 4; #P<0.05 vs stage 3. (B, C) The progression-free survival (PFS) and overall survival (OS) rates were retrieved from the TCGA dataset (probe 214856_at) and compared between the low-SPTBN1 and high-SPTBN1 ovarian cancer patients. The low-SPTBN1 group had shorter PFS than the high-SPTBN1 group. (D) Expression of SPTBN1 in 8 ovarian cancer cell lines. The expression of SPTBN1 in high-metastasis HO8910-PM cells was lower than that in conventional HO8910 cells, suggesting that the decrease in SPTBN1 expression may be correlated with the increased incidence of malignancy of epithelial ovarian cancer.

  
  
  

  

  SPTBN1 inhibits the growth of epithelial ovarian cancer cells in vitro and in vivo. (A, B) The protein level of SPTBN1 was detected by western blot. (C, D) The mRNA level of SPTBN1 was analyzed by real-time PCR. SPTBN1 was overexpressed in HO8910 cells but decreased in A2780 cells after transfection with the SPTBN1-V5 or SPTBN1-sh plasmid, respectively. *P < 0.05 vs Con-V5, **P < 0.01 vs Con-sh, n=3. (E, F) Cell viability was evaluated by CCK8 assay. **P < 0.01 vs Con-V5, *P < 0.05 vs Con-sh, n=3. (G) Cell proliferation was assessed by colony formation assay. Loss of SPTBN1 promotes the proliferation of A2780 and HO8910 cells. *P < 0.05 vs Con-sh, n=3; (H–J) Mouse xenograft tumors derived from A2780 cells. Loss of SPTBN1 promotes the growth of epithelial ovarian cancer cells in vivo. (H) Images of mice with tumors (upper) and harvested tumors for each treatment group (lower). (I) Tumor growth curves. (J) Tumor weight at sacrifice. *P < 0.05; **P < 0.01 vs LV-Con.

  
  
  

  

  SPTBN1 inhibits the migration and EMT of epithelial ovarian cancer cells. (A, B) Assessments of cell migration. Overexpression of SPTBN1 inhibits the migration of HO8910 cells (A), while downregulation of SPTBN1 promotes the migration of A2780 cells (B). (C, D) Comparison of EMT-related markers at the protein level (C) and mRNA level (D). * P < 0.05, **P < 0.01 vs Con-sh, n=3.

  
  
  

  

  Positive correlation between the expression of SPTBN1 and SOCS3 in epithelial ovarian cancer patients and cells. (A, B) Positive correlation between SPTBN1 and SOCS3 expression was investigated by GEPIA (gene expression profiling interactive analysis, http://gepia.cancer-pku.cn) based on the TCGA dataset (A) and observed by IHC staining of ovarian cancer patient samples (n=11) with 40× magnification (B). (C) The protein level of SOCS3 was decreased, and the phosphorylated STAT3 level was increased when SPTBN1 was downregulated. (D) SPTBN1 regulated SOCS3 expression at the mRNA level. * P < 0.05, ** P < 0.01 vs LV-RFP. (E) ChIP-qPCR was performed in HO8910 cells with anti-SPTBN1 and anti-Smads2/3 antibodies to determine the enrichment of SOCS3 promoter region sequences in the obtained ChIP DNA. *P < 0.05 vs Con-si.

  
  
  

  

  Loss of SPTBN1 activates the JAK/STAT3 signaling pathway through downregulation of SOCS3. (A) Assessments of JAK/STAT signaling pathway-associated proteins by western blot after SPTBN1 knockdown cooperated with SOCS3 overexpression in HO8910 cells (left), and SPTBN1 overexpression cooperated with SOCS3 knockdown (right). (B) DNA gel electrophoresis after PCR. Mouse embryonic fibroblasts (MEFs) were cultured from SPTBN1^-/- embryos (n=2), SPTBN1^+/- embryos (n=3) and wild-type embryos (n=2). The genotypes of MEFs were identified by PCR and DNA gel electrophoresis. Lanes 1 and 2: SPTBN1^-/- MEFs; lanes 3, 4, and 5: SPTBN1^+/- MEFs; lanes 6 and 7: SPTBN1^-/- MEFs. (C) Assessments of EMT and JAK/STAT3 signaling pathway-associated proteins by western blot in SPTBN1^-/-, SPTBN1^+/-, and SPTBN1^+/+ MEFs. Lanes 1 and 2: SPTBN1^-/- MEFs; lanes 3, 4, and 5: SPTBN1^+/- MEFs; lanes 6 and 7: SPTBN1^-/- MEFs. Loss of SPTBN1 can promote EMT, inhibit SOCS3 and activate the JAK/STAT signaling pathway.

  
  
  

  

  SOCS3 overexpression or the JAK2 inhibitor reverses the inhibitory effects of SPTBN1 on cell viability and migration. (A, B) In vitro cell migration assay. * P < 0.05 vs LV-RFP+LV-GFP, #P < 0.05 vs LV-SPTBN1sh+LV-GFP, n=3. (C, D) Comparison of protein (C) and mRNA (D) levels of the EMT-related proteins E-cadherin (E-cad) and Vimentin (Vim). The expression of SOCS3 and E-cadherin was decreased and Vimentin was increased by the loss of SPTBN1, while SOCS3 overexpression reversed the effects of the loss of SPTBN1. *P<0.05 **P<0.01 vs LV-RFP+LV-GFP, ##P<0.01 vs LV-SPTBN1sh+LV-GFP, n=3. (E–H) Cell viability was determined by CCK8 assay. SOCS3 overexpression reversed the enhanced cell viability due to the loss of SPTBN1 in A2780 (E) and HO8910 cells (F). #P<0.05, ##P<0.01 vs LV-RFP+LV-GFP, *P<0.05 vs LV-SPTBN1sh+LV-GFP, n=3. The JAK2 inhibitor Ag490 or tofacitinib (Tofa) inhibited cell viability and reversed the promoting effect of the loss of SPTBN1 in A2780 (G) and HO8910 cells (H). *P<0.05, **P<0.01 vs LV-RFP, #P<0.05 vs LV-SPTBN1sh, n = 3.

  
  
  

• Research Paper Volume 12, Issue 10 pp 9066-9084

  Overexpression of miR-142-5p inhibits the progression of nonalcoholic steatohepatitis by targeting TSLP and inhibiting JAK-STAT signaling pathway

  Relevance score: 12.89825

  Chao Zhou, Pu Wang, Lei Lei, Yi Huang, Yue Wu

  Keywords: non-alcoholic steatohepatitis, miR142-5p, TSLP, JAK-STAT signaling pathway

  Published in Aging on May 15, 2020

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  This study aimed to figure out the underlying mechanism of miR-142-5p in the non-alcoholic steatohepatitis (NASH). Bioinformatics, luciferase assay and Western blot were performed. The NASH mouse model was established through feeding a high fat diet (HFD). Relative expressions of miR-142-5p, thymic stromal lymphopoietin (TSLP), inflammatory factors were detected by qRT-PCR. The injury level of liver was assessed via measurement of serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST). H&E staining and Masson’s trichrome staining examine the liver fatty degeneration and fibrosis. MiR-142-5p and TSLP were differentially expressed and JAK-STAT signaling pathway was activated in the NASH group. Luciferase assay identified that TSLP was the downstream target of miR-142-5p. Through overexpression of miR-142-5p, ALT and AST in serum were inhibited, pro-inflammatory factors, liver fatty degeneration and fibrosis in liver tissues were decreased, while anti-inflammatory factors were increased. Overexpression of TSLP and JAK-STAT signaling pathway activation could reverse the effects of miR-142-5p on NASH. Taken together, overexpression of miR-142-5p could attenuate NASH progression via inhibiting TSLP and JAK-STAT pathway. MiR-142-5p might be a novel latent target for NASH therapy.

  

  TSLP was up-regulated and miR-142-5p was downregulated in NASH. (A) Heatmap showing differentially expressed genes between health and NASH group. (B) Heatmap showing differentially expressed miRNAs between health and NASH group. The expression fold in NASH groups was calculated compared with the health groups. Log₂|FC|>1 and adj. P < 0.05 was considered statistically significant.

  
  
  

  

  JAK-STAT pathway was actived in NASH patients and verification of the target gene. (A) Dotplot suggest the distributions of some biological pathway gene sets. The size of the circle represents the count value. The gradient changes of color represented adjust P values. (B) KEGG analysis of JAK-STAT pathway in NASH. (C) Expression of TSLP in NASH disease model. ^*P < 0.05, compared with control group or WT group. (D) Venn diagram showing the overlap between dysregulated miRNAs and miRNAs that target at TSLP. (E) RIP assays of the enrichment of Ago2 on TSLP and miR-142-5p relative to IgG in liver. ^**P < 0.01, compared with IgG group. (F) The miR-142-5p binding sites on TSLP were predicted by bioinformatics. TSLP wild-type form (TSLP-wt) and mutated form (TSLP-mut) were displayed on the left panel. Dual-luciferase reporter assay was conducted to identify the target relationship between miR-142-5p and TSLP. All data were means ± SD.

  
  
  

  

  The expression of miR-142-5p and TSLP and the function of miR-142-5p to TSLP in the liver of mouse model. (A) The relative expression of miR-142-5p in the liver of different group. (B) Relative mRNA expression of TSLP in the liver of different mouse group. (C) Protein expression of TSLP in the liver of different mouse group; WT group: wild-type mice fed a chow diet (CD); blank group: Ldlr−/− mice fed an high-fat diet (HFD) treated with phosphate buffer saline (PBS); NC group: Ldlr−/− mice fed an HFD treated with control agonis; A142 group: Ldlr−/− mice fed an HFD treated with agonist of miR-142-5p; A142-C group: Ldlr−/− mice fed an HFD treated with miR-142-5p agonist and JAK-STAT signaling pathway activator-colivelin; A142-TSLP group: Ldlr−/− mice fed an HFD treated with miR-142-5p agonist and AAV/TSLP. AAV: adeno-associated virus. n=6. ^*P < 0.05; ^**P < 0.01, compared with WT group; ^#P< 0.05; ^##P < 0.01, compared with NC group; ^{^}P < 0.05, compared with A142 group. All data were means ± SD.

  
  
  

  

  The expression of inflammatory factors and JAK-STAT signaling pathway biomarkers in the NASH mouse model. (A) The mRNA expression level of tumor necrosis factor (TNF)-α. (B) The mRNA expression level of interferon (IFN)-β. (C) The mRNA expression level of liver monocyte chemoattractant protein-1 (MCP1). (D) The mRNA expression level of IL-4. (E) The mRNA expression level of IL-6. (F) The expression level of TG in the liver. (G) The protein expression of the biomarkers of JAK-STAT signaling pathway. All data were means ± SD. ^*P < 0.05, compared with WT group; ^#P < 0.05, compared with NC group; ^{^}P < 0.05, compared with A142 group.

  
  
  

  

  miR-142-5p reduced liver injury, liver fatty degeneration and liver fibrosis, which could be rescued by JAK-STAT signaling pathway activator or AAV/TSLP. (A) The level of alanine aminotransferase (ALT) secreted into serum. (B) The level of aspartate aminotransferase (AST) secreted into serum. (C, E) H&E staining for liver fatty degeneration. Original magnification ×200. (D, F) The representative images of collagen deposition in Masson’s trichrome. The expression of fibrosis-related genes. Original magnification ×200. (G) The mRNA expression level of Tgfβ and (H) The mRNA expression level of Collagen-1 α2. ^*P < 0.05, compared with WT group; ^#P < 0.05, compared with NC group; ^{^}P < 0.05, compared with A142 group. All data were means ± SD.

  
  
  

  

  The effect of molecular interference sequences on the expression of miR-142-5p and TSLP. (A) Relative expression level of miR-142-5p in different groups. (B) The relative mRNA expression of TSLP. (C) The protein expression level of TSLP. WT group: wild-type mice fed a chow diet; blank group: Ldlr−/− mice fed an HFD treated with phosphate buffer saline; NC group: Ldlr−/− mice fed an HFD treated with sh-control; sh-TSLP group: Ldlr−/− mice fed an HFD treated with sh-TSLP; sh-TSLP-C group: Ldlr−/− mice fed an HFD treated with sh-TSLP and colivelin (JAK-STAT signaling pathway activator). Each group contained 6 mice. ^*P < 0.05, ^**P < 0.01, compared with WT group; ^#P < 0.05, compared with NC group. All data were means ± SD.

  
  
  

  

  Downregulating TSLP inhibited the expression of inflammatory factors and JAK-STAT pathway biomarkers, which could be rescued by JAK-STAT signaling pathway activator. (A) The mRNA expression level of tumor necrosis factor (TNF)-α. (B) The mRNA expression level of interferon (IFN)-β. (C) The mRNA expression level of liver monocyte chemoattractant protein-1 (MCP1). (D) The mRNA expression level of IL-4. (E) The mRNA expression level of IL-6. (F) The expression level of TG in the liver. (G) The protein expression of the biomarkers of JAK-STAT signaling pathway. ^*P < 0.05; ^**P < 0.01, compared with WT group; ^#P < 0.05; ^##P < 0.01, compared with NC group; ^&P < 0.05; ^&&P < 0.01, compared with sh-TSLP group. All data were means ± SD.

  
  
  

  

  Knockdown TSLP reduced liver injury, liver fatty degeneration and liver fibrosis, which could be rescued by JAK-STAT signaling pathway activator. (A) The level of alanine aminotransferase (ALT) secreted into serum. (B) The level of aspartate aminotransferase (AST) secreted into serum. (C) Liver fatty degeneration was detected by H&E staining; (D) The degree of fibrosis was shown by Masson’s trichrome Original magnification ×200. (E) The mRNA expression level of Tgfβ and (F) The mRNA expression level of Collagen-1α2. ^*P < 0.05, compared with WT group; ^#P < 0.05, compared with NC group; ^&P < 0.05, compared with sh-TSLP group. All data were mean ± SD.

  
  
  

• Research Paper Volume 9, Issue 11 pp 2411-2435

  TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion

  Relevance score: 12.064303

  Renuka Kandhaya-Pillai, Francesc Miro-Mur, Jaume Alijotas-Reig, Tamara Tchkonia, James L. Kirkland, Simo Schwartz

  Keywords: senescence, inflammation, DNA-damage, interferon response genes, JAK/STAT pathway

  Published in Aging on November 22, 2017

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  Cellular senescence is a cell fate program that entails essentially irreversible proliferative arrest in response to damage signals. Tumor necrosis factor-alpha (TNFα), an important pro-inflammatory cytokine secreted by some types of senescent cells, can induce senescence in mouse and human cells. However, downstream signaling pathways linking TNFα-related inflammation to senescence are not fully characterized. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that TNFα induces permanent growth arrest and increases p21CIP1, p16INK4A, and SA-β-gal, accompanied by persistent DNA damage and ROS production. By gene expression profiling, we identified the crucial involvement of inflammatory and JAK/STAT pathways in TNFα-mediated senescence. We found that TNFα activates a STAT-dependent autocrine loop that sustains cytokine secretion and an interferon signature to lock cells into senescence. Furthermore, we show STAT1/3 activation is necessary for cytokine and ROS production during TNFα-induced senescence. However, inhibition of STAT1/3 did not rescue cells from proliferative arrest, but rather suppressed cell cycle regulatory genes and altered TNFα-induced senescence. Our findings suggest a positive feedback mechanism via the STAT pathway that sustains cytokine production and reveal a reciprocal regulatory role of JAK/STAT in TNFα-mediated senescence

  

  TNFα induces senescence and DNA damage in HUVECs. (A) Long-term growth curve of cells exposed to recombinant human TNFα (5ng/ml). Untreated cells were used as controls. Population doubling and doubling times were calculated based on cell density at confluence. Data represent mean values from 3 independent experiments. (B) The percentage of BrdU-positive cells was determined by FACS analysis in cells untreated or chronically treated with TNFα at the concentration indicated. (C) Western blot analysis of p21, p16, and actin in cells treated with TNFα 5ng/ml for the indicated times. (D) SA-β-gal activity in TNFα (5ng/ml)-treated or control cells for the indicated number of days. (E) Percentages of SA-β-gal-positive cells in control or TNFα-treated cultures. The data represent 2 independent counts of 200 cells from 3 independent experiments. (F) Intracellular ROS levels were monitored by 2',7’-dichlorodihydrofluorescein diacetate staining followed by flow cytometry. Bar graph represents percentage of DCFDA-positive cells treated with TNFα or medium alone. (G) Immunofluorescence detection of γH2AX foci in controls or cells treated with TNFα (5ng/ml) for indicated days. Data in A, B, E, and F represent mean value ± standard deviation (s.d.) from n=3, 2, 3, and 2 independent experiments, respectively.

  
  
  

  

  Top canonical pathways identified by IPA analysis in TNFα-induced senescence. (A) Bar chart represents the top canonical signaling pathways that were influenced during TNFα-induced senescence. p-values were determined using Fisher´s exact test with a threshold value of >0.05. Ratios represent the number of genes that mapped to a specific canonical pathway divided by the total number of genes that make up the respective pathway. (B) Relative mRNA expression of SASP components in cells treated with TNFα at 6, 16, or 26 days compared to untreated cells. (C) Relative mRNA expression of p16 and p21 in cells treated with TNFα (5ng/ml) compared to untreated cells. Results are mean ± standard deviation of n=2 independent experiments.

  
  
  

  

  Activation of the canonical JAK/ STAT pathway. Pathway analysis using IPA reveals canonical activation of STAT1 acts as a central regulator for JAK/ STAT-mediated interferon response genes during TNFα-induced senescence. The red symbols represent up-regulated genes, with the intensity of node color indicating degree of up-regulation and white indicating genes absent from the list. Shape of nodes denotes functions of gene products.

  
  
  

  

  Prolonged activation of JAK/ STAT signaling in TNFα-induced senescence. (A) Immunoblot detection of p-Ser727-STAT1 and total STAT1 in cells exposed to TNFα 20ng/ml for the indicated times. (B) SA-β-gal activity in TNFα (20ng/ml)-treated or control cells for 3 or 6 days. (C) Immunoblot detection of p-Ser727-STAT1, total STAT1, p-Tyr705-STAT3, and total STAT3 in cells exposed to TNFα (5ng/ml) for the indicated intervals. (D) Immunoblot detection of pSTAT1, total STAT1, pSTAT3, and total STAT3 in cells stimulated with IL6 (10ng/ml) or IFNγ (1ng/ml) for the indicated intervals. (E) Secretion of IFNγ/IL6 quantified by ELISA in conditioned medium collected in the presence or absence of TNFα. (F) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, STAT1, and STAT3 in cells treated with conditioned medium (CM) (cell free-culture supernatants from control and cells stimulated with TNFα for 3 days transferred after 1:4 dilution with fresh culture medium) from TNFα-induced senescent cells or from non-senescent cells for the indicated times

  
  
  

  

  Persistent activation of STAT1/3. Cells were exposed to TNFα (20ng/ml) for 3 days, then washed to remove the residual TNFα and cultured for 3 days in the absence of exogenous TNFα (TNFα-PST). Parallel cultures were exposed to exogenous TNFα throughout the experiment. (A) Levels of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT3 proteins were quantified by immunoblot. (B) Secretion of IL-6/IFNγ was assessed in culture supernatants from cells treated with TNFα as indicated. (C) Immunodetection of ROS production and γH2AX foci in control or cells treated with TNFα or TNFα-post-stimulated (PST), as indicated. (D) Real-time gene expression of IRF1 and MX1 in cells exposed to TNFα or TNFα-PST for 3 days. Results were normalized to internal control TBP and are shown relative to untreated cells. (E) SA-β-gal activity in TNFα-treated cells for 3 days or in cells treated with TNFα (20ng/ml) for three days, then washed to remove residual TNFα and left untreated for another 3 days. (F) mRNA expression of p21 and p16 quantified by real-time PCR in cells exposed to TNFα or TNFα-PST for 3 days. Data in D and F represent mean value of ± sd from 2 independent experiments.

  
  
  

  

  JAK2 inhibitor decreases TNFα-mediated inflammation, ROS levels, and interferon signature. Cells were exposed to TNFα alone, TNFα in combination with AG490 (30μM), or AG490 alone for 3 days. (A) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT1 and STAT3. (B) Secretion of IL6 was estimated by ELISA in culture supernatants from cells treated with TNFα alone, or in combination with AG490, or AG490 alone for 3 days. (C) FACS analysis of ROS levels in cells stimulated either with TNFα, TNFα along with AG490, or AG490 alone for 3 days using DCFDA staining 2',7’-dichlorofluorescein (DCF) positive cells were analyzed. Inhibition of STAT signals modulates senescence. (D) Cell cycle analysis using BrdU and 7- aminoactinomycin D (7-AAD) staining in cells exposed to TNFα, TNFα in combination with AG490, or AG490 alone for 3 days. (E) Percentage of SA-β-gal-positive cells. Quantification of SA-β-gal activity in cells stimulated with TNFα 20ng/ml, TNFα in combination with AG490, or AG490 alone for 3 days. The data represent means of 3 independent counts of 200 cells from 2 independent experiments. (F) Effect of AG490 on cell cycle regulatory proteins. Western analysis performed using cells treated with AG490 for 3 days and blotted against anti-p21, CDK2, and NDC80. Actin serves as loading control.

  
  
  

  

  Model of mechanisms involved in TNFα-induced senescence of HUVECs. (A) TNFα activates JAK/STAT and p38 signaling pathways, which mediate increased expression of STAT1/3 phosphorylation. Activation of the JAK pathway leads to persistent phosphorylation of STAT1/3 signaling, which together with ROS, interferon genes, and other SASP components, drives a positive auto-regulatory loop, leading to sustained inflammation and stable senescence. (B) Inhibition of STAT1/3 with the JAK inhibitor AG490 decreased ROS and IL-6 production and decreased expression of interferon response genes. On the other hand, blockade of STAT1/3 expression decreased S phase entry of cells and increased p21 expression, leading to senescence.