Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, PDGF-BB induces time-dependent NF-κB activation. Quiescent SMC were incubated with PDGF-BB (10 ng/ml). At the indicated time periods, activation of NF-κB was analyzed by immunoblotting using antibodies that specifically detect phosphorylated p65 at Ser536. B, PDGF-BB induces time-dependent AP-1 activation. Quiescent SMC incubated as in A were analyzed for AP-1 activation by immunoblotting using antibodies that specifically detect phosphorylated c-Jun at Ser73. C, Silencing IKKβ or pre-treatment with minocycline inhibit PDGF-BB-induced NF-κB activation. SMC incubated with lentiviral IKKβ shRNA (moi0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 μM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of NF-κB was analyzed as in A. D, Silencing JNK2 or pre-treatment with minocycline inhibits PDGF-BB-induced AP-1 activation. SMC incubated with lentiviral JNK2 shRNA (moi 0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 mM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of AP-1 was analyzed as in B. Silencing IKKβ and JNK2 was confirmed by immunoblotting. JNK2 and IKKβ served as off-targets in IKKβ and JNK2 silenced cells, respectively (right hand panels in C and D). Tubulin served as a loading control. Bar graphs at the bottom of panels in A-D represent densitometric analyses from three independent experiments. *P<0.05 control, †P<0.05 versus PDGF-BB (n=3).

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Activation Assay, Incubation, Western Blot, shRNA

Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, B, PDGF-BB induces miR-221 (A) and miR-222 (B) expression via IKKβ, NF-κB, JNK and AP-1. Quiescent SMC incubated with PDGF-BB (10 ng/ml) for lh were analyzed for miR-221 (A) and miR-222 (B) expression by TaqMan® Advanced miRNA assays. The results were normalized to corresponding U6 expression. In a subset of experiments, SMC were incubated with lentiviral IKKβ, p65, JNK2 or c-Jun shRNA (moi0.5 for 48 h), made quiescent and then treated with PDGF-BB addition. C, D, miR-221 and miR-222 mediate PDGF-induced SMC migration (C) and proliferation (D), without affecting cell viability (E). SMC were transduced with miR-221 or miR-222 inhibitors prior to the addition of PDGF-BB (10 ng/ml). Cell migration was analyzed after 18 h using transwell migration assays (C). Cell proliferation was analyzed after 48h by CyQUANT® Cell Proliferation Assay (D). Cleaved caspase-3 levels, indicative of cells undergoing apoptosis, was analyzed after 8 h by immunoblotting using antibodies that detect both total and cleaved caspase-3 levels (E). Hydrogen peroxide (H2O2, 100 μM) served as a positive control. *P

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Expressing, Incubation, shRNA, Migration, Transduction, CyQUANT Assay, Proliferation Assay, Western Blot, Positive Control

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 4. Deoxynyboquinone (DNQ) activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements (ARE) signal pathway in the presence or absence of lipopolysaccharides (LPS)-treated RAW264.7 cells. (A) DNQ increased the stabilization and nucleus translocation of Nrf2. RAW264.7 cells were treated with DNQ with or without of LPS for 6 h, then the cells were subjected to immunofluorescence staining (blue, DAPI; red, Nrf2). (B) DNQ induced the high expression of ARE reporter in HEK293T cells. HEK293T cells were transfected with ARE reporter, then incubated with DNQ for 24 h, following by dual luciferase assay with a dual-luciferase reporter assay system (n ¼ 3). (C) DNQ induced genes expression in Nrf2 downstream. RAW264.7 cells were treated with DNQ for 6 h in the presence or absence of LPS, then the mRNA expression of genes (Hmox1, Nqo1, Gclm, and Pdg) in cells were detected by quantitative polymerase chain reaction (qPCR) (n ¼ 5). (D,E) DNQ induced the decrease of Keap1 and increase of Nrf2, heme oxygenase 1 (HO-1) and GCLM in RAW264.7 cells in a time- (D) and dose-dependent (E) manner. $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. Ctrl group. Ctrl: control; DAPI: 40,6-diamidino-2-phenylindole.

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Staining, Expressing, Transfection, Incubation, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Control

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 5. Deoxynyboquinone (DNQ) activated Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway in BV2 microglial cells and peritoneal macrophages. (A) DNQ increased the stabilization and nucleus translocation of Nrf2. BV2 cells were subjected to immunofluorescence staining (blue, DAPI; red, Nrf2) after treatment with DNQ in the presence or absence of lipopolysaccharides (LPS) for 6 h. (B) DNQ induced genes expression in Nrf2 downstream. BV2 cells were treated with DNQ for 6 h in the presence or absence of LPS, then the mRNA expressions of genes (Hmox1, Nqo1 and Gclm) in cells were detected by quantitative polymerase chain reaction (qPCR) (n ¼ 5). (C) DNQ induced the decrease of Keap1 and increase of Nrf2, heme oxygenase 1 (HO-1) and GCLM in BV2 cells in the presence or absence of LPS for 12 h. (D) mRNA levels of genes (Hmox1, Nqo1 and Gclm; n ¼ 5) and (E) expression of proteins (Nrf2, Keap1, HO-1 and GCLM; n ¼ 3) in the peritoneal macrophages derived from mice were detected. Male C57/BL6 mice (n ¼ 6) were intraperitoneally administered with vehicle (25% PEG40) or DNQ (1 mg/kg DNQ in 25% PEG40) for 6 h, then the peritoneal macrophages were collected for qPCR and Western blot assays. $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. Ctrl group. Ctrl: control; DAPI: 40,6-diamidino-2-phenylindole.

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Staining, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Control

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 6. Deoxynyboquinone (DNQ) upregulated heme oxygenase 1 (HO-1) and inhibited inflammation in a nuclear factor erythroid 2-related factor 2 (Nrf2) dependent manner. (A) BV2 cells were seeded in 24-well plates and cultured to a 50% cell confluence, then cells were transfected with Nrf2 or HO-1 shRNA lentiviral particles for 24 h and sub-cultured in a 6-well plate. Subsequently, the cells were treated with DNQ in the presence or absence of lipopolysaccharides (LPS) for 12 h. (B) The supernatants were collected for nitric oxide (NO) release assay (n ¼ 5), and the cells were subjected to (A) protein (Nrf2, HO-1) and (C) IL6 mRNA (n ¼ 5) assays. (D) DNQ partially inhibited the NO production in the presence of ZnPP (HO-1 inhibitor) in LPS-stimulated RAW264.7 cells for 12 h (n ¼ 5).

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Transfection, shRNA, Release Assay

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 7. Deoxynyboquinone (DNQ) exerted anti-inflammatory effects through activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway via directly targeting Keap1. (A) DNQ bound with human recombinant Kelch-like ECH-associated protein 1 (Keap1) protein with a binding constant (Kd) ¼ 1.5±0.125 mM by isothermal titration calo- rimetry experiment. (B) Cellular thermal shift assay suggested the interaction between DNQ and Keap1. AML12 cells lysates were incubated with DNQ (1 mM) at 4 C overnight, then 50 mL of cellular lysates were aliquoted into fresh tubes and subjected to thermal treatment for 2.5 min at varying temperatures (4878 C), utilizing Eppendorf ThermoMixer C, then the Keap1 in the supernatants was detected by Western blot (n ¼ 3). (C) Schemes for the synthesis of DNQ-L probe (i) and functionalization with biotin-PEG3-azide in cell lysates (ii). (D) DNQ-Linker (DNQ-L) probe was able to “Fishhook” the Keap1 protein from the cell lysates. BV2 cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail, and the supernatants of the lysates were divided into two parts. One part was used for the input analysis, and the other part was incubated with DNQ-L probe in the presence or absence of N-acetylcysteine (NAC) at 4 C for 12 h. Then the biotin-PEG3-azide was added into the cell lysates to bind with the DNQ-L through a click chemistry reaction. Thereafter, the avidin agaroses were added into the cell lysates to bind with the biotin at 4 C for 12 h under

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Recombinant, Binding Assay, Titration, Thermal Shift Assay, Incubation, Western Blot, Radio Immunoprecipitation, Protease Inhibitor, Avidin-Biotin Assay

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 8. Cys489 was the indispensable site for deoxynyboquinone (DNQ) to activate Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway and inhibit inflammation. (A) Tandem mass spectrometry (MS/MS) data of DNQ-modified peptide at Cys489 from Keap1. The human recombinant Keap1 was incubated with DNQ (protein-to-compound molar ratio, 1:10) at 37 C for 12 h prior to MS analysis (left: MS/MS spectrum; right: list of the main fragments in MS/MS spectrum). (B) Successful overexpression of Flag-Keap1 proteins in HEK293T cells. HEK293T cells were transfected with vector, wild type (WT) and mutant Keap1 plasmids for 24 h at a 6-well plate, and the expression of proteins in cells were detected by Western blot. (C) Binding capacity of DNQ to different cysteines-mutant Keap1 by pull-down assay with DNQ-Linker (DNQ-L) probe. HEK293T cells, successfully transfected with WT or mutant Keap1 plasmids, were subjected to lysis utilizing radioimmunoprecipitation assay (RIPA) lysis buffer. Following centrifugation, the resulting supernatants were subjected to incubation with DNQ-L at 4 C for 12 h. Subsequently, biotin-PEG3-azide was introduced into the mixture to facilitate click chemistry reaction. Then, avidin agarose beads were introduced into the cellular lysates to isolate the biotin-DNQ-protein complex. Following centrifugation, the supernatants and precipitates were subjected to boiling with 5 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer at 95 C for 10 min prior to Western blot analysis. (D) DNQ failed to activate Keap1/Nrf2 in Cys489-mutated group. The HEK293T cells were transfected with vector, WT and mutant Keap1 plasmids for 24 h at a 6-well plate, and the cells were sub-cultured in two 6-well plates. After incubation in the presence or absence of DNQ for 6 h, the expression of proteins in cells was detected by Western blot. (E) DNQ failed to inhibit lipopolysaccharides (LPS)-induced IL6 mRNA expression in Cys489-mutated group. The HEK293T cells were transfected with vector, WT and mutant Keap1 plasmids for 24 h at a 6-well plate. The cells were sub-cultured in three 6-well plates and then incubated with DNQ for 6 h in the presence or absence of LPS, and the IL6 mRNA was analyzed by qPCR (n ¼ 6). HO-1: heme oxygenase 1.

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Recombinant, Incubation, Over Expression, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Western Blot, Binding Assay, Pull Down Assay, Lysis, Radio Immunoprecipitation, Centrifugation, Avidin-Biotin Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Cell Culture

Journal: Journal of pharmaceutical analysis

Article Title: Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy.

doi: 10.1016/j.jpha.2023.07.009

Figure Lengend Snippet: Fig. 9. Deoxynyboquinone (DNQ)-mediated alkylation of Kelch-like ECH-associated protein 1 (Keap1)-Cys489 triggered the dissociation of Keap1-nuclear factor erythroid 2-related factor 2 (Nrf2) complex and the degradation of Keap1 by driving Keap1-ubiquitination. (A) DNQ failed to trigger the dissociation of Keap1-Nrf2 complex in the Keap1-Cys489 mutant HEK293T cells. HEK293T cells were seeded in 100 mm culture dish and transfected with Myc-Nrf2 plasmid together with wild type (WT) Flag-Keap1 or Flag-Keap1-C489A plasmid for 24 h and then sub-cultured into two dishes. The sub-cultured cells were treated in the presence or absence of DNQ for 12 h, then total protein expression (Input) in the cell lysates was measured with the anti-Nrf2, anti-Keap1, anti-Myc and anti-Flag antibodies by immunoblot (IB). The Keap1 proteins associated with Nrf2 were detected with an anti-Keap1 antibody after being immunoprecipitated with anti-Myc magnetic beads (IP: Myc) or protein A/G PLUS-agarose (IP: Nrf2). (B) DNQ induced the ubiquitination of Keap1 but decreased the ubiquitination of Nrf2. RAW264.7 cells were treated with DNQ (0, 0.25, 0.5 or 1 mM) for 12 h following treatment with 10 mM MG132 for 5 h. Cells were harvested in reduced buffer (containing 2% sodium dodecyl sulphate (SDS), 150 mM NaCl, 10 mM Tris-HCl pH 8.0, and 1 mM dithiothreitol (DTT)) then immediately boiled and diluted five-fold in buffer without SDS. The total protein of Nrf2 and Keap1 (Input) in the cell lysates was detected with anti-Nrf2 and anti-Keap1 antibodies. The ubiquitination levels of Nrf2 and Keap1 in the immunoprecipitants by protein A/G PLUS-agarose (IP: Nrf2, Keap1) were analyzed through IB with an antibody against the ubiquitin. (C) DNQ failed to trigger the ubiquitination of Keap1 in the Keap1-C489 mutant HEK293T cells. HEK293T cells were transfected with Myc3-Nrf2 and mRFP-Ub plasmids together with WT-Flag-Keap1 or Flag- Keap1-C489 mutant plasmid for 24 h and treated with 1 mM DNQ for 6 h, following treatment with 10 mM MG132 for 5 h. Cells were harvested in reduced buffer then immediately boiled and diluted five-fold in buffer without SDS. The total protein (Input) of Myc and Flag in the cell lysates were detected with anti-Myc and anti-Flag antibodies, and the ubiquitination levels of Myc-Nrf2 and Flag-Keap1 in the immunoprecipitants by anti-Myc or anti-Flag magnetic beads were evaluated using the anti-Ub antibody with IB.

Article Snippet: The Nrf2 shRNA (m) lentiviral particles (# sc-37049-V), heme oxygenase 1 shRNA (m) lentiviral particles (#sc-35555-V), control shRNA lentiviral particles (#sc-108080) and polybrene (#sc134220) were procured from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Ubiquitin Proteomics, Mutagenesis, Transfection, Plasmid Preparation, Cell Culture, Expressing, Western Blot, Immunoprecipitation, Magnetic Beads

Journal: Cell Death & Disease

Article Title: Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression

doi: 10.1038/cddis.2017.45

Figure Lengend Snippet: Effect of IFN- γ R α and iNOS knockdown MSCs on CD25 expression. To knock down IFN- γ R α , MSCs were infected with shRNA-harboring lentiviral particles ( a – f ). IFN- γ R α KD MSCs were stimulated with IFN- γ (20 ng/ml) and TNF- α (10 ng/ml). IFN- γ R α and iNOS expression was measured after 24 h by ( a ) RT-PCR or ( b ) western blotting. IFN- γ R α KD MSCs were cultured with lymphocytes for 48 h. ( c ) NO and ( d ) T-cell proliferation were measured by ELISA and thymidine incorporation, respectively. ( e ) CD25 cell surface expression was measured at 48 h by flow cytometry. ( f ) IL-2 secretion into cell culture media was measured at 48 h by ELISA. To knock down iNOS, MSCs were transfected with siRNA ( g – k ). iNOS KD MSCs were stimulated with IFN- γ (20 ng/ml) and TNF- α (10 ng/ml). iNOS mRNA and protein were measured after 24 h by ( g ) qRT-PCR or ( h ) western blotting. iNOS KD MSCs were cultured with lymphocytes for 48 h. ( i ) NO and ( j ) CD25 cell surface expression were measured by ELISA and flow cytometry, respectively. ( k ) IL-2 level in culture media was measured at 48 h by ELISA. Similar results were obtained in two independent experiments. WT, wild type; KD, knockdown; I, IFN- γ , T; TNF- α ; M, MSCs, * P <0.05, ** P <0.01 compared to the controls

Article Snippet: IFN- γ R α knockdown was induced using shRNA lentiviral particles (SC-35636-v; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Infection, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Transfection, Quantitative RT-PCR