Journal: The European Respiratory Journal

Article Title: Pharmacological inhibition of Epac1 protects against pulmonary fibrosis by blocking FoxO3a neddylation

doi: 10.1183/13993003.02250-2024

Figure Lengend Snippet: Molecular responses to AM-001 and neddylation inhibition in lung fibroblasts (FBs). a) The transcript levels of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-activating enzyme E1 ( NAE1 ), ubiquitin conjugating enzyme E2 M ( UBE2M ) and NEDD8 in lung tissue samples from healthy donors (n=7) and idiopathic pulmonary fibrosis (IPF) patients (n=7) were assessed by quantitative reverse-transcriptase PCR (qRT-PCR). b) Co-immunostaining of NEDD8 (red) and α-smooth muscle actin (α-SMA) (green) in lung tissue sections from both healthy donors and IPF patients. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. c) Forkhead box protein O3 ( FoxO3a ) mRNA levels (n=8). d) Representative images of co-immunostaining of FoxO3a (red) with α-SMA (green) in lung tissue sections from healthy donors and IPF patients. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. e) Representative immunoblot analysis of FoxO3a, UBE2M and NEDD8 in normal human lung (NHL) FBs and IPF-FBs (left panel). Right panels show the ratio quantification using GAPDH as the loading control (n=3). f) Transcript levels of NAE1 , NEDD8 and UBE2M in NHL-FBs and IPF-FBs after 48 h of treatment with either vehicle or AM-001 (20 μM) for 48 h (n=3). g) FoxO3a mRNA expression in NHL-FBs and IPF-FBs after 48 h of treatment with either vehicle or AM-001 (20 μM) for 48 h (n=3). h) Representative immunoblot analysis showing the levels of phosphorylated FoxO3a at Thr32 (p-FoxO3a Thr32 ), total FoxO3a and NEDD8 under the indicated conditions. GAPDH was used as a loading control. i) mRNA expression levels of fibrosis markers transforming growth factor β1 ( TGF-β1 ), collagen type 1 ɑ1 ( COL1A1 ), collagen type 3 ɑ1 ( COL3A1 ) and connective tissue growth factor ( CTGF ) were measured by qRT-PCR in NHL-FBs and IPF-FBs treated with MLN4924 (5 µM) alone, a small molecule inhibitor of NAE, or in combination with AM-001 (20 μM) for 48 h (n=3). j) Representative immunoblot analysis of p-FoxO3a Thr32 , total FoxO3a and NEDD8 protein levels in NHL-FBs or IPF-FBs treated with vehicle, MLN4924 alone or in combination with AM-001 for 48 h. k) Immunoprecipitation of haemagglutinin (HA)-tagged NEDD8 (HA-NEDD8) followed by immunoblot analysis of NEDD8 and total FoxO3a in IPF-FBs transfected with HA-NEDD8, in IPF-FBs pretreated with proteasome inhibitor MG132 (10 µM) to inhibit protein degradation, and under the indicated conditions. Data are presented as mean± sem . 8-CPT: Sp-8-(4-chlorophenylthio)-2′-O-methyl-cAMP; IB: immunoblot; IgG: immunoglobulin G; ns : nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Article Snippet: Pevonedistat (MLN4924) is a potent and selective inhibitor of the NEDD8-activating enzyme (NAE) with a half-maximal inhibitory concentration of 4.7 nM, obtained from MedChemExpress (Monmouth Junction, NJ, USA; catalogue number: HY-70062).

Techniques: Inhibition, Ubiquitin Proteomics, Reverse Transcription, Quantitative RT-PCR, Immunostaining, Western Blot, Control, Expressing, Immunoprecipitation, Transfection

Journal: The European Respiratory Journal

Article Title: Pharmacological inhibition of Epac1 protects against pulmonary fibrosis by blocking FoxO3a neddylation

doi: 10.1183/13993003.02250-2024

Figure Lengend Snippet: Pharmacological inhibition of exchange factor directly activated by cAMP 1 (Epac1) by AM-001 attenuates bleomycin (BLM)-induced pulmonary fibrosis (PF). a) Schematic outlining the experimental design. C57BL/6 mice received a single intratracheal (IT) delivery of BLM (4 U·kg −1 ) for 28 days (∼4 weeks). AM-001 was then administered via intraperitoneal (IP) injection every other day at a dose of 10 mg·kg −1 for an additional 2 weeks. b) Representative images of Masson's trichrome staining (left panel) of lung sections from control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001. The right panel shows the quantification of the fibrotic area, expressed as the fold change relative to the saline control group (n=5–7). c) Lung hydroxyproline content, an index of collagen deposition, was measured in saline-treated control mice and BLM-challenged mice administered either vehicle or AM-001 (n=5–7). d) mRNA expression levels of profibrotic markers transforming growth factor β1 ( TGF-β1 ), collagen type 1 ɑ1 ( COL1A1 ), collagen type 3 ɑ1 ( COL3A1 ) and connective tissue growth factor ( CTGF ) in lung tissues were measured by quantitative reverse-transcriptase PCR (qRT-PCR) in lung tissue from the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001 (n=3–7). e) The transcript levels of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-activating enzyme E1 ( NAE1 ), ubiquitin conjugating enzyme E2 M ( UBE2M ) and NEDD8 in lung tissues from both the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001 were analysed by qRT-PCR (n=3–7). f) Co-immunostaining of NEDD8 (red), forkhead box protein O3 (FoxO3a) (red) and α-smooth muscle actin (α-SMA) (green) in lung sections from the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001. Nuclei were stained with DAPI (blue). Scale bars: 100 μm. g) Representative immunoblot analysis and h) quantification of Epac1, phosphorylated FoxO3a (p-FoxO3a), total FoxO3a, NEDD8, phosphorylated SMAD2/3 (p-SMAD2/3) and total SMAD2/3 in lung homogenates from the indicated group of mice (n=3). The data are presented as mean± sem . ns : nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Article Snippet: Pevonedistat (MLN4924) is a potent and selective inhibitor of the NEDD8-activating enzyme (NAE) with a half-maximal inhibitory concentration of 4.7 nM, obtained from MedChemExpress (Monmouth Junction, NJ, USA; catalogue number: HY-70062).

Techniques: Inhibition, Injection, Staining, Control, Saline, Expressing, Reverse Transcription, Quantitative RT-PCR, Ubiquitin Proteomics, Immunostaining, Western Blot

Journal: Molecules

Article Title: The Large Molecular Weight Polysaccharide from Wild Cordyceps and Its Antitumor Activity on H22 Tumor-Bearing Mice

doi: 10.3390/molecules28083351

Figure Lengend Snippet: WCP promotes Cyto-c/Caspase8/3 and inhibits IL-10/STAT3/Bcl2 pathway. ( A – F ) Relative mRNA expression of IL-6, IL-Iβ, NF-κB, TNF-α, Bax, and Bcl2. * p < 0.05 compared to model group.

Article Snippet: Rabbit monoclonal antibodies against Caspase8 (PTM-6085) antibody were purchased from PTM Biolab Co., Ltd. (Hangzhou, China), and signal transducer and activator of transcription3 (Stat3, BM4052), phosphorylated signal transducer and activator of transcription3 (p-STAT3 Y705, BM4835) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Molecules

Article Title: The Large Molecular Weight Polysaccharide from Wild Cordyceps and Its Antitumor Activity on H22 Tumor-Bearing Mice

doi: 10.3390/molecules28083351

Figure Lengend Snippet: WCP promotes Cyto-c/Caspase8/3 and inhibits IL-10/STAT3/Bcl2pathway. ( A , F ) Photographs of the proteins in each group. ( B – E , G – I ) Relative protein expression of Cyto-c, Caspase8, Caspase3, p-STAT3, Bcl2, Bax, and Bax/Bcl2. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to model group.

Article Snippet: Rabbit monoclonal antibodies against Caspase8 (PTM-6085) antibody were purchased from PTM Biolab Co., Ltd. (Hangzhou, China), and signal transducer and activator of transcription3 (Stat3, BM4052), phosphorylated signal transducer and activator of transcription3 (p-STAT3 Y705, BM4835) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Antioxidants

Article Title: The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

doi: 10.3390/antiox9050452

Figure Lengend Snippet: Post-translational modification of proteins by neddylation. The ubiquitin-like protein Nedd8 is covalently bound to substrate proteins by a cascade of E1 activating, E2 conjugating and E3 ligase enzymes through a process termed neddylation. Deneddylation regulates this process and is catalyzed by the c-Jun activation domain-binding protein-1 (Jab1)/Csn5 subunit active site of the COP9-signalosome, which cleaves the isopeptide linkage to release neural precursor cell expressed developmentally down-regulated 8 (Nedd8) from the substrate protein. ~, thioester intermediate. —, isopeptide linkage.

Article Snippet: Antibodies used included: anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA), anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA), anti-Cul-1 antibody (Novus, Littleton, CO, USA), anti-Nedd8 antibody (Boster Bio, Pleasanton, CA, USA), anti-P27 antibody (Proteintech Group, Rosemont, IL, USA), anti-β actin antibody (Abcam, Cambridge, UK), and HRP-conjugated secondary antibodies for Western blot analyses (Rabbit anti-mouse and Goat anti-rabbit) (Bio-Rad, Hercules, CA, USA).

Techniques: Modification, Ubiquitin Proteomics, Activation Assay, Binding Assay

Journal: Antioxidants

Article Title: The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

doi: 10.3390/antiox9050452

Figure Lengend Snippet: In vivo regulation of Jab1 activity by MsrA through monitoring Cul-1 neddylation levels in mouse brain and liver extracts. ( A ), a–d. Mouse extracts from 6 months old mice ( n = 3) were made in PBS and in the presence of protease inhibitors cocktail (Sigma-Aldrich). Immunoprecipitation (IP) experiments were performed on brain and liver extracts (500 µg of protein per extract) using either anti-Cul-1 antibody or anti-Nedd8 antibody followed by Western blot (WB) analyses using anti-Nedd8 antibody or anti-Cul-1 antibody as the primary antibody, respectively. Ae. In a unique experiment, Jab1 was used as a “fishing” probe in an IP experiment using brain extracts, followed by Western blot analysis using anti-Cul-1antibody. Only the 100-kDa neddylated Cul-1 protein was identified due to the specific pull-down of the neddylated form of Cul-1 by Jab1. ( B ) Quantification of each band identified by Western blot analyses described in Panel ( A ) a–e, as determined by using the NIH Image-J program. All the observed differences in the observed band levels between the WT and MT pairs were statistically significant as judged by student t -test analysis (* p < 0.01, n = 3 per strain). ( C ) Loading controls for the liver and brain protein levels, following Coomassie blue staining (to confirm the use of equal amount of proteins from each mouse strain per tissue in the IP experiments. WT, wild type; M, MsrA KO. ( D ) Western blot analysis of the mouse brain extracts using anti Jab1 antibody as the primary antibody and quantified by the NIH Image-J program. WT, wild type; MT, MsrA KO. Arrows shown in ( A ) a–e indicate the position of the neddylated 100-kDa Cul-1 (deneddylated form runs as ~90 kDa protein, not detected). The shown WB and Coomassie blue staining experiments are representatives of three independent experiments.

Article Snippet: Antibodies used included: anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA), anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA), anti-Cul-1 antibody (Novus, Littleton, CO, USA), anti-Nedd8 antibody (Boster Bio, Pleasanton, CA, USA), anti-P27 antibody (Proteintech Group, Rosemont, IL, USA), anti-β actin antibody (Abcam, Cambridge, UK), and HRP-conjugated secondary antibodies for Western blot analyses (Rabbit anti-mouse and Goat anti-rabbit) (Bio-Rad, Hercules, CA, USA).

Techniques: In Vivo, Activity Assay, Immunoprecipitation, Western Blot, Staining

Journal: Antioxidants

Article Title: The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

doi: 10.3390/antiox9050452

Figure Lengend Snippet: MsrA alters the kinetics of Jab1 deneddylase activity in vitro. (A) Western blot pattern of control group (time 0′). Total protein neddylation and Nedd8 levels are shown using the primary anti-His antibody. We focused on lowest molecular weight Nedd8 positive bands (Low M.W.Nedd8) as they represent the best end-product of the Nedd8 conjugates through the deneddylation process. ( B ) Quantification of lowest molecular weight Nedd8 positive bands at different time points following incubation with WT and MsrA KO mouse brain extracts ( n = 3, females, 6 months old). ( C ) Time curve of lowest molecular weight Nedd8 levels. All data were normalized to 0 min time. The shown WB are representatives of three repeated experiments. WT, Wild-type; KO, MsrA knockout; The difference between the two animal groups was statistically significant from the 5–30 min time points ( t -test, p < 0.001).

Article Snippet: Antibodies used included: anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA), anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA), anti-Cul-1 antibody (Novus, Littleton, CO, USA), anti-Nedd8 antibody (Boster Bio, Pleasanton, CA, USA), anti-P27 antibody (Proteintech Group, Rosemont, IL, USA), anti-β actin antibody (Abcam, Cambridge, UK), and HRP-conjugated secondary antibodies for Western blot analyses (Rabbit anti-mouse and Goat anti-rabbit) (Bio-Rad, Hercules, CA, USA).

Techniques: Activity Assay, In Vitro, Western Blot, Control, Molecular Weight, Incubation, Knock-Out

Journal: International journal of oncology

Article Title: Knockdown of SNHG15 suppresses renal cell carcinoma proliferation and EMT by regulating the NF-κB signaling pathway.

doi: 10.3892/ijo.2018.4395

Figure Lengend Snippet: Figure 5. Knockdown of SNHG15 affects NF‑κB entry into the nucleus. (A) Knockdown of SNHG15 reduced the protein expression levels of EMT‑associated transcription factors (Snail1, Slug and ZEB1). However, there was no difference in the total protein expression levels of NF‑κB between the SNHG15 siRNA and NC groups. *P<0.01; nsP>0.05. (B) Nuclear/cytoplasmic NF‑κB expression in ACHN and 786‑O cells transfected with siRNAs. *P<0.01. (C) Following stimulation with TNF‑α for 6 h, EMT markers were examined among the various groups. *P<0.01.

Article Snippet: Monoclonal primary antibodies were specific to Snail (1:1,000, 3879), Slug (1:1,000, 9585), Vimentin (1:1,000, 5741), E-cadherin (1:1,000, 14472), N-cadherin (1:1,000, 13116) and Histone H3 (1:2,000, 4499) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), ZEB1 (1:500, BA2871‐2; Wuhan Boster Biological Technology, Ltd., Wuhan, China), β-actin (1:5,000, A1978), GAPDH (1:5,000, G9545) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and NF-κB1 (p50) (1:1,000, 14220-1-AP; Wuhan Sanying Biotechnology, Wuhan, China).

Techniques: Knockdown, Expressing, Transfection

Journal: International journal of oncology

Article Title: Knockdown of SNHG15 suppresses renal cell carcinoma proliferation and EMT by regulating the NF-κB signaling pathway.

doi: 10.3892/ijo.2018.4395

Figure Lengend Snippet: Figure 5. Continued. (D) Nuclear immunofluorescence intensity of NF‑κB was reduced in the SNHG15 siRNA groups compared with in the NC groups. (E) Cell migration and invasion were altered among the various groups following TNF‑α stimulation and siRNA transfection (magnification, x40). *P<0.01. EMT, epithelial‑mesenchymal transition; NC, negative control; NF‑κB, nuclear factor‑κB; siRNA, small interfering RNA; SNHG15, small nucleolar RNA host gene 15; TNF‑α, tumor necrosis factor‑α; ZEB1, zinc finger E‑box‑binding homeobox 1.

Article Snippet: Monoclonal primary antibodies were specific to Snail (1:1,000, 3879), Slug (1:1,000, 9585), Vimentin (1:1,000, 5741), E-cadherin (1:1,000, 14472), N-cadherin (1:1,000, 13116) and Histone H3 (1:2,000, 4499) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), ZEB1 (1:500, BA2871‐2; Wuhan Boster Biological Technology, Ltd., Wuhan, China), β-actin (1:5,000, A1978), GAPDH (1:5,000, G9545) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and NF-κB1 (p50) (1:1,000, 14220-1-AP; Wuhan Sanying Biotechnology, Wuhan, China).

Techniques: Immunofluorescence, Migration, Transfection, Negative Control, Small Interfering RNA

Journal: Cells

Article Title: Current Advancements in Spinal Cord Injury Research—Glial Scar Formation and Neural Regeneration

doi: 10.3390/cells12060853

Figure Lengend Snippet: A list of recent clinical trials for SCI.

Article Snippet: A Single Center, Open Label, Single Group, Phase 1/2a Clinical Study to Evaluate the Safety and Exploratory Efficacy of Transplantation Therapy Using PSA-NCAM(+) NPC Derived From hESC Line in AIS-A Level of Sub-acute SCI(From 7 to 60 Days) , Neural precursor cells derived from human embryonic stem cell line , 2021–2023 , I/IIa , recruiting , S.Biomedics Co., Ltd..

Techniques: Transplantation Assay, Injection, Derivative Assay, Cell Culture