Journal: bioRxiv

Article Title: STING contributes to pulmonary hypertension by targeting the interferon and BMPR2 signaling through targeting F2RL3

doi: 10.1101/2024.02.20.578386

Figure Lengend Snippet: A , Relative mRNA expression profiles of cGAS-STING pathway genes ( cGAS, STING, IRF3, IRF7, IFIT1, IFIT2, IFIT3 and CXCL10 ) in total lung tissues from PH patients and healthy controls (n=5-11). B , Protein expression levels of STING in the total lung tissues of PH patients and healthy controls (n=4). C , Representative colocalization staining of STING (red), vWF (red) and α-SMA (green) in pulmonary arteries of healthy controls and PH patients (n=4), Scale bar=20μm. D , Protein expression levels of STING in PAECs and PASMCs (n=4). E, Relative mRNA expression of STING in PAEC exposure to 1% O 2 hypoxia for 24h (n=4). F, Relative mRNA expression of Sting in lung microvascular endothelial cells from hypoxia and SuHx mouse PH models (n=4). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.01. Statistical analysis was done using Unpaired t test for A, B, D, and E. Statistical analysis was done using One-way ANOVA for F.

Article Snippet: Human pulmonary artery endothelial cells (PAECs) were purchased from Promcell (C-12241).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: STING contributes to pulmonary hypertension by targeting the interferon and BMPR2 signaling through targeting F2RL3

doi: 10.1101/2024.02.20.578386

Figure Lengend Snippet: A , Relative mRNA expression profiles of cGAS-STING pathway genes ( cGAS, STING, IRF3, IRF7, IFIT1, IFIT2, IFIT3 and CXCL10 ) in PAECs with TNF-α (2ng/ml) treatment for 24h (n=3). B-D , Protein expression levels of cGAS, p-STING/STING, p-IRF3/IRF3, and p-TBK1/TBK1 in PAECs with TNF-α (2ng/ml) treatment for 24 h (n=3). E , Protein expression levels of p-IRF3/IRF3, and p-TBK1/TBK1 in PAECs with siRNA STING and siRNA negative control (NC) transfection and followed by TNF-α (2ng/ml) treatment for 24 h (n=3). P , Protein expression levels of p-IRF3/IRF3, and p-TBK1/TBK1 in PAEC with H-151 (DMSO) treatment and followed by TNF-α (2ng/ml) treatment for 24 h (n=3). G , Relative mRNA expression profiles of interferon-stimulated genes (ISGs) ( IRF3, IRF7, IFIT1, IFIT2, IFIT3 and CXCL10 ) in PAECs transfected by siRNA STING and siRNA negative control (NC) and followed with or without TNF-α (2ng/ml) treatment for 24 h (n=3). H , Relative mRNA expression profiles of interferon-stimulated genes (ISGs) ( IRF3, IRF7, IFIT1, IFIT2, IFIT3 and CXCL10 ) in PAECs with H-151 (DMSO) treatment and followed with or without TNF-α (2ng/ml) treatment for 24 h (n=3). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.01. Statistical analysis was done using unpaired two-tailed t test for A-F. Statistical analysis was done using a two-way ANOVA with multiple comparisons for G-H.

Article Snippet: Human pulmonary artery endothelial cells (PAECs) were purchased from Promcell (C-12241).

Techniques: Expressing, Negative Control, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: STING contributes to pulmonary hypertension by targeting the interferon and BMPR2 signaling through targeting F2RL3

doi: 10.1101/2024.02.20.578386

Figure Lengend Snippet: A and B , The positive EdU staining of proliferating PAECs transfected with siRNA STING and siRNA NC were detected by fluorescent microscopy and absorbance measurement (n =3 and 6). C and D , The positive EdU staining of proliferating PAECs treated with H-151 (DMSO) were detected by fluorescent microscopy and absorbance measurement (n =3 and 6). E and F , Protein expression levels of PCNA in PAECs transfected with siRNA STING (NC) or treated with H-151(DMSO) (n=3). G and H , the relative migration distances were analyzed by wound healing assay in PAECs transfected with siRNA STING (NC) or treated with H-151(DMSO) (n=3). I , The protein levels ICAM-1 and STING in PAECs transfected with siRNA STING (NC) and stimulated with or without TNF-α (2ng/ml) for 24 h (n=3). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.01. Statistical analysis was done using unpaired two-tailed t test for A-H. Statistical analysis was done using a two-way ANOVA with multiple comparisons for I.

Article Snippet: Human pulmonary artery endothelial cells (PAECs) were purchased from Promcell (C-12241).

Techniques: Staining, Transfection, Microscopy, Expressing, Migration, Wound Healing Assay, Two Tailed Test

Journal: bioRxiv

Article Title: STING contributes to pulmonary hypertension by targeting the interferon and BMPR2 signaling through targeting F2RL3

doi: 10.1101/2024.02.20.578386

Figure Lengend Snippet: A and B , Downregulated Gene ontology (GO) terms and KEGG pathways analysis from the bulk RNA-sequencing (RNA-seq) in PAECs with STING silencing and followed by TNF-α (2ng/ml) treatment for 24 hours. C , Heatmap showing dysregulated gene expression and highlighting the F2RL3 in Complement and coagulation cascades. D , Volcano plot showing the differentially expressed genes in PAECs and highlights the F2RL3. E , Relative mRNA expression of STING and F2RL3 in PAECs treated with siSTING versus siNC (siNegative Control) (n=3). F , The protein levels of F2RL3 and STING in PAECs with siRNA STING transfection (n=3). G, The protein levels of phosphorylation of NF-κB p65 in PAECs treated with siSTING versus siNC±TNF-α (2ng/ml) treatment for 24 h (n=3). H, Schematic illustration shows the potential NF-κB binding sites of the F2RL3 promoter region, and agarose gel electrophoresis to assess the PCR reactions from the ChIP samples. I, Dual luciferase assay showing the transcriptional regulation of NF-κB on F2RL3 WT and mutants in 293T cells. J, Co-immunoprecipitation (Co-IP) in PAECs to assess the interaction between STING and F2RL3. K, Relative mRNA expression of F2RL3 in lung tissues from PH patients and healthy controls (n=5-12). L, Representative colocalization staining of F2RL3 (red), vWF (red) and α-SMA (green) in pulmonary arteries of healthy controls and PH patients (n=4), Scale bar=20μm. M, The protein levels of p-STING/STING and p-TBK1/TBK1 in PAECs treated with siF2RL3 versus siNC±TNF-α (2ng/ml) treatment for 24 h (n=3). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.01. Statistical analysis was done using unpaired two-tailed t test for E,F,and K. Statistical analysis was done using a two-way ANOVA with multiple comparisons for G,I, and M.

Article Snippet: Human pulmonary artery endothelial cells (PAECs) were purchased from Promcell (C-12241).

Techniques: RNA Sequencing, Gene Expression, Coagulation, Expressing, Control, Transfection, Phospho-proteomics, Binding Assay, Agarose Gel Electrophoresis, Luciferase, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: STING contributes to pulmonary hypertension by targeting the interferon and BMPR2 signaling through targeting F2RL3

doi: 10.1101/2024.02.20.578386

Figure Lengend Snippet: A , Heatmap showing the dysregulated gene expression associated with the BMP signaling pathway. B , Relative mRNA expression of BMPR1A , BMPR2 and ID3 in PAECs treated with siSTING versus siNC (siNegative Control) +BMP9 (10ng/ml) treatment for 24 h (n=3). C-D , The protein levels of BMPR2 and p-Smad1/5/9 in PAECs treated with siSTING versus siNC±BMP9 (10ng/ml) treatment (n=3). E-F , The protein levels of p-Smad1/5/9, Smad1, F2RL3 and STING in PAECs treated with siF2RL3 versus siNC±BMP9 (10ng/ml) treatment for 24 h (n=3). G , Relative mRNA expression of STING , F2RL3 , BMPR2 , ID1 and ID3 in PAECs with BMP9 (10ng/ml) (n=3). H-I , The protein levels of F2RL3 and STING in PAEC with BMP9 (10ng/ml) treatment (n=3). J-K , The protein levels of BMPR2 and F2RL3 in PAECs transfected with siRNA BMPR2 and siRNA NC (n=3). L, Relative mRNA expression of BMPR2 and F2RL3 in PAECs transfected with siRNA BMPR2 and siRNA NC (n=3). M-N , The protein levels of BMPR2, p-Smad1/5/9, STING, and F2RL3 in PAECs transfected with siRNA STING and siRNA NC followed by lentivirus NC (LV-NC) and lentivirus F2RL3 (LV-F2RL3) infection 48 h (n=3). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.01. Statistical analysis was done using unpaired two-tailed t test for B, G, I, K, and L. Statistical analysis was done using a two-way ANOVA with multiple comparisons for D, F, and N.

Article Snippet: Human pulmonary artery endothelial cells (PAECs) were purchased from Promcell (C-12241).

Techniques: Gene Expression, Expressing, Control, Transfection, Infection, Two Tailed Test