Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Control, Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The relative expression of BCAT1 in Ishikawa and HEC-1A cells. BCAT1 expression in Ishikawa and HEC-1A cells transfected with a blank control, shCtrl and shBCAT1. The y-axis indicates the relative expression of BCAT1 in each group tested using RT-qPCR.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: A. Heat map showing the changes in the concentrations (Z-score) of 26 amino acids and their derivatives after BCAT1 knockdown in HEC-1A cells. B. Intracellular amino acids and their derivative levels in shCtrl cells and shBCAT1 cells.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: USP14 promotes pyroptosis of human annulus fibrosus cells derived from patients with intervertebral disc degeneration through deubiquitination of NLRP3

doi: 10.3724/abbs.2022171

Figure Lengend Snippet: USP14 knockdown inhibits AF cell pyroptosis AF cells collected from IVDD patients were transduced with shUSP14 or shNC, and the expression of USP14 was measured by quantitative RT-PCR (A) and western blot analysis (B,C). (D) Cell pyroptosis was assessed by flow cytometry. (E) LDH release. (F,H) The expressions of NLRP3, active Caspase-1 (p20), MMP3, ADAMTS-5 and Aggrecan were examined by western blot analysis. (G) The concentrations of IL-1β and IL-18 were determined by ELISA. *** P<0.001 compared with shNC.

Article Snippet: USP14 shRNAs (shUSP14#1 position 787–805, 5′-GGAAGCAATAGAGGATGAT-3′; shUSP14#2 position 1057–1075, 5′-GCAAAGAAATGCCTTGTAT-3′; shUSP14#3 position 1467–1485, 5′-GGAAACAAGATGAATGGAT-3′) or scramble shRNA (5′-GGAATGAATGGAAGAAGAT-3′) as a negative control (shNC) were also purchased from RiboBio (Guangzhou, China).

Techniques: Transduction, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: USP14 promotes pyroptosis of human annulus fibrosus cells derived from patients with intervertebral disc degeneration through deubiquitination of NLRP3

doi: 10.3724/abbs.2022171

Figure Lengend Snippet: USP14 interacts with NLRP3 and inhibits ubiquitination of NLRP3 (A) The interaction between USP14 and NLRP3 in AF cells was determined by coimmunoprecipitation. (B) The interaction between HA-USP14 and Flag-NLRP3 in transfected AF cells was determined by coimmunoprecipitation. (C) Effects of MG132 (10 μM) or vehicle on shUSP14-mediated NLRP3 expression. (D) AF cells were transduced with shUSP14 or shNC, and NLRP3 was immunoprecipitated and then immunoblotted with the indicated antibodies. * P<0.05, *** P<0.001 compared with shNC+vehicle.

Article Snippet: USP14 shRNAs (shUSP14#1 position 787–805, 5′-GGAAGCAATAGAGGATGAT-3′; shUSP14#2 position 1057–1075, 5′-GCAAAGAAATGCCTTGTAT-3′; shUSP14#3 position 1467–1485, 5′-GGAAACAAGATGAATGGAT-3′) or scramble shRNA (5′-GGAATGAATGGAAGAAGAT-3′) as a negative control (shNC) were also purchased from RiboBio (Guangzhou, China).

Techniques: Transfection, Expressing, Transduction, Immunoprecipitation