Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 interacts with TRAF3 and IRF3 upon VSV infection. (A) Mouse peritoneal macrophages were infected with VSV for indicated hours. Immunoblot analysis of endogenous TRAF3, IRF3, Parkin, RIG-I, TBK1, and MAVS immunoprecipitated with antibody to PINK1. IgG was as control. Numbers between two blots indicate densitometry of TRAF3, IRF3, or Parkin relative to that of PINK1 in immunoprecipitates. (B,C) HEK293T cells were transfected with PINK1 expressing plasmid together with Flag-TRAF3 or HA-IRF3 plasmid. Cells were lysated 24 h after transfection for immunoblot analysis of indicated proteins immunoprecipitated with antibody to Flag (B) or HA (C) tag. (D) Confocal microscopy of HEK293T co-transfected with Myc-PINK1 and Flag-TRAF3 plasmids followed by VSV infection for 4 h. MitoTracker (Mito) was used to probe the mitochondrion (red). DAPI served as a marker of nuclei (blue). Scale bar, 5 μm. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Control, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Marker

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 promotes RLR-triggered immune response via kinase domain dependent manner. (A) Immunoblot analysis of HEK293T cells that cotransfected with GFP-TRAF3 plus Flag-PINK1, Flag-PINK1 mutants vectors followed by VSV infection for 4 h, then immunoprecipitated with antibody to Flag tag. (B) Immunoblot analysis of RAW264.7 cells stably overexpressed with empty vector or vector encoding PINK1 and its mutants. (C) qPCR analysis of IFN-β, IL-6, and VSV-G mRNA in stably transfected RAW264.7 cells expression of PINK1 variants, infected with VSV for indicated times. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Western Blot, Infection, Immunoprecipitation, FLAG-tag, Stable Transfection, Plasmid Preparation, Transfection, Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 inhibits TRAF3 degradation. Mouse peritoneal macrophages transfected with scrambled negative control siRNA (siNC) or PINK1 specific siRNA (30 nM) (A) , or RAW264.7 cells transfected with PINK1 plasmid (B) , were infected with VSV for indicated hours. TRAF3 proteins in lysates were detected by western blot. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). (C) HEK293T cells were transfected with GFP-TRAF3, HA-Ub, Flag-Parkin, and varying doses of Flag-PINK1 (0, 0.5, and 1 μg) and infected with VSV for 4 h. Cells were treated with MG132 (10 uM) and harvested for immunoblot analysis of K48-Ub and K63-Ub immunoprecipitated with antibody to GFP tag. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Transfection, Negative Control, Plasmid Preparation, Infection, Western Blot, Expressing, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 positively regulates the RLR-triggered antiviral immune response by inhibiting TRAF3 degradation and relieving YAP-mediated inhibition of the cellular antiviral response. (1) Virus infection down-regulates PINK1 expression in macrophages. (2) PINK1 promotes RLR-triggered IRF3 activation via inhibiting Parkin-mediated K48-linked ubiquitination of TRAF3, (3) and PINK1 inhibits YAP1/IRF3 complex formation, which ultimately promotes RIG-I triggered antiviral immune response.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Inhibition, Virus, Infection, Expressing, Activation Assay, Ubiquitin Proteomics

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH upregulates TRAF3IP2 expression. At 70–80% confluency, SMC were made quiescent, and exposed to IH for the indicated number of cycles. Nx represents time equivalent to 50 cycles of IH. TRAF3IP2 expression was analyzed by Western blotting using 20 μg of cleared whole cell lysates (n=3). Tubulin served as a loading control. B, IH stimulates superoxide generation via TRAF3IP2 and Nox2. Quiescent SMC exposed to 50 cycles of IH were analyzed for superoxide generation by the lucigenin-enhanced chemiluminescence assay. In a subset of experiments, SMC were exposed to NAC or gp91 ds-tat or transduced with Ad.TRAF3IP2-shRNA prior to IH. sgp91 ds-tat or GFP shRNA served as controls (n=6). Knockdown of TRAF3IP2 was confirmed by Western blotting as shown on the right. The adapter molecule MyD88 served as an off target. C, IH stimulated H 2 O 2 production via TRAF3IP2 and Nox4. SMC were treated as in C , but with GKT137831 or NAC or transduced with Ad.TRAF3IP2-shRNA, and then analyzed for H 2 O 2 production by Amplex Red assay (n=6). D, IH stimulated nitric oxide generation. Quiescent SMC exposed to IH as in C , but pretreated with 1400W or AMT, or transduced with Ad.TRAF3IP2-shRNA were analyzed for nitric oxide generation by the Greiss reaction, and the data were presented as cumulative nitrite production in μM (n=6). E, IH upregulates TRAF3IP2 expression via nitroxidative stress. Quiescent SMC treated as in C , D and E were analyzed for TRAF3IP2 expression by Western blotting (n=3). F, IH stimulates SMC proliferation via TRAF3IP2 and nitroxidative stress. Quiescent SMC treated as in C - E were analyzed for proliferation by the CyQUANT ™ assay after 48h (n=6) G. The pharmacological inhibitors and the shRNA used did not compromise cell viability as analyzed by a colorimetric LDH release assay and activation of caspase-3 by Western blotting. A, though a representative Western blot is shown, changes in target protein expression from three independent experiments was semi-quantified by densitometry and presented on the right as fold change over Nx, which was set at a value of 1. The numbers at the bottom in panels F and G denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Control, Chemiluminescence Immunoassay, Transduction, shRNA, Knockdown, Amplex Red Assay, CyQUANT Assay, Lactate Dehydrogenase Assay, Activation Assay

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH activates NF-κB via TRAF3IP2 and nitroxidative stress. Quiescent SMC were treated with NAC, gp91 ds-tat, GKT137831, 1400W or AMT prior to IH (50 cycles), and analyzed for NF-κB activation by Western blotting using equal amounts of nuclear protein extracts (10 μg) and activation-specific p65 antibodies (Ser 536 ). Lamin A/C served as a loading control. In a subset of experiments, SMC were transduced with Ad.TRAF3IP2 shRNA, made quiescent, and then exposed to IH (right hand panel). B, IH activates HIF-1α via TRAF3IP2 and nitroxidative stress. Quiescent SMC treated as in A were analyzed for HIF-1α activation by Western blotting using cleared whole cell lysates (20 μg). Tubulin served as a loading control. C, IH induces HIF-1α activation via NF-κB. Quiescent SMC were treated with the NF-κB inhibitor SN-50 or the proteasomal inhibitor MG-132 prior to IH and analyzed for HIF-1α activation as in B . Inhibition of NF-κB activation was confirmed by Western blotting (right hand panel) as in A . D, Targeting NF-κB and HIF-1α inhibit IH-induced SMC proliferation without affecting cell viability. SMC transduced with lentiviral shRNA against NF-κBp65 or HIF-1α were made quiescent and exposed to IH. Cell proliferation was analyzed by the CyQUANT ™ assay after 48h (D; n=6). Cell viability was analyzed by a colorimetric LDH release assay and activation of caspase-3 by Western blotting (E). A,B,C,E, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry, and presented at the bottom of respective panels as a fold change over control, which was set at a value of 1. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Activation Assay, Western Blot, Control, Transduction, shRNA, Inhibition, CyQUANT Assay, Lactate Dehydrogenase Assay, Expressing

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A-C, IH upregulates IL-6 mRNA and protein expression via TRAF3IP2, NF-κB and HIF-1α. SMC were transduced with NF-κBp65, HIF-1α or TRAF3IP2 shRNA using viral vectors, made quiescent, and exposed to 50 cycles of IH. GFP shRNA served as a control. IL-6 mRNA was analyzed by RT-qPCR (A) and protein expression by Western blotting (B). Knockdown of NF-κBp65 and HIF-1α was confirmed by Western blotting (C). ASK1 served as a non-targeting control. Tubulin served as a loading control. D, IL-6 activates STAT3 in a concentration- and time-dependent manner. Quiescent SMC treated with the indicated concentrations of IL-6 (left hand panel) and for up to 120 min at 30 ng/ml (middle panel) were analyzed for total and p-STAT3 levels by Western blotting using cleared whole cell lysates (20 μg). In a subset of experiments, SMC were exposed to Polymyxin B sulphate for 2 h prior to incubation with IL-6 (30 ng/ml for 15 min). STAT3 activation was analyzed by Western blotting (right hand panel). E, F, IL-6 induces STAT3 phosphorylation via IL-6R, gp130 and JAK. Quiescent SMC treated with the gp130 inhibitor SC144, JAK inhibitor HO-3867 and the STAT3 inhibitor Tofacitinib prior to IH were analyzed for STAT3 phosphorylation as in D . In a subset of experiments, SMC were transduced with IL-6R or gp130 shRNA, made quiescent, and then exposed to IH (right hand panel). Knockdown of IL-6R and gp130 was confirmed by Western blotting (F). B-F, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry and presented at the bottom or side of respective panels as a fold change over control, which was set at a value of 1. The numbers at the bottom of panels denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Transduction, shRNA, Control, Quantitative RT-PCR, Western Blot, Knockdown, Concentration Assay, Incubation, Activation Assay, Phospho-proteomics

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, Quiescent SMC treated with the gp130, JAK or STAT3 inhibitor prior to IL-6 addition were analyzed for proliferation at 48 h by the CyQUANT ™ assay. In a subset of experiments, SMC were transduced with IL-6 or gp130 or TRAF3IP2 shRNA, made quiescent, and then exposed to IL-6 (n=6). B, Pharmacological inhibitors or shRNA-mediated knockdown of targets described in A did not negatively impact SMC viability. Cell viability was analyzed by LDH release by the LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting (bottom panel; n=3). In LDH cytotoxicity assay, treatment of SMC with Triton X-100 served as a positive control and was set to 100% LDH release. H 2 O 2 (100 μM) served as a positive control in Western blotting. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: CyQUANT Assay, Transduction, shRNA, Knockdown, Cytotoxicity Assay, Activation Assay, Western Blot, LDH Cytotoxicity Assay, Positive Control, Saline

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IL-6 upregulates SGLT2 expression. Quiescent SMC exposed to IL-6 (30 ng/ml for 4h) were analyzed for SGLT2 expression by Western blotting using equal amounts of whole cell lysates (20 μg). Cell lysates from the human proximal tubule epithelial cells HK-2 and human kidney extracts served as positive controls (20 μg). B, IL-6 upregulates SGLT2 expression via STAT3. Quiescent SMC exposed to IL-6 (30 ng/ml) for up to 6h (left hand panel) were analyzed for SGLT2 expression as in A. In a subset of experiments, quiescent SMC were treated with the STAT3 inhibitor HO-3867 prior to IL-6 addition (30 mg/ml for 4 h). C, Empagliflozin inhibits IL-6-induced STAT3 phosphorylation via SGLT2. Quiescent SMC were exposed to empagliflozin (1 μM for 15 min) prior to IL-6 addition (30 ng/ml for 15 min). Total and phospho-STAT3 levels were analyzed by Western blotting using whole cell lysates. In a subset of experiments, SMC were transduced with SGLT2 shRNA by lentiviral transduction, made quiescent, and then treated with IL-6. Knockdown of SGLT2 was confirmed by Western blotting. TRAF3IP2 served as an off target. D, Empagliflozin or SGLT2 knockdown failed to affect cell viability. Cell viability was analyzed by LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting as in . A-D, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry, and presented at the bottom or side of respective panels as a fold change over control, which was set at a value of 1. The numbers at the bottom of panels denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Phospho-proteomics, Transduction, shRNA, Knockdown, Cytotoxicity Assay, Activation Assay, Control, Saline

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH upregulates SGLT2, and targeting SGLT2 by empagliflozin inhibits IH-induced TRAF3IP2 expression. Quiescent SMC were exposed to IH or Nx for 50 cycles, and analyzed for SGLT2 expression by Western blotting. Human kidney homogenate served as a positive control. In a subset of experiments, quiescent SMC were exposed to empagliflozin (1 μM for 15 min) prior to IH (50 cycles), and then analyzed for TRAF3IP2 expression by Western blotting (n=3). B, Empagliflozin inhibits IH-induced nitrooxidative stress. Quiescent SMC exposed to empagliflozin as in A were analyzed for superoxide, H 2 O 2 and nitrate+nitrite levels by lucigenin-enhanced chemiluminescence assay, Amplex Red assay and Greiss reaction (n=6). C, Empagliflozin inhibits IH-induced NF-κB and HIF-1α activation. Quiescent SMC exposed to empagliflozin and IH as in A were analyzed for NF-κB activation by Western blotting using equal amounts of nuclear protein extracts and activation-specific anti-p65 antibodies. Total and phospho-HIF-1α levels were analyzed in whole cell lysates by Western blotting. D, empagliflozin inhibits IH-induced IL-6 expression and STAT3 phosphorylation. Quiescent SMC exposed to empagliflozin followed by IH were analyzed for IL-6 expression and phospho-STAT3 levels by Western blotting using cleared whole cell lysates. E, Empagliflozin inhibits IH-induced SMC proliferation without affecting cell viability. Quiescent SMC treated as in A were analyzed for proliferation by the CyQUANT ™ assay after 48h (n=6). Cell viability was analyzed by LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting as in . *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Positive Control, Chemiluminescence Immunoassay, Amplex Red Assay, Activation Assay, Phospho-proteomics, CyQUANT Assay, Cytotoxicity Assay

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: IH induces primary human aortic SMC proliferation via the crosstalk between TRAF3IP2 and nitrooxidative stress, NF-κB and HIF-1α activation, and induction of IL-6 and activation of its downstream signaling. Moreover, IL-6 induced SGLT2 expression in part via STAT3 and targeting SGLT2 by empagliflozin attenuated STAT3 phosphorylation and SMC proliferation without affecting cell viability. IH also upregulated SGLT2 expression and targeting SGLT2 by empagliflozin attenuated IH-induced TRAF3IP2 expression, NF-κB and HIF-1α activation, IL-6 expression, STAT3 phosphorylation and SMC proliferation. Together, these mechanistic in vitro results suggest the therapeutic potential of empagliflozin in vascular proliferative diseases.

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Activation Assay, Expressing, Phospho-proteomics, In Vitro

Journal: Experimental & molecular medicine

Article Title: IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

doi: 10.1038/emm.2016.117

Figure Lengend Snippet: Figure 2 IL-17A further activates the inflammatory signaling pathway Act1/TRAF6/IKK/NF-κB in both HG-treated retinal Müller cells and Akita mouse retinal tissue. (a) Expression of IL-17A-related signaling molecules in primarily cultured retinal Müller cells. Retinal Müller cells were exposed to HG (25 mM) for 24 h and subsequently treated with IL-17A (25 ng ml−1) and/or the IKK inhibitor Wedel (10 μM) for 24 h. For silencing the Act1 gene, Müller cells were infected with Ad-Act1-shRNA (or Ad-GFP as a control) for 24 h in the HG condition; the adenoviruses were subsequently removed, and the cells were cultured for an additional 24 h. Following the treatments, the proteins of the cells were extracted for western blot analysis. (b) Expression of IL-17RA downstream signaling molecules in the retinas. At 3 months after the onset of diabetes, Akita mice were subjected to intravitreal injection of IL-17A (8 or 40 ng per 2 μl in PBS per eye) or Ad-Act1-shRNA (or Ad-GFP as a control). At 2 days after the IL-17A intravitreal injection or 2 weeks after the Ad-Act1-shRNA intravitreal injection, the proteins of the retinas were extracted for western blot analysis. **Po0.01 vs control or wild type (WT); +Po0.05, ++Po0.01 vs HG or Akita; &&Po0.01 vs HG+IL-17A+Ad-GFP (or HG+IL-17A) or Akita+Ad-GFP; NS (no significance) vs HG+IL-17A or Akita. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; HG, high glucose; IL, interleukin; NF-κB nuclear factor-κB; PBS, phosphate-buffered saline; TRAF6, tumor necrosis factor receptor-associated factor-6.

Article Snippet: Western blot assays were performed according to previously described methods.35 The following primary antibodies were used: IL-17A (1:200; Santa Cruz Biotechnology, Dallas, TX, USA); Act1 (1:200; OriGene Technologies, Rockville, MD, USA); p-p65 and p65 (both 1:500; Cell Signaling, Danvers, MA, USA); IL-17RA (1:200), GFAP (1:10 000), VEGF (1:1000), GS (1:1000), EAAT1 (1:1000), TRAF6 (1:2000), caspase-3 (1:1000) and β-actin (1:5000; all from Abcam, Cambridge, MA, USA).

Techniques: Expressing, Cell Culture, Infection, shRNA, Control, Western Blot, Injection, Plasmid Preparation, Saline

Journal: Experimental & molecular medicine

Article Title: IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

doi: 10.1038/emm.2016.117

Figure Lengend Snippet: Figure 3 IL-17A exacerbates HG-induced Müller cell activation and dysfunction via Act1/IKK signaling. (a) Expression of proteins related to Müller cell activation (GFAP) and function (VEGF, GS and EAAT1) in cultured Müller cells. (b) GFAP content in cultured Müller cell lysates assessed via ELISA. (c) VEGF concentration in cultured Müller cell supernatants assessed via ELISA. (d) Glutamate content in Müller cell lysates measured by HPLC. The treatment time periods of IL-17A, Ad-Act1-shRNA and the IKK inhibitor Wedel were the same as described in Figure 2. The cutline at the lower left of this figure is shared by (a–d). **Po0.01 vs control; +Po0.05, ++Po0.01 vs HG; &&Po0.01 vs HG+IL-17A+Ad-GFP or HG+IL-17A; NS (no significance) vs HG+IL-17A. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; EAAT1, excitatory amino acid transporter-1; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HG, high glucose; HPLC, high-performance liquid chromatography; IL, interleukin; VEGF, vascular endothelial growth factor.

Article Snippet: Western blot assays were performed according to previously described methods.35 The following primary antibodies were used: IL-17A (1:200; Santa Cruz Biotechnology, Dallas, TX, USA); Act1 (1:200; OriGene Technologies, Rockville, MD, USA); p-p65 and p65 (both 1:500; Cell Signaling, Danvers, MA, USA); IL-17RA (1:200), GFAP (1:10 000), VEGF (1:1000), GS (1:1000), EAAT1 (1:1000), TRAF6 (1:2000), caspase-3 (1:1000) and β-actin (1:5000; all from Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, shRNA, Control, Plasmid Preparation, High Performance Liquid Chromatography

Journal: Experimental & molecular medicine

Article Title: IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

doi: 10.1038/emm.2016.117

Figure Lengend Snippet: Figure 4 IL-17A aggravates in vivo Müller cell activation and dysfunction in Akita mouse retina via Act1 signaling. At 3 months after the onset of diabetes, Akita mice were injected in the vitreous cavity with IL-17A, Ad-Act1-shRNA (or Ad-GFP as a control) or anti-IL-17A mAb. At 2 days after the intravitreal injections of IL-17A or anti-IL-17A mAb or 2 weeks after the intravitreal injection of Ad-Act1-shRNA, the tests were performed. (a) Expression of proteins related to Müller cell activation (GFAP) and function (VEGF, GS and EAAT1) in the retina. (b, d) Glutamate content in the retina assessed via HPLC. (c) Immunofluorescent staining on retinal cross-sections indicates a coexpression of GS that labels Müller cells and VEGF. **Po0.01 vs WT mice; +Po0.05, ++Po0.01 vs Akita or Akita+PBS; &&Po0.01 vs Akita+Ad-GFP; NS (no significance) vs Akita. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; EAAT1, excitatory amino acid transporter-1; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HPLC, high-performance liquid chromatography; IL, interleukin; INL, inner nuclear layer; mAb, monoclonal antibody; ONL, outer nuclear layer; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Article Snippet: Western blot assays were performed according to previously described methods.35 The following primary antibodies were used: IL-17A (1:200; Santa Cruz Biotechnology, Dallas, TX, USA); Act1 (1:200; OriGene Technologies, Rockville, MD, USA); p-p65 and p65 (both 1:500; Cell Signaling, Danvers, MA, USA); IL-17RA (1:200), GFAP (1:10 000), VEGF (1:1000), GS (1:1000), EAAT1 (1:1000), TRAF6 (1:2000), caspase-3 (1:1000) and β-actin (1:5000; all from Abcam, Cambridge, MA, USA).

Techniques: In Vivo, Activation Assay, Injection, shRNA, Control, Expressing, Staining, Plasmid Preparation, High Performance Liquid Chromatography, Saline

Journal: Experimental & molecular medicine

Article Title: IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

doi: 10.1038/emm.2016.117

Figure Lengend Snippet: Figure 5 IL-17A exacerbates BRB breakdown in Akita mice via Act1 signaling. (a) Retinal flat-mounted images indicate vascular leukostasis. The treatment time periods of IL-17A, Ad-Act1-shRNA and anti-IL-17A mAb were the same as described in Figure 4. The adherent leukocytes labeled by FITC-concanavalin A within retinal vessels were counted in each retina. **Po0.01 vs wild-type (WT) mice; ++Po0.01 vs Akita mice; &&Po0.01 vs Akita+Ad-GFP; NS (no significance) vs Akita. (b) Retinal angiography indicates retinal vascular leakage. The retinal angiography was performed 2 weeks after the intravitreal treatments with IL-17A, Ad-Act1-shRNA or anti-IL-17A mAb. The arrows indicate the vascular FITC–dextran exudation in the flat-mounted retinas. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; BRB, blood–retinal barrier; FITC, fluorescein isothiocyanate; IL, interleukin; mAb, monoclonal antibody.

Article Snippet: Western blot assays were performed according to previously described methods.35 The following primary antibodies were used: IL-17A (1:200; Santa Cruz Biotechnology, Dallas, TX, USA); Act1 (1:200; OriGene Technologies, Rockville, MD, USA); p-p65 and p65 (both 1:500; Cell Signaling, Danvers, MA, USA); IL-17RA (1:200), GFAP (1:10 000), VEGF (1:1000), GS (1:1000), EAAT1 (1:1000), TRAF6 (1:2000), caspase-3 (1:1000) and β-actin (1:5000; all from Abcam, Cambridge, MA, USA).

Techniques: shRNA, Labeling, Plasmid Preparation

Journal: Experimental & molecular medicine

Article Title: IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

doi: 10.1038/emm.2016.117

Figure Lengend Snippet: Figure 6 IL-17A increases retinal ganglion cell apoptosis in Akita mice via Act1 signaling. The treatment time periods of IL-17A, Ad-Act1- shRNA and anti-IL-17A mAb were the same as described in Figure 4. (a) Immunofluorescent staining of retinal cross-sections indicates NeuN/TUNEL double-positive cells in the GCL. (b) Immunofluorescent staining of retinal cross-sections indicates NeuN/Active-Cas (Active-caspase-3) double-positive cells in the GCL. The arrows in (a, b) indicate representative double-labeled cells. (c) Statistical histogram for (a, b). The data were obtained as described in the Materials and methods. (d) Caspase-3 activity in the retina that was determined by the ratio of active-caspase-3 (Active-Cas) and pro-caspase-3 (Pro-Cas). **Po0.01 vs wild-type (WT) mice; ++Po0.01 vs Akita mice or Akita+PBS; &&Po0.01 vs Akita+Ad-GFP; NS (no significance) vs Akita. Ad-Act1-shRNA, adenoviral vector that expressed short hairpin RNA targeting Act1; Ad-GFP, adenoviral vector that expressed green fluorescent protein; GCL, ganglion cell layer; IL, interleukin; mAb, monoclonal antibody; TUNEL, TdT-mediated dUTP nick end labeling.

Article Snippet: Western blot assays were performed according to previously described methods.35 The following primary antibodies were used: IL-17A (1:200; Santa Cruz Biotechnology, Dallas, TX, USA); Act1 (1:200; OriGene Technologies, Rockville, MD, USA); p-p65 and p65 (both 1:500; Cell Signaling, Danvers, MA, USA); IL-17RA (1:200), GFAP (1:10 000), VEGF (1:1000), GS (1:1000), EAAT1 (1:1000), TRAF6 (1:2000), caspase-3 (1:1000) and β-actin (1:5000; all from Abcam, Cambridge, MA, USA).

Techniques: shRNA, Staining, TUNEL Assay, Labeling, Activity Assay, Plasmid Preparation, End Labeling

Journal: Cardiovascular Diabetology

Article Title: Dipeptidyl peptidase-4 (DPP-4) inhibition with linagliptin reduces western diet-induced myocardial TRAF3IP2 expression, inflammation and fibrosis in female mice

doi: 10.1186/s12933-017-0544-4

Figure Lengend Snippet: Linagliptin prevents WD-induced increases in TRAF3IP2 expression. a Western blot shows myocardial TRAF3IP2 expression and accompanying bar graphs b show quantitative analysis of protein and mRNA expression as fold change from baseline in the CD group. c Immunofluorescent localization of TRAF3IP2 in the myocardium. The left panels show co-localization of phalloidin (cardiomyocytes, green ) and TRAF3IP2 ( red ). The right panels show colocalization of CD31 (endothelial cells,) and TRAF3IP2 ( yellow ). d Bar graphs show quantitative analysis of TRAF3IP2 immunofluorescence in the myocardium ( top bar graph ) and in the coronary endothelium ( bottom bar graph ) expressed relative to CD. *p < 0.05 vs CD and † p < 0.05 vs WD. N = 5–6/group

Article Snippet: Total RNA was isolated from frozen LV tissue using Trizol reagent (Sigma) and 0.5 μg of RNA was reverse transcribed into cDNA using a reverse transcription kit (Agilent Technologies). mRNA expression was quantified by RT-qPCR using the following Applied Biosystems™ TaqMan™ probes: ANP (Assay ID: Mm01255748), TRAF3IP2 (Assay ID: Mm00506094_m1), IL-18 (Assay ID: Mm00434226), IL-6 (Assay ID: Mm00446191), IL-17A (Assay ID: Mm00439618-m1), IL-17F (Assay ID: Mm00521423-m1), Ccl2/MCP-1 (Assay ID: Mm00441242-m1), CD68 (Assay ID: Mm03047343-m1), AGTR1a/AT1 (Assay ID: Mm01957722-s1), ColIα1 (Assay ID: Mm00801666), ColIIIα1 (Assay ID: Mm1254476), CTGF (Assay ID: Mm01192932_g1), and LOX (Assay ID: Mm00495386).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cardiovascular Diabetology

Article Title: Dipeptidyl peptidase-4 (DPP-4) inhibition with linagliptin reduces western diet-induced myocardial TRAF3IP2 expression, inflammation and fibrosis in female mice

doi: 10.1186/s12933-017-0544-4

Figure Lengend Snippet: Linagliptin inhibits aldosterone (Aldo)-induced cardiac fibroblast activation and migration. The MR agonist, Aldo upregulated TRAF3IP2 expression in a dose-dependent manner ( a ) and pretreatment with the MR antagonist spironolactone and silencing MR each attenuated Aldo-induced TRAF3IP2 expression ( b and c ). Further, linagliptin inhibited Aldo-induced oxidative stress as evidenced by reduced H 2 O 2 generation ( d ), and the induction of CTGF, MCP-1, and IL-18 ( e ). Moreover, linagliptin inhibited upregulation in extracellular matrix proteins collagens Iα1 and IIIα1, and AT1R ( f ). These results were recapitulated by TRAF3IP2 knockdown ( e and f ). Importantly, linagliptin inhibited CF activation and migration ( g ), the hallmarks of cardiac fibrosis. These in vitro experiments were performed at least three times, and a representative immunoblot is shown

Article Snippet: Total RNA was isolated from frozen LV tissue using Trizol reagent (Sigma) and 0.5 μg of RNA was reverse transcribed into cDNA using a reverse transcription kit (Agilent Technologies). mRNA expression was quantified by RT-qPCR using the following Applied Biosystems™ TaqMan™ probes: ANP (Assay ID: Mm01255748), TRAF3IP2 (Assay ID: Mm00506094_m1), IL-18 (Assay ID: Mm00434226), IL-6 (Assay ID: Mm00446191), IL-17A (Assay ID: Mm00439618-m1), IL-17F (Assay ID: Mm00521423-m1), Ccl2/MCP-1 (Assay ID: Mm00441242-m1), CD68 (Assay ID: Mm03047343-m1), AGTR1a/AT1 (Assay ID: Mm01957722-s1), ColIα1 (Assay ID: Mm00801666), ColIIIα1 (Assay ID: Mm1254476), CTGF (Assay ID: Mm01192932_g1), and LOX (Assay ID: Mm00495386).

Techniques: Activation Assay, Migration, Expressing, In Vitro, Western Blot

Journal: Cardiovascular Diabetology

Article Title: Dipeptidyl peptidase-4 (DPP-4) inhibition with linagliptin reduces western diet-induced myocardial TRAF3IP2 expression, inflammation and fibrosis in female mice

doi: 10.1186/s12933-017-0544-4

Figure Lengend Snippet: Schematic illustrates a possible causal role of TRAF3IP2 in western diet induced oxidative stress, inflammation, fibrosis and diastolic dysfunction, and the efficacy of linagliptin in reducing these cardiac impairments. Area within the dotted grey box summarizes novel data presented in this investigation

Article Snippet: Total RNA was isolated from frozen LV tissue using Trizol reagent (Sigma) and 0.5 μg of RNA was reverse transcribed into cDNA using a reverse transcription kit (Agilent Technologies). mRNA expression was quantified by RT-qPCR using the following Applied Biosystems™ TaqMan™ probes: ANP (Assay ID: Mm01255748), TRAF3IP2 (Assay ID: Mm00506094_m1), IL-18 (Assay ID: Mm00434226), IL-6 (Assay ID: Mm00446191), IL-17A (Assay ID: Mm00439618-m1), IL-17F (Assay ID: Mm00521423-m1), Ccl2/MCP-1 (Assay ID: Mm00441242-m1), CD68 (Assay ID: Mm03047343-m1), AGTR1a/AT1 (Assay ID: Mm01957722-s1), ColIα1 (Assay ID: Mm00801666), ColIIIα1 (Assay ID: Mm1254476), CTGF (Assay ID: Mm01192932_g1), and LOX (Assay ID: Mm00495386).

Techniques: Western Blot