Supplementary_Materials_2 – Supplemental material for Berberine reduces neuroglia activation and inflammation in streptozotocin-induced diabetic mice

Supplemental material, Supplementary_Materials_2 for Berberine reduces neuroglia activation and inflammation in streptozotocin-induced diabetic mice by Mei Liu, Linlin Gao and Na Zhang in International Journal of Immunopathology and Pharmacology</p

Image1_Impaired FGF10 Signaling and Epithelial Development in Experimental Lung Hypoplasia With Esophageal Atresia.tiff

<p>Patients with esophageal atresia (EA) and tracheoesophageal fistula (TEF) often experience persistent respiratory tract disease. In experimental models, doxorubicin-induced developmental lung abnormalities may result from downregulation of branching morphogenesis factor fibroblast growth factor (Fgf10). This study investigated the temporospatial expression of Fgf10 pathway components and lung epithelial factors in an doxorubicin-induced EA-TEF model by quantitative polymerase chain reaction, immunohistochemistry, and immunoblotting. Epigenetic regulation of gene expression by histone deacetylation was also investigated. Bone morphogenetic protein (Bmp) 4 and Cathepsin H (Ctsh), downstream targets of Fgf10, were significantly downregulated in the EA-TEF model during the saccular stage, consistent with Fgf10 expression. The developmental expression pattern of P2x7 receptor (ATI-cell marker), Sftpa, and Sftpb in lung epithelial cells was not affected. Sftpc (ATII-cell Marker) and Scgb1a1 (Clara cell marker) were significantly downregulated at the canalicular stage. Meanwhile, histone deacetylase (Hdac) 1 was upregulated and subsequently decreased acetylation of histone H3 Lys56 in the EA-TEF model, which returned to a normal level at the saccular stage. In conclusion, disturbed molecular signaling involving Fgf10/Ctsh was associated with impaired airway branching and epithelial cell development in lung morphogenesis, as evidenced by downregulated Sftpc and Scgb1a1 protein expression. The influence of Hdac1 activity on gene and protein expression in lung epithelial cells deserves further study.</p

Table_1_Maternal Protein Restriction Induces Alterations in Hepatic Unfolded Protein Response-Related Molecules in Adult Rat Offspring.DOCX

Intrauterine growth restriction (IUGR) leads to the development of metabolic syndrome in adulthood. To explore the potential mechanisms of metabolic imprinting, we investigated the effect of malnutrition in utero on hepatic unfolded protein response (UPR)-related genes in IUGR offspring. An IUGR rat model was developed by feeding a low-protein diet to pregnant rats. The expression levels and activity of hepatic UPR genes were analysed by quantitative PCR (qPCR) arrays and western blotting. The hepatic UPR molecules heat-shock 70-kDa protein 4l (Hspa4l), mitogen-activated protein kinase 10 (Mapk10), and endoplasmic reticulum to nucleus signalling 2 (Ern2) were markedly downregulated in IUGR foetuses, but the expression of Mapk10 and Ern2 returned to normal levels at 3 weeks postnatal. In contrast, cAMP responsive element binding protein 3-like 3 (Creb3l3) was upregulated in hepatic tissues at embryo 20(E20), then restored to normal in adulthood (12 weeks). The protein levels of activating transcription factor 2 (Atf2) and Atf6, two key factors of the UPR pathway, were upregulated in the livers of IUGR foetuses, and the latter remained upregulated until 12 weeks. Combined with our previous findings showing an increase in hepatic gluconeogenesis enzymes in IUGR offspring, we speculated that aberrant intrauterine milieu impaired UPR signalling in hepatic tissues; these alterations early in life might contribute to the predisposition of IUGR foetuses to adult metabolic disorders.</p

RNF11 disrupts the interaction between TRAF3 and TBK1/IKKi.

<p>(A, C) 293T cells were transfected with 1 μg of either HA-TRAF3, Flag-IKKi, Flag-TBK1 or RNF11-GFP. Co-IPs were conducted using anti-Flag followed by immunoblotting with anti-HA and anti-Flag. Immunoblotting was performed with lysates using anti-Flag, anti-HA, anti-GFP and anti-Actin. (B, D) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and 1 µg of RNF11, TRAF3, IKKi or TBK1. Dual luciferase assays were performed with protein lysates 24 h later. *, p<0.05. <i>Error bars, S.D.</i></p

RNF11 is a negative regulator of virus-induced IFN-β production.

<p>(A) Micrographs of 293T cells transfected with either empty vector or Myc-RNF11 and then infected with VSV-GFP (MOI of 0.1) 24 h later. Pictures were taken 24 h post-infection. Immunoblotting was conducted with protein lysates using anti-Myc, anti-GFP and anti-Actin (right panel). (B) MEFs were transfected with either empty vector or Myc-RNF11 and were transfected again with poly(I:C) (15 μg) 24 h later. An IFN-β ELISA was performed 16 h later using supernatants. Immunoblotting was conducted with anti-Myc and anti-Actin. (C) 293T cells were transfected with an IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 µg) or RNF11-GFP (1 µg). Cells were transfected 24 h later with poly(I:C) (15 µg) and dual luciferase assays were performed after 16 h. Immunoblotting was conducted with protein lysates using anti-GFP and anti-Actin. (D) 293T cells were transfected with RNF11-GFP (1 μg) and Myc-A20 (1 μg) and then transfected 24 h later with poly(I:C) (20 µg). Immunoblotting was performed with anti-p-IRF3, anti-Myc, anti-GFP, anti-IRF3 and anti-Actin.</p

RNF11 inhibits IFN-β production at the level of TBK1/IKKi.

<p>(A–E) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 μg), RNF11-GFP (1 μg) and either 0.5 μg of ΔRIG-I (A), MDA5 (B), IPS-1 (C), TBK1 (D) or IRF3-SA (E). Dual luciferase assays were performed with protein lysates 24 h later. (F, G) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 24 h, cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and either Flag-TBK1 or Flag-IKKi (0.5 μg) and dual luciferase assays were performed 24 h later. (H) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 48 h, RT-PCR was performed to detect RNF11 and Actin transcripts. *, p<0.05. <i>Error bars, S.D.</i></p

RNF11 requires TAX1BP1 to inhibit antiviral signaling.

<p>(A) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), RNF11 (1 µg) and either control scrambled or TAX1BP1 siRNA (60 pmol). After 24 h, cells were transfected with poly(I:C) (15 µg) and dual luciferase assays were performed 16 h later. The data is presented as percent inhibition by RNF11 of poly(I:C)-induced IFN-β promoter induction with either control scrambled or TAX1BP1 siRNA (left panel). Knockdown of TAX1BP1 was confirmed by immunoblotting using anti-TAX1BP1 and anti-Actin (right panel). (B) <i>Tax1bp1</i>^+/– and <i>Tax1bp1</i>^–/– MEFS were transfected with poly(I:C) (20 μg), and co-IPs were performed with anti-RNF11 followed by immunoblotting with anti-IKKi (left panel) or anti-TBK1 (right panel). Immunoblots were performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. *, p<0.05. <i>Error bars, S.D.</i></p

RNF11 interacts with TBK1/IKKi and blocks their Lys63-linked polyubiquitination.

<p>(A) 293T cells were transfected with 1 µg of RNF11-GFP, Flag-IKKi and Flag-TBK1. Co-IPs were conducted using anti-Flag for IP followed by immunoblotting with anti-GFP and anti-Flag. Immunoblotting was performed with lysates using anti-GFP, anti-Flag and anti-Actin. (B) 293T cells were transfected with poly(I:C) (20 μg) and co-IPs were performed with anti-RNF11 or isotype control IgG followed by immunoblotting with anti-IKKi (top panel) or anti-TBK1 (lower panel). Immunoblots were also performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. (C) 293T cells were transfected with empty vector, Flag-RNF11 or RNF11-GFP (1 µg) and HA-Ub-Lys63-only (500 ng). Cells were transfected again 24 h later with poly(I:C) (20 μg) and co-IPs were conducted the next day using anti-TBK1 (left panel) or anti-IKKi (right panel) followed by immunoblotting with anti-HA, anti-TBK1 (left panel) and anti-IKKi (right panel). Immunoblotting was performed with lysates with anti-Flag, anti-GFP and anti-Actin.</p

RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

<div><p>A key feature of the innate antiviral immune response is a rapid nonspecific response to virus infection largely mediated by the induction and extracellular secretion of type I interferons (IFNs) that restrict virus replication. Cytoplasmic sensors such as RIG-I recognize viral RNA and trigger antiviral signaling pathways that upregulate IFN transcription. However, it remains largely unknown how antiviral signaling is negatively regulated to maintain homeostasis after the elimination of virus. In this report, we have identified the RING domain-containing protein RING finger 11 (RNF11) as a novel negative regulator of innate antiviral signaling. Overexpression of RNF11 downregulated IFN-β expression and enhanced viral replication whereas siRNA-mediated knockdown of RNF11 suppressed viral replication. RNF11 interacted with the noncanonical IKK kinases TBK1/IKKi and attenuated their Lys63-linked polyubiquitination by blocking interactions with the E3 ligase TRAF3. The inhibitory function of RNF11 was dependent on the ubiquitin-binding adaptor molecule TAX1BP1 which was required for RNF11 to target TBK1/IKKi. Collectively, these results indicate that RNF11 functions together with TAX1BP1 to restrict antiviral signaling and IFN-β production.</p> </div

AgBr Nanoparticles Anchored on CdS Nanorods as Photocatalysts for H₂ Evolution

We designed and fabricated an AgBr/CdS S-scheme heterojunction with Ag–S bonds. It was found that the formed Ag–S bond served as a bridge to transfer photoinduced electrons from CdS to AgBr. The as-prepared AgBr/CdS materials showed remarkably enhanced performance and good stability for photocatalytic hydrogen evolution using triethanolamine (TEOA) as the sacrificial agent. Under visible light illumination, the 10% AgBr/CdS sample exhibited optimal activity (5406 μmol h–1 g–1) among all samples, which was 85 times higher than that of the pristine CdS (64 μmol h–1 g–1). Because of the formation of the AgBr/CdS S-scheme heterojunction and the rapid electron-transfer channel provided by Ag–S bonds, high charge separation and utilization efficiency were achieved, which contributed to its superior performance