Journal: EBioMedicine

Article Title: TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM

doi: 10.1016/j.ebiom.2019.03.058

Figure Lengend Snippet: Phosphorylation of p65 mediated TGF-β1-induced MAT2A expression. (a) Representative Western blot result of the total p65 and phospho-p65 (p-p65) in the control, CCl 4 -treated or NPLC0393-treated mice (n = 6 per group). (b) LX-2 cells were pre-treated with 10 μM NPLC0393 or 10 μM SB431542 for 24 h and then stimulated with 5 ng/mL TGF-β1 for another 2 h. The expression levels of the total p65 and p-p65 were analyzed by Western blot. (c) LX-2 cotransfected with the 1 μg/mL NF-κB reporter gene and 1 μg/mL pRL-TK plasmid were treated with vehicle, NPLC0393 or 10 μM SB431542 for 12 h and then stimulated with 5 ng/mL TGF-β1 for another 24 h. The Luciferase activity of NF-κB was normalized against that of the cotransfected pRL-TK. The result was shown as fold changes over untreated transfectants obtained from three independent tests. (d) LX-2 cells cotransfected with the 1 μg/mL MAT2A promoter construct and 1 μg/mL pRL-TK plasmid were treated with NPLC0393 or 50 ng/mL SN50 for 12 h and then stimulated with 5 ng/mL TGF-β1 for another 24 h. Relative luciferase activities were analyzed as above. (e) LX-2 cells infected with Ad-CMV-p65 (Ad-p65) or Ad-empty vector (Ad-EV) were treated with or without NPLC0393 for 12 h and then stimulated with 5 ng/mL TGF-β1 for another 24 h and the expression levels of p-p65, MAT2A, COL1A1 and α-SMA proteins were analyzed by Western blot. (f) LX-2 cells transfected with NC siRNA or PP2Cα siRNA were treated as (e) and the expression levels of p-p65, MAT2A and α-SMA proteins were analyzed by Western blot. * P < .05, ** P < .01 and *** P < .001. Data are representative of three independent experiments.

Article Snippet: The adenovirus vector encoding RELA (Ad-p65) and the empty vector (Ad-EV) were purchased from Vigene Biosciences (Jinan, China).

Techniques: Expressing, Western Blot, Plasmid Preparation, Luciferase, Activity Assay, Construct, Infection, Transfection

Journal: EBioMedicine

Article Title: TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM

doi: 10.1016/j.ebiom.2019.03.058

Figure Lengend Snippet: Schematic representation of TGF-β1/p65/MAT2A signaling pathway and inhibitory effect of NPLC0393 on this pathway. In HSCs, TGF-β1 induces MAT2A expression via p65 phosphorylation, which results in decrease of SAM concentration and consequential increase of α-SMA and COL1A1 expressions. PP2Cα small molecular activator NPLC0393 downregulates MAT2A by inhibiting phosphorylation of p65, thereby maintaining intracellular SAM concentration.

Article Snippet: The adenovirus vector encoding RELA (Ad-p65) and the empty vector (Ad-EV) were purchased from Vigene Biosciences (Jinan, China).

Techniques: Expressing, Concentration Assay

Journal: Journal of Medical Microbiology

Article Title: LPS stimulation during HCV infection induces MMP/TIMP1 imbalance in macrophages

doi: 10.1099/jmm.0.001185

Figure Lengend Snippet: NF-κB and TGF-β signal activation during TIMP1 upregulation. Macrophages from healthy donors were isolated. Cells were preincubated with HCVcc supernatant and then stimulated with LPS. (a) MMP1, MMP8 and TIMP1 expression was analysed by RT-PCR at different time points. (b) TIMP1 and MMP1 expression was analysed by Western blot. (c) The activities of NF-κB, TGF-β and Erk signalling were tested regarding p65, Smad2 and Erk1/2 phosphorylation; additionally, the inhibitors, including A20, IRAK-M and SHIP1, were examined. *, P <0.05.

Article Snippet: A20 adenovirus overexpression vectors (ad-A20) were purchased from Genechem Co. Ltd (Shanghai, PR China).

Techniques: Activation Assay, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Phospho-proteomics

Journal: Journal of Medical Microbiology

Article Title: LPS stimulation during HCV infection induces MMP/TIMP1 imbalance in macrophages

doi: 10.1099/jmm.0.001185

Figure Lengend Snippet: TIMP1 was negatively regulated by A20 through the TGF-β/Erk pathway. Macrophages were preincubated with HCVcc supernatant and then stimulated with LPS. Each stimulated cell was co-incubated with TGF-β inhibitor, Erk inhibitor, or transfected with A20 expressing vector during LPS stimulation. (a) p-p65, p-smad2 and p-Erk were examined regarding the NF-κB, TGF-β and Erk pathways. (b) TIMP1 expression was tested in different signal inhibition groups. (c) A20 overexpression significantly suppressed TIMP1 expression during LPS stimulation. (d) Erk phosphorylation was suppressed by A20 overexpression but not NF-κB inhibition, suggesting that A20 suppression of Erk signal is independent of A20 negative regulation of NF-κB. *, P <0.05.

Article Snippet: A20 adenovirus overexpression vectors (ad-A20) were purchased from Genechem Co. Ltd (Shanghai, PR China).

Techniques: Incubation, Transfection, Expressing, Plasmid Preparation, Inhibition, Over Expression, Phospho-proteomics

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Expression of plasma exosomal PTENP1 in patients with bladder cancer. Plasma Exosomes (Exos), exos isolated from the plasma of cases and controls. a Micrographs of exos isolated from the plasma of cases (left) and controls (right, bars =100 nm). b Western blots of TSG101 and CD63 in circulating exos. c qRT-PCR detection of PTENP1 in exos from plasma. d ROC curves analysis of exosomal PTENP1 signature. e The expression of exosomal PTENP1 was detected after placing plasma samples at room temperature 0 h, 4 h, 8 h, and 24 h. f The expression of exosomal PTENP1 was detected after freezing and thawing plasma samples repeatedly 0 cycle, 2 cycles, 4 cycles and 8 cycles. Results are presented as mean ± SD. * P < 0.05

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Clinical Proteomics, Isolation, Western Blot, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Effect of PTENP1 on bladder cancer cellular phenotype. EJ and J82 cells were transfected with PTENP1 -expressing plasmid or NC vector. a qRT-PCR detection of the PTENP1 mRNA level. b A CCK8 assay detection of cell viability. c A colony-forming growth assay detection of cell colony formation ability. The colonies were counted and captured. d Representative images of invasion assays of EJ (upper) and J82 cells (lower). The number of cells were counted. e Representative images of migration assays of EJ (upper) and J82 cells (lower). The number of cells were counted. f Flow cytometry detection of the apoptosis of EJ (upper) and J82 cells (lower). g Flow cytometry detection of cell cycle of EJ (upper) and J82 cells (lower). Results are presented as mean ± SD. * P < 0.05. All of the experiments were performed in triplicate

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay, Growth Assay, Migration, Flow Cytometry

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Exosomal PTENP1 serve as a mediator in intercellular communication. Exosomes (Exos) isolated from the medium of 293A, J82 and EJ cells. a qRT-PCR detection of the normalized expression of PTENP1 in the medium of 293A, J82 and EJ cells treated with RNase (2 μg/ml) alone or combined with Triton X-100 (0.1%) for 20 min. b Micrographs of exos isolated from 293A (left), J82 (middle) and EJ cells (right, bars =100 nm). c Western blots of TSG101 and CD63 in exos of cell lines. d qRT-PCR detection of the fold change of PTENP1 between exos of 293A, J82 and EJ and their producer cells. e Exos of 293A cells were labeled with PKH67; green represents PKH67, and blue represents nuclear DNA staining by DAPI. J82 and EJ cells were incubated with exos derived from 293A cells for 3 h. Results are presented as mean ± SD. * P < 0.05. All of the experiments were performed in triplicate

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Labeling, Staining, Incubation, Derivative Assay

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Effect of exosomal PTENP1 on bladder cancer cellular phenotype. Exosomes (Exos) were isolated from 293A cells transfected with PTENP1 -expressing plasmid or NC vector, namely PTENP1 -Exos and NC-Exos, respectively. Their exosomes were extracted and added to the EJ and J82 cells for 24 h. a A CCK8 assay detection of cell viability. b qRT-PCR detection of the PTENP1 mRNA level. c A colony-forming growth assay detection of cell colony formation ability. The colonies were counted and captured. d Representative images of invasion assays of EJ (upper) and J82 cells (lower). The number of cells were counted. e Representative images of migration assays of EJ (upper) and J82 cells (lower). The number of cells were counted. f Flow cytometry detection of the apoptosis of EJ (upper) and J82 cells (lower). g Flow cytometry detection of cell cycle of EJ (upper) and J82 cells (lower). Results are presented as mean ± SD. * P < 0.05. All of the experiments were performed in triplicate

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Isolation, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Quantitative RT-PCR, Growth Assay, Migration, Flow Cytometry

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Effect of PTENP1 overexpression and exosomal PTENP1 on tumor in vivo. PTENP1 / NC lentiviral vector was transfected into EJ cells, namely PTENP1 vector and NC, respectively. Exosomes (Exos) were isolated from 293A cells transfected with PTENP1 / NC lentiviral vector, namely PTENP1 -Exos and NC-Exos, respectively. a. Burdened nude mice inoculated in NC, PTENP1 -Exos, PTENP1 vector and NC-Exos. Red arrows show position of tumor. b. The xenografts from nude mice inoculated in NC, PTENP1 -Exos, PTENP1 vector and NC-Exos. c. The tumor volumes were measured every two days after injection. d. The tumor weights in nude mice at the 15 day were determined. e. Detection of PTENP1 and PTEN expression in tumor tissues of nude mice treated with NC, PTENP1 vector, PTENP1 -Exos and NC-Exos by qRT-PCR. f. H&E stained images and immunohistochemistry analysis of ki67 and PTEN expression in tumor tissues. Results are presented as mean ± SD. * P < 0.05

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Over Expression, In Vivo, Plasmid Preparation, Transfection, Isolation, Injection, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Exosomal PTENP1 regulates PTEN expression via miR-17. a. Western blots of PTEN in EJ and J82 cells were transfected with PTENP1 -expressing plasmid or NC vector. b. Western blots of PTEN in EJ and J82 cells were treated with PTENP1 -Exos or NC-Exos. c. Putative miR-17 binding sequence in the 3′-UTR of PTEN mRNA. d. Western blots of PTEN in EJ and J82 cells with PTENP1 -Exos and/ or miR-17 mimics. Results are presented as mean ± SD. * P < 0.05. All of the experiments were performed in triplicate

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Binding Assay, Sequencing

Journal: Molecular Cancer

Article Title: Exosome–transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression

doi: 10.1186/s12943-018-0880-3

Figure Lengend Snippet: Schematic diagram of exosomal PTENP1 -mediated bladder cancer progression. Exosomal PTENP1 derived from normal cells transfected with PTENP1 vector enhance PTENP1 expression of bladder cancer cells. Exosomal PTENP1 suppresses the progression of bladder cancer by acting as a ceRNA to competitively bind to miR-17 and regulate PTEN expression

Article Snippet: The lentiviral vectors containing PTENP1 /NC were synthesized by GeneChem (Shanghai, China).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Excessive branched-chain amino acid accumulation restricts mesenchymal stem cell-based therapy efficacy in myocardial infarction

doi: 10.1038/s41392-022-00971-7

Figure Lengend Snippet: The BCAA catabolic capability of ADSCs determined their adaptation to the extracellular high BCAA milieu. a Schematic diagram of genes related to BCAA uptake, transportation, and catabolism. b mRNA levels of genes involved in BCAA metabolism measured by RT-PCR. Water was used as the negative control and Actb was used as the positive control. c ADSCs were transfected with control adenovirus (Ad-Ctrl), adenovirus overexpressing PP2Cm (Ad-PP2Cm) or adenovirus overexpressing BCKDK (Ad-BCKDK) and treated with or without BCAA (3.432 mM) in the presence of hydrogen peroxide (100 μM). Representative blots and quantification of p-mTOR (S2448), mTOR, p-S6K1 (T389), S6K1, DUX4, KDM4E, and β-tubulin as determined by western blot analysis. β-Tubulin was used as the loading control. d Representative blots and quantification of H3K9me3, histone H3, and β-tubulin. e Representative immunostaining images and quantification of H3K9me3. f ADSCs viability was determined by CCK-8 assay. g Representative blots of cleaved caspase-3, caspase-3, and β-tubulin. β-Tubulin was used as the loading control. h The mRNA levels of P16 , P21 , Il1b , and Il6 were analysed by RT-qPCR. i ADSC premature senescence was induced as methods described. Representative images and quantification of SA-β-gal positive cells. The data are shown as the means ± SD. The data were analysed by two-way ANOVA followed by Bonferroni post hoc test. ADSCs adipose tissue-derived mesenchymal stem cells, BCAA branched chain amino acids, BCKDK BCKDHA kinase, DUX4 double homeobox protein 4, H3K9me3 histone H3K9 trimethylation, KDM4E lysine-specific demethylase 4E, mTOR the mechanistic target of rapamycin, PP2Cm mitochondrial matrix-targeted protein phosphatase 2C family member, SA-β-gal senescence-associated β-galactosidase, S6K1 ribosomal protein S6 kinase polypeptide 1

Article Snippet: Adenovirus vectors carrying plasmids overexpressing PP2Cm (Ad-PP2Cm) constructed by Hanbio Co., Ltd. (Shanghai, China) or BCKDK (Ad-BCKDK) constructed by GeneChem Co., Ltd. (Shanghai, China) were used to transfect ADSCs that had reached 60% confluence.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Transfection, Control, Western Blot, Immunostaining, CCK-8 Assay, Quantitative RT-PCR, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Excessive branched-chain amino acid accumulation restricts mesenchymal stem cell-based therapy efficacy in myocardial infarction

doi: 10.1038/s41392-022-00971-7

Figure Lengend Snippet: The BCAA catabolic capability of ADSCs determined their adaptation and cardioprotective efficacy in the post-infarction heart. a Retention of ADSCs labeled by the cell tracker CM-DiI (red) was visualized by immunostaining of heart tissues 1, 3, and 7 d post-MI. Troponin T staining (green) indicates cardiomyocytes. Left panel, representative images; right panel, ratios of the number of engrafted ADSCs to the number of cardiomyocytes. ADSC-Ctrl ADSCs transfected with adenovirus carrying empty plasmids, ADSC-PP2Cm ADSCs transfected with adenovirus overexpressing PP2Cm, ADSC-BCKDK ADSCs transfected with adenovirus overexpressing BCKDK. b Top panel, representative echocardiographic images 28 d post-MI; bottom panel, quantification of LVEF at baseline and 7, 14, and 28 d after MI. c Representative images of TUNEL staining (top) and CD31 immunostaining in the infarct border zone 3-d post-MI (middle). Bottom panel, quantification of TUNEL and CD31 staining intensity. d Left panel, representative images of Masson’s trichrome stained tissue from the bottom to the apex of hearts 28 d post-MI. right panel, quantification values of infarcted area as determined with ImageJ software. e HW/BW ratios 28 d post-MI; f LW/BW ratios 28 d post-MI. The data are shown as the means ± SD. Data were analysed by one-way ANOVA followed by a Bonferroni post hoc test. ADSCs adipose tissue-derived mesenchymal stem cells, AutoFluo autofluorescence, BCAA branched-chain amino acids, BCKDK BCKDHA kinase, HW/BW heart weight/body weight ratio, LW/BW lung weight/body weight ratio, MI myocardial infarction, PP2Cm mitochondrial matrix-targeted protein phosphatase 2C family member, TUNEL transferase-mediated dUTP nick-end labeling

Article Snippet: Adenovirus vectors carrying plasmids overexpressing PP2Cm (Ad-PP2Cm) constructed by Hanbio Co., Ltd. (Shanghai, China) or BCKDK (Ad-BCKDK) constructed by GeneChem Co., Ltd. (Shanghai, China) were used to transfect ADSCs that had reached 60% confluence.

Techniques: Labeling, Immunostaining, Staining, Transfection, TUNEL Assay, Software, Derivative Assay, End Labeling