Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmid coding for E1 Ubiquitin-Activating Enzyme 1 (UBA1) (pCMV3-UBA1-c-Myc) was purchased from SiNo Biological (Düsseldorfer, Eschborn, Germany).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Cotransfection

Journal: Viruses

Article Title: Ubiquitin and Not Only Unfolded Domains Drives Toscana Virus Non-Structural NSs Protein Degradation

doi: 10.3390/v12101153

Figure Lengend Snippet: TOSV NSs undergoes ubiquitination. NSs ubiquitination was evaluated by immunoblotting. ( A ) Lenti-X 293T cells were transfected with wt-, ΔC-, or ΔN-expressing plasmids, along with the plasmid expressing HA-tagged wild-type ubiquitin (Ub). Cells treated with the proteasome inhibitor MG-132 were collected at 48 h post-transfection and NSs protein enrichment was performed on cell lysates by pull-down (PD) experiments using Ni-NTA agarose beads. 6×His-NSs-enriched samples were subjected to immunoblotting for Ub (α-HA) or NSs (α-6×His) detection. The ubiquitinated status of the three NSs forms was evaluated as a modification of the targeted substrate, causing a shift in MW of ~10 kDa (mono-ubiquitination) or multiples. Asterisk represents ubiquitinated NSs forms. ( B ) The rate on K 48 -and K 63 -moiety ubiquitination was assessed by PD assay and immunoblotting. Lenti-X 293T cells were transfected with wt-, ΔC, or ΔN NSs expressing plasmids, along with the plasmid expressing HA-tagged K 48 -only or K 63 -only ubiquitin mutants. Twenty-four hours later, cells were treated with MG-132 and collected after additional 24 h. Lysates were prepared and PD with Ni-NTA agarose beads. Isolated proteins were separated by SDS-PAGE and probed by immunoblotting for NSs (α-6×His) and Ub-K 48 and Ub-K 63 (α-HA) detection. Asterisk indicates ubiquitinated forms of the NSs proteins.

Article Snippet: HA-tagged ubiquitin expressing plasmid was a kind gift of D. Arnoult (Inserm, France) while K48-only and K63-only ubiquitin plasmids were purchased from Addgene (Teddington, UK).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Protein Enrichment, Modification, Isolation, SDS Page

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 is a driver of cardiac dysfunction and myocardium infarct post-MI. WT mice were treated with rAAV9-cTnT-GFP or rAAV9-cTnT-WWP1 by intravenous injection of tail two weeks before suffering from sham or LAD ligation, and after additional one day, mice were sacrificed. A Infection efficiency was detected by Western blot. n = 3. B-H Cardiac function indices were measured by echocardiography. EF% ejection fraction, FS% fractional shortening, LVEDV left ventricular end-diastolic volume, LVESV left ventricular end-systolic volume, LVIDd left ventricular internal dimension diastole, LVIDs left ventricular internal dimension systole. n = 6. I, J Infarct size was determined by TTC staining and expressed as the percentage of infarct over ventricular areas. n = 3. K-U Mice were treated with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1 by intravenous injection of tail 2 weeks before suffering from sham or LAD ligation, and after additional 3 days, mice were sacrificed. K Immunofluorescence co-staining for cTnI with WWP1 and DAPI in mice hearts infected with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1 at day 3 post-MI. Scale bar = 200 μm. n = 3. L, M WWP1 protein level was detected by Western blots. Corresponding statistic of WWP1 was shown. n = 4. N-T Cardiac function indices were measured by echocardiography. n = 4, 4, 7, 6, respectively. U Infarct size was determined by TTC staining and expressed as the percentage of infarct over ventricular areas. n = 3. The data are shown as the means ± SD. The data shown in A, C-H, J, M, O-T, and U were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Infection, Western Blot, Staining, Immunofluorescence

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: The regulatory function of WWP1 on cardiac ischemic injury post-MI is dependent on KLF15. Mice were injected with rAAV9-cTnT-WWP1 with or without rAAV9-cTnT-KLF15 before they subjected to MI. A-C Cardiac function indices were measured by echocardiography at day 3 post-MI. n = 6. D-H mRNA levels of IL-6, IL-1β, TNF-α, VCAM-1, and MCP-1 in infarcted or healthy hearts were tested by qRT-PCR at day 3 post-MI. n = 4. I-K Cardiac function indices were measured by echocardiography at day 21 post-MI. n = 6. L Ratio of heart weight/ body weight (HW/BW). n = 6. M-Q qRT-PCR was used to test the mRNA levels of ANP, BNP, β-MHC, collagen I, collagen III. n = 3. R, S Sirius red staining were performed to detect fibrosis. n = 4. The data are shown as the means ± SD. The data shown in B-H, J-Q, and S were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Quantitative RT-PCR, Staining

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: I3C pre-treatment did not further improved cardiac function and remodeling after MI in WWP1-knockdown mice. A-C Mice were administered with vehicle or I3C (20 mg/kg) three times a week for a month. When mice were pretreated by I3C for two weeks, they were randomly assigned to rAAV9-cTnT-shWWP1 or rAAV9-cTnT-shScramble injection, and all of which were subjected to LAD ligation after two weeks of virus injection. All mice sacrificed at day 21 after induction of MI. cardiac function indices were measured by echocardiography. EF% ejection fraction, FS% fractional shortening. n = 4, 5, 5, 4, 5, 5, respectively. D Ratio of heart weight/ body weight (HW/BW). n = 4, 5, 5, 4, 5, 5, respectively. E Treatment regimen. F Masson's trichrome staining was performed to detect the fibrosis in left ventricle. n = 3. G Sirius red staining and hematoxylin (top) and eosin (H&E) staining (bottom) were performed to detect fibrosis and hypertrophy in non-infarct zone. n = 3. H, I Corresponding fibrosis area and cardiomyocyte cross-sectional area were calculated. n = 3. J-N qRT-PCR was used to test the mRNA levels of collagen I, collagen III, ANP, BNP, β-MHC. n = 3. The data are shown as the means ± SD. The data shown in B-D, H-N were analysed by two-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Staining, Quantitative RT-PCR

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: Inhibition of WWP1 expression in cardiomyocytes restrains NF-κB and MAPK activation involving degrading KLF15 in the infarcted myocardium. Mice were treated with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1 by intravenous injection of tail two weeks before suffered from sham or MI surgery, and after additional one day, mice were sacrificed. A Ubiquitination of KLF15 was detected by immunoprecipitation with anti-KLF15 antibody followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. B Immunohistochemistry for KLF15 (brown) in sham or infarcted hearts. Scale bar = 50 μm. n = 3. C Immunofluorescence co-staining for cTnI with KLF15 and DAPI in mice hearts post-MI. Scale bar = 40 μm. n = 3. D-F Protein expression of KLF15 in cytoplasm and nuclear lysates harvested from infarct areas post-MI was detected by Western blots. Corresponding statistics of KLF15 were shown. n = 3. G Western blot analysis and statistical results of expression of P65-AcK310 in nucleus. n = 3. H The levels of phosphorylated P38 and ERK1/2 were examined by Western blots, and statistical results were shown. n = 3. The data are shown as the means ± SD. The data shown in E-H were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Inhibition, Expressing, Activation Assay, Injection, Immunoprecipitation, Western Blot, Negative Control, Immunohistochemistry, Immunofluorescence, Staining

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 expression is increased in cardiomyocytes in response to myocardial infarction. A Immunoblot analysis of WWP1 (n = 6, 14) and Bax (n = 3, 4) expression in the infarct areas (infarct and border zone) of wild type (WT) mice at day 1 post-MI as well as in the sham control. Corresponding statistics of WWP1 and Bax were shown. B Immunoblot analysis of WWP1 (n = 6, 14) and Bax (n = 3, 4) expressions in non-infarct areas at day 1 post-MI as well as in the sham control. Corresponding statistics of WWP1 and Bax were shown. C Immunohistochemistry for WWP1 (brown) in the sham heart or infarcted heart tissue. n = 4. D Immunofluorescence co-staining for cTnI with WWP1 and DAPI in the heart post-MI. n = 4. E Neonatal rat cardiac myocytes (NRCMs) were treated by hypoxia for 6h. WWP1 and Bcl2 expressions were examined by Western blots. Corresponding statistics of WWP1 and Bcl2 were shown. n = 3. The data are shown as the means ± SD. The data shown in A, B, and E were analysed by unpaired Student's t-test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 induces cardiomyocyte apoptosis post-MI. A-D WT mice were treated with rAAV9-cTnT-GFP or rAAV9-cTnT-WWP1 by intravenous injection of tail two weeks before suffering from sham or LAD ligation, and after additional one day, mice were sacrificed. Representative Western blots and statistical results of Bax, Bcl2 and cleaved-caspase3 in the infarct zone. n = 3. E-H Mice were treated with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1 by intravenous injection of tail two weeks before suffering from sham or LAD ligation, and after additional three days, mice were sacrificed. Representative Western blots and statistical results of Bax, Bcl2 and cleaved-caspase3 in the infarct zone. n = 3. I TUNEL assay and immunofluorescence staining with cTnI were performed to detect cardiomyocyte apoptosis in the myocardium infected with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1. Scale bar = 100 μm. The enlarged image represents a digital enlargement of the area indicated within the box. n = 3. J Statistical results of TUNEL+ cardiomyocytes at day 3 post-MI. K-N NRCMs infected with Adv-GFP or Adv-WWP1 for 48 h followed by hypoxic stimulation for 6 h. Representative Western blots and statistical results of Bax, Bcl2 and cleaved-caspase3 in NRCMs. n = 3. O, P TUNEL assay and statistical result of TUNEL + cardiomyocytes. Scale bar = 400 μm. n = 3. The data are shown as the means ± SD. The data shown in B-D, F-H, J, L-N, and P were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Western Blot, TUNEL Assay, Immunofluorescence, Staining, Infection

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 triggers cardiomyocyte inflammatory response post-MI. A WT mice were treated with rAAV9-cTnT-GFP or rAAV9-cTnT-WWP1 by intravenous injection of tail two weeks before suffering from sham or LAD ligation, and after additional one day, mice were sacrificed. Representative Western blot and statistical result of Ly6G in the infarct zone. n = 3. B-F mRNA levels of IL-6, IL-1β, TNF-α, VCAM-1, and MCP-1 in infarcted hearts or healthy hearts were tested by qRT-PCR. n = 3. G, H Mice were treated with rAAV9-cTnT-shScramble or rAAV9-cTnT-shWWP1 by intravenous injection of tail two weeks before suffered from sham or LAD ligation, and after additional three day, mice were sacrificed. Immunofluorescence co-staining for cTnI with F4/80 and DAPI in infarcted hearts. Scale bar = 100 μm. The enlarged image represents a digital enlargement of the area indicated within the box. n = 3. I-M mRNA levels of IL-6, IL-1β, TNF-α, VCAM-1, and MCP-1 in infarcted or healthy hearts were tested by qRT-PCR. n = 3, 3, 5, 5, respectively. N-P H9C2 cells were transfected with Si-NC or Si-WWP1 for 48 h followed by hypoxic stimulation for 6 h. mRNA levels of IL6, IL-1β, and TNF-α were tested by qRT-PCR. n = 3. Q-S NRCMs infected with Adv-GFP or Adv-WWP1 for 48 h followed by hypoxic stimulation for 6 h. mRNA levels of IL6, IL-1β, and TNF-α were tested by qRT-PCR. n = 3. T NRCMs infected with Adv-GFP or Adv-WWP1 for 48 h followed by hypoxic stimulation for 6 h. Conditioned media was collected and used to treat RAW264 cells. Transwell assay was performed to test the chemotaxis of RAW264 cells. Scale bar = 200 μm. n = 3. The data are shown as the means ± SD. The data shown in A-U were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Transfection, Infection, Transwell Assay, Chemotaxis Assay

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 promotes KLF15-ubiquitination mediated degradation in hypoxia-induced cardiomyocytes. A NRCMs were infected with Adv-WWP1 or Adv-GFP for 48 h before the cells were treated with hypoxia and MG132 (5 μM) for 6 h, synchronously. Cellular proteins were isolated for immunoprecipitation with anti-KLF15 antibody followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. B Total expression level of KLF15 in NRCMs were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. C H9C2 cells were transfected with Si-NC or Si-WWP1 for 48 h followed by hypoxic stimulation for 6 h. Total expression level of KLF15 were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. D Expression levels of KLF15 in cytoplasm and nucleus in NRCMs were examined by Western blots. Corresponding statistics of KLF15 in cytoplasm and nucleus were shown. n = 3. E HEK293T cells transfected with vectors for His-Ub, GFP-KLF15, and either Flag-WWP1 or HA-C8886A mutant form of WWP1 were subjected to immunoprecipitation with the antibody against KLF15, and followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. F H9C2 cells were transfected with Flag-WWP1 plasmid or HA-C886A plasmid for 36 h before synchronous administration with hypoxia and MG132 (5 μM) for 6 h. Immunoprecipitation with the antibody against KLF15, and followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. G H9C2 cells were transfected with Flag-WWP1 plasmid or HA-C886A plasmid for 36 h before administration with hypoxia for 6 h. Total expression level of KLF15 were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. The data are shown as the means ± SD. The data shown in B-D, and G were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

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

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: WWP1 promotes inflammatory activation signals of NF‑κB and MAPK in hypoxia-induced cardiomyocytes. A Interaction of p300 with p65 was determined by immunoprecipitation with anti-p300 antibody followed by immunoblot with anti-p65 antibody. IgG as a negative control. n = 3. B, C Western blot analysis and statistical results of expression of P65-AcK310 in nucleus. n = 3. D-F The levels of phosphorylated P38 and ERK1/2 were examined by Western blots, and statistical results were shown. n = 3. G, H H9C2 cells were transfected with Si-NC or Si-WWP1 for 48 h followed by hypoxic stimulation for 6 h. Western blot analysis and statistical results of expression of P65-AcK310 in nucleus. n = 3. I-K The levels of phosphorylated P38 and ERK1/2 in H9C2 cells under the condition of WWP1 knockdown were examined by Western blots, and statistical results were shown. n = 3. The data are shown as the means ± SD. The data shown in C, E, F, H, J, and K were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Expressing, Transfection

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: I3C pre-treatment suppresses WWP1-mediated excessive myocardial inflammation post-MI. Mice were administered with vehicle or I3C (20 mg/kg) three times a week for a month. When mice were pre-treated by I3C for two weeks, they were randomly assigned to rAAV9-cTnT-WWP1 or rAAV9-cTnT-GFP injection, and all of which were subjected to LAD ligation after two weeks of virus injection. All mice sacrificed 1day after induction of MI. A-E mRNA levels of IL-6, IL-1β, TNF-α, VCAM-1, and MCP-1 in infarcted or healthy hearts were tested by qRT-PCR. n = 4. F Representative Western blot and statistical result of Ly6G and KLF15 in the infarct zone. n = 3. G Western blot analysis and statistical results of expression of P65-AcK310 in nucleus. n = 3. H-J H9C2 cells were infected with Adv-WWP1 or Adv-GFP for 24 h followed by I3C (50 μM) treatment for 24 h, and then the cells were treated with hypoxia for 6 h. mRNA levels of IL-6, IL-1β, TNF-α were tested by qRT-PCR. n = 3. K Western blot analysis and statistical results of expression of P65-AcK310 in nucleus in H9C2 cells. n = 3. The data are shown as the means ± SD. The data shown in A-G were analysed by two-way ANOVA followed by Bonferroni post hoc test. H-K were analysed by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Quantitative RT-PCR, Western Blot, Expressing, Infection

Journal: Theranostics

Article Title: Targeting WWP1 ameliorates cardiac ischemic injury by suppressing KLF15-ubiquitination mediated myocardial inflammation

doi: 10.7150/thno.77694

Figure Lengend Snippet: I3C pre-treatment protects against WWP1-mediated cardiac dysfunction and remodeling after MI. A-C Mice were administered with vehicle or I3C (20 mg/kg) three times a week for a month. When mice were pre-treated by I3C for two weeks, they were randomly assigned to rAAV9-cTnT-WWP1 or rAAV9-cTnT-GFP injection, and all of which were subjected to LAD ligation after two weeks of virus injection. All mice sacrificed two weeks after induction of MI. cardiac function indices were measured by echocardiography. EF% ejection fraction, FS% fractional shortening. n = 5, 4, 8, 6, respectively. D Ratio of heart weight/ body weight (HW/BW). n = 5, 4, 8, 6, respectively. E Treatment regimen. F Masson's trichrome staining was performed to detect the fibrosis in left ventricle. n = 4. G Sirius red staining and hematoxylin (top) and eosin (H&E) staining (bottom) were performed to detect fibrosis and hypertrophy in non-infarct zone. n = 4. H, I Corresponding fibrosis area and cardiomyocyte cross-sectional area were calculated. n = 4. J-N qRT-PCR was used to test the mRNA levels of collagen I, collagen III, ANP, BNP, β-MHC. n = 3. The data are shown as the means ± SD. The data shown in B-D, H-N were analysed by two-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Mouse expression plasmids encoding His-tagged ubiquitin, GFP-tagged KLF15, Flag-tagged WWP1, and HA-tagged WWP1 (C886A), in which Cystein-886 (C886A) was replaced by alanine, were purchased from Genechem (Shanghai, China).

Techniques: Injection, Ligation, Staining, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA-129/FBW7/NF-κB, a Novel Regulatory Pathway in Inflammatory Bowel Disease

doi: 10.1016/j.omtn.2019.10.048

Figure Lengend Snippet: FBW7 Decreases IκBα Expression and Promotes NF-κB Activation (A) Caco-2 cells were transfected with FBW7 siRNA (20 nM) or negative siRNA (negative) for 48 h. FBW7 mRNA expression was determined by RT-PCR. (B) The cells were infected with adenovirus encoding FBW7-GFP (FBW7-GFP, 50 MOI) or GFP for 48 h. RT-PCR analysis of FBW7 mRNA expression. **p < 0.01 versus control, n = 6. (C and D) Western blotting analysis of FBW7, IκBα, and p65 expression and IκBα phosphorylation in cells treated with FBW7 siRNA (C) or FBW7-GFP adenovirus (D). FBW7 knockdown significantly increased IκBα expression and decreased IκBα phosphorylation and p65 expression, whereas FBW7 upregulation reduced IκBα expression and increased IκBα phosphorylation and p65 expression. Representative images from six independent repetitions were shown. (E–H) The mRNA expression of TNF-α (E), IL-1β (F), IL-6 (G), and IL-8 (H) was examined by RT-PCR. **p < 0.01 versus negative siRNA or GFP adenovirus, n = 6. (I and J) Caco-2 cells were treated with FBW7 siRNA (I) and FBW7-GFP adenovirus (J) for 48 h, and then cycloheximide (CHX) was added at 10 μg/mL for the indicated times. IκBα expression was determined by western blotting. The degradation of IκBα-induced cycloheximide was significantly inhibited by FBW7 downregulation but enhanced by FBW7 overexpression; n = 4. (K and L) Western blotting analysis of IκBα expression in Caco-2 cells pretreated with MG132 (10 μg/mL) (K) or chloroquine (10 μg/mL) (L) for 30 min, followed by FBW7 siRNA for another 48 h. MG132, but not chloroquine, significantly reversed the inhibitory effect of FBW7 knockdown on IκBα expression. n = 5. (M) Cell lysates were immunoprecipitated with IκBα antibody, and immunoprecipitated proteins were blotted with FBW7 antibody; n = 4. (N) Caco-2 cells were cotransfected with HA-IκBα plasmid and FBW7-GFP adenovirus. HA-IκBα was immunoprecipitated (IP) and immunoblotted (IB) with GFP antibody; n = 4. (O and P) Cell were cotransfected with myc-ubiquitin (myc-Ub), HA-IκBα, and FBW7 siRNA (O) or FBW7-GFP adenovirus (P). After immunoprecipitation with HA antibody, proteins were immunoblotted with myc antibody to show IκBα ubiquitination; n = 6.

Article Snippet: Mammalian expression plasmids for myc-tagged ubiquitin and HA-tagged IκBα were constructed by BioVector NTCC (Beijing, China).

Techniques: Expressing, Activation Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Western Blot, Phospho-proteomics, Knockdown, Over Expression, Immunoprecipitation, Plasmid Preparation, Ubiquitin Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA-129/FBW7/NF-κB, a Novel Regulatory Pathway in Inflammatory Bowel Disease

doi: 10.1016/j.omtn.2019.10.048

Figure Lengend Snippet: miR-129 Suppresses IκBα Ubiquitination and Increases IκBα Expression (A) Caco-2 cells were transfected with miR-129 mimics (MiR-129-m) or mimics negative control (NC-m) in the presence or absence of FBW7-GFP adenovirus treatment for 48 h. IκBα protein expression was examined by western blotting. (B) miR-129 inhibitor (MiR-129-i; 10 nM) or inhibitor negative control (NC-i) was cotransfected with or without FBW7 siRNA into cells. IκBα protein expression was determined. **p < 0.01 versus mimics negative control or inhibitor negative control; ##p < 0.01 versus miR-129 mimics or inhibitor; n = 6. (C and D) Caco-2 cells transfected with myc-ubiquitin (myc-Ub) and HA-IκBα were treated as above. HA-IκBα was immunoprecipitated (IP) and immunoblotted (IB) with myc antibody. Upregulation of FBW7 reversed the miR-129-induced decrease in ubiquitination of IκBα (C), while miR-129 inhibitor failed to induce ubiquitination of IκBα in FBW7 siRNA-treated cells (D); n = 5.

Article Snippet: Mammalian expression plasmids for myc-tagged ubiquitin and HA-tagged IκBα were constructed by BioVector NTCC (Beijing, China).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Negative Control, Western Blot, Immunoprecipitation

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA-129/FBW7/NF-κB, a Novel Regulatory Pathway in Inflammatory Bowel Disease

doi: 10.1016/j.omtn.2019.10.048

Figure Lengend Snippet: miR-129 Prevents TNBS-Induced Colitis in Mouse (A) The expression of miR-129 in non-IBD individuals (n = 189) and patients with Crohn’s disease (CD; n = 172) and patients with ulcerative colitis (UC; n = 147) was determined by RT-PCR. **p < 0.01 versus non-IBD individuals. (B) Colonic miR-129 expression in mice treated with TNBS was analyzed by RT-PCR. (C) Mice were treated with miR-129 mimics (129-m) or mimics negative control (NC-m) for 7 days prior to TNBS-induced colitis. The expression of miR-129 was determined. **p < 0.01 versus control; ##p < 0.01 versus TNBS + mimics negative control; n = 10 in each group. (D) Survival curves were recorded until day 14 after the start of TNBS treatment; n = 20 in each group. (E) The change of body weight of mice. *p < 0.05, **p < 0.01 versus TNBS + mimics negative control; n = 15 in each group. (F and G) Mice were sacrificed on day 7 (F), and the colon length was measured (G). MiR-129 overexpression inhbited TNBS-induced the shorting of colon length. **p < 0.01 versus mimics negative control; ##p < 0.01 versus TNBS + mimics negative control; n = 6 in each group. (H) Histopathological changes in colon tissue were examined by hematoxylin and eosin staining. Scale bar, 50 μm. (I) Semiquantitative scoring of histopathology was performed. **p < 0.01 versus TNBS + mimics negative control; n = 6 in each group. (J) The activity of myeloperoxidase (MPO) was assessed. (K–N) The colonic mRNA expression of TNF-α (K), IL-1β (L), IL-6 (M), and IL-8 (N) was examined by RT-PCR. (O–Q) Western blotting analysis of FBW7 (O), IκBα (P), and p65 (Q) protein expression in colon tissues. *p < 0.01, **p < 0.01 versus mimics negative control; ##p < 0.01 versus TNBS + mimics negative control; n = 6 in each group. (R) Lysates of colon tissues were immunoprecipitated with IκBα antibody, and proteins were blotted with ubiquitin antibody to detect the ubiquitination of IκBα. n = 5.

Article Snippet: Mammalian expression plasmids for myc-tagged ubiquitin and HA-tagged IκBα were constructed by BioVector NTCC (Beijing, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Control, Over Expression, Staining, Histopathology, Activity Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Virology

Article Title: HSV-1 UL56 protein recruits cellular NEDD4-family ubiquitin ligases to suppress CD1d expression and NKT cell function

doi: 10.1128/jvi.02140-24

Figure Lengend Snippet: UL56 interacts with NEDD4-family E3 ubiquitin ligases and may recruit to suppress CD1d expression. ( A ) Cellular proteins associated with HSV-1 UL56 protein were purified from cell lysates of transfected 293T.CD1d cells expressing GST-fused UL56 (1–215) by GST-pulldown. The high-molecular-weight proteins co-purified with GST-UL56 protein were subjected to mass spectrometry analysis to identify these proteins. Cells expressing GST protein were used as a control. ( B–D ) Individual members of FLAG-tagged NEDD4-family E3 ubiquitin ligases were co-transfected with pTracer plasmid in 293T.CD1d cells. Transfected cells were subjected to western blotting to verify the expression of individual E3 ubiquitin ligases ( B ) or stained with anti-CD1d antibody, CD1d.51.1 and cell surface CD1d expression was analyzed by flow cytometry ( C, D ). Transfected and untransfected cells were gated as GFP-positive and GFP-negative cells, respectively. ( D ) Quantitation of CD1d downregulation upon expression of NEDD4-family E3 ubiquitin ligases. The relative CD1d MFI was calculated as (CD1d MFI of CD1d in GFP-positive cells)/(CD1d MFI in GFP-negative cells). The average and standard deviations of relative CD1d MFI were calculated from three independently repeated experiments and plotted. Statistical analysis by one-way ANOVA with Duncan post hoc multiple comparisons. n.s., not significant. * P < 0.05, ** P < 0.01.

Article Snippet: The plasmid constructs expressing NEDD4 ubiquitin ligase family protein constructs expressed in the pCIneo vector (Promega) were generously provided by Dr. Wesley Sundquist at University of Utah and reported previously ( ).

Techniques: Ubiquitin Proteomics, Expressing, Purification, Transfection, High Molecular Weight, Mass Spectrometry, Control, Plasmid Preparation, Western Blot, Staining, Flow Cytometry, Quantitation Assay

Journal: Journal of Virology

Article Title: HSV-1 UL56 protein recruits cellular NEDD4-family ubiquitin ligases to suppress CD1d expression and NKT cell function

doi: 10.1128/jvi.02140-24

Figure Lengend Snippet: NEDD4L is the major NEDD4-family ubiquitin ligase interacting with UL56 and cooperating with UL56 to suppress CD1d expression. ( A, B ) 293T.CD1d cells were transfected with plasmids expressing UL56 alone or together with FLAG-tagged NEDD4L and subjected to co-immunoprecipitation with anti-UL56 antibodies or anti-FLAG antibodies. Immunoprecipitates were western blotted with anti-FLAG or anti-UL56 antibodies, respectively. Cellular Grp94 protein was used as a loading control. ( C–E ) 293T.CD1d were transfected with either pTracer alone or together with plasmids to express UL56 alone, NEDD4L alone, UL56 plus NEDD4L, or KSHV K5 proteins and subjected to western blotting ( C ) or cell surface staining for CD1d expression ( D ). Transfected and untransfected cells were gated as GFP-positive and GFP-negative cells, respectively. ( E ) Quantitation of CD1d downregulation. The relative CD1d MFI was calculated as (CD1d MFI of CD1d in GFP-positive cells)/(CD1d MFI in GFP-negative cells). The average and standard deviations of relative CD1d MFI were calculated from three independently repeated experiments and plotted. The experiments were repeated three times and results were summarized. Statistical analysis by one-way ANOVA with Duncan post hoc multiple comparisons. n.s., not significant. * P < 0.05, ** P < 0.01.

Article Snippet: The plasmid constructs expressing NEDD4 ubiquitin ligase family protein constructs expressed in the pCIneo vector (Promega) were generously provided by Dr. Wesley Sundquist at University of Utah and reported previously ( ).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Immunoprecipitation, Western Blot, Control, Staining, Quantitation Assay