Journal: Oncogene

Article Title: Inhibition of autophagy and tumor growth in colon cancer by miR-502

doi: 10.1038/onc.2012.167

Figure Lengend Snippet: (A) A putative miR-502 binding site exists in the 3’-UTR of Rab1B mRNA and two point mutations were generated in the binding site. Ectopic expression of miR-502 or siRNAs against Rab1B (siRab1B) in HCT116 and SW480 cells decreased Rab1B protein levels by (B) Western blot analysis and (C) mRNA level by real-time qRT-PCR. (D) Transfection of miR-502 inhibited firefly luciferase activity of pMIR-REPORT-3UTRRab1B (wt) and such inhibition was absent with mutations in the miR-502 binding site (mut). The negative miRNA was used as the negative control in all experiments. The impact of miR-502 and siRab1B on Rab1B expression was normalized and compared to those of negative miRNA (n=3, p < 0.001).

Article Snippet: 10 pmole of miR-502 or negative miRNA was transfected into cells together with 100ng of pMIR-REPORT-3UTRRab1B and 1ng of Renilla luciferase plasmid pRL-SV40 (Promega) by DharmaFect Duo (Dharmacon).

Techniques: Binding Assay, Generated, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Inhibition, Negative Control

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 downregulates HCV Core levels by upregulating E6AP levels in a p53-dependent manner. ( a – c ) Cells were transfected with the specified plasmids for 24 h and treated with H 2 O 2 for an extra 24 h, followed by Western blotting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Transfection, Western Blot

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 stimulates E6AP expression through promoter hypomethylation in the presence of p53 and HCV Core. Cells were transfected with the specified plasmids for 24 h and subsequently treated with H 2 O 2 for an extra 24 h. ( a , b ) DNMT activity from cells was determined ( n = 3). Levels of the indicated proteins were measured by Western blotting. ( c , d ) Methylation-specific PCR (MSP) was performed to determine whether the CpG sites in the E6AP promoter are unmethylated (U) or methylated (M). ( e ) Cells were treated with the specified concentration of 5-Aza-2′dC for 24 h before harvesting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Expressing, Transfection, Activity Assay, Western Blot, Methylation, Concentration Assay

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 triggers the E6AP-mediated ubiquitination and proteasomal degradation of HCV Core in a p53-dependent manner. ( a ) Cells prepared as in a,b were subjected to treatment with 50 μM cycloheximide (CHX) for the specified duration before harvesting. The quantification of each band was performed using Image J image-analysis software (NIH, USA) to determine the half-life (t 1/2 ) of HCV Core. The presented values represent the levels of HCV Core relative to the loading control (γ-tubulin). ( b ) Cells were transfected with the specified plasmids for 24 h and treated with H 2 O 2 for an extra 24 h. The transfection mixtures included the HA-Ub expression plasmid. Total HCV Core proteins in cell lysates were immunoprecipitated using an anti-HCV Core antibody and subsequently analyzed by Western blotting. The membranes were probed with antibodies against p53, HCV Core, E6AP, and HA to detect p53, HCV Core, E6AP, and HA-Ub-complexed HCV Core, respectively. Additionally, the input shows the levels of the specified proteins in the cell lysates. ( c ) Cells were either mock-treated or treated with MG132 for 4 h before harvesting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Software, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 inhibits HCV replication in vitro through the E6AP-mediated downregulation of HCV Core levels. ( a ) Cells were transfected with the designated plasmids for 24 h and infected with HCV for an extra 24 h in the absence or presence of H 2 O 2 , followed by Western blot analysis. ( b ) The levels of HCV particles in the supernatants, as prepared in ( a ), were determined using both q-RT-PCR ( n = 4) and conventional RT-PCR. ( c ) Cells cultured in a differentiation medium (Biopredic International) for 2 weeks were infected with HCV for 24 h and treated with H 2 O 2 for an extra 24 h, followed by Western blotting. ( d ) The levels of HCV particles in the supernatants prepared in ( c ) were determined using q-RT-PCR ( n = 3) and conventional RT-PCR. ( e ) Cells were transfected with the specified plasmids for 24 h and subsequently infected with HCV for an extra 24 h, either in the absence or presence of H 2 O 2 , followed by co-IP, as described in b.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: In Vitro, Transfection, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Co-Immunoprecipitation Assay

Journal: Molecular Medicine Reports

Article Title: TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

doi: 10.3892/mmr.2020.11152

Figure Lengend Snippet: DKK1 mRNA levels in MC3T3-E1 cells and primary calvarial osteoblasts treated with TNF-α. (A) DKK1 mRNA levels in MC3T3-E1 cells and primary calvarial osteoblasts treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h. *P<0.05 vs. the 0-h group, n=3. (B) DKK1 mRNA levels in MC3T3-E1 cells and primary calvarial osteoblasts treated with 0, 15 and 50 ng/ml TNF-α for 48 h. *P<0.05 vs. the 0-ng/ml group, n=3. DKK1, Dickkopf WNT signaling pathway inhibitor 1; TNF-α, tumor necrosis factor-α.

Article Snippet: For transient transfection, MC3T3-E1 cells were cultured in 12-well plates at a density of 5×10 4 cells per ml overnight, and co-transfected with 900 ng pMIR-REPORT-DKK1 UTR and 100 ng pRL-TK Vector (Promega Corporation) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques:

Journal: Molecular Medicine Reports

Article Title: TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

doi: 10.3892/mmr.2020.11152

Figure Lengend Snippet: DKK1 protein levels in MC3T3-E1 cells and primary calvarial osteoblasts treated with TNF-α. (A) Protein levels of DKK1 in MC3T3-E1 cells and primary calvarial osteoblasts treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h. *P<0.05 vs. the 0-h group, n=3. (B) Protein levels of DKK1 in MC3T3-E1 cells and primary calvarial osteoblasts treated with 0, 15 and 50 ng/ml TNF-α for 48 h. *P<0.05 vs. the 0-ng/ml group, n=3. DKK1, Dickkopf WNT signaling pathway inhibitor 1; TNF-α, tumor necrosis factor-α.

Article Snippet: For transient transfection, MC3T3-E1 cells were cultured in 12-well plates at a density of 5×10 4 cells per ml overnight, and co-transfected with 900 ng pMIR-REPORT-DKK1 UTR and 100 ng pRL-TK Vector (Promega Corporation) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques:

Journal: Molecular Medicine Reports

Article Title: TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

doi: 10.3892/mmr.2020.11152

Figure Lengend Snippet: Role of the NF-κB signaling pathway in mediating TNF-α-regulated expression of DKK1 in MC3T3-E1 cells and primary calvarial osteoblasts. MC3T3-E1 cells and primary calvarial osteoblasts were treated with 15 ng/ml TNF-α and/or 1 µM BAY 11-7082 for 48 h, and the untreated cells served as controls. The DKK1 (A) mRNA levels and (B) protein levels in these cells were determined. *P<0.05 vs. the Control group; # P<0.05 vs. the TNF-α group; & P<0.05 vs. the TNF-α + BAY 11-7082 group, n=3. TNF-α, tumor necrosis factor-α; DKK1, Dickkopf WNT signaling pathway inhibitor 1.

Article Snippet: For transient transfection, MC3T3-E1 cells were cultured in 12-well plates at a density of 5×10 4 cells per ml overnight, and co-transfected with 900 ng pMIR-REPORT-DKK1 UTR and 100 ng pRL-TK Vector (Promega Corporation) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

doi: 10.3892/mmr.2020.11152

Figure Lengend Snippet: TNF-α treatment exhibits no effects on the inhibitory action of miR-335-5p via targeting of DKK1 3′UTR. MC3T3-E1 cells were co-transfected with pMIR-REPORT-DKK1 UTR and pRL-TK Vector, and cells co-transfected with pMIR-REPORT and pRL-TK served as controls. (A) Transfected MC3T3-E1 cells were treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h, and the luciferase levels were determined. *P<0.05 vs. the pMIR-REPORT group, n=3. (B) Transfected MC3T3-E1 cells were treated with 0, 15 and 50 ng/ml TNF-α for 48 h, and the luciferase levels were determined. *P<0.05, vs. the pMIR-REPORT group, n=3. (C) mmu-miR-335-5p mimic or mirVana™ miRNA Mimic Negative Control #1 were transfected into the MC3T3-E1 cells concurrently, and the luciferase levels were determined. The transfected cells were treated with or without 15 ng/ml TNF-α for 48 h. The luciferase levels were determined. *P<0.05, vs. the pMIR-REPORT group, n=3. mimic NC= miRNA Mimic negative Control. (D) Transfected MC3T3-E1 cells were treated with or without 15 ng/ml TNF-α and/or 1 µM BAY 11-7082 for 48 h. *P<0.05 vs. the pMIR-REPORT group, n=3. TNF-α, tumor necrosis factor-α; miR, microRNA; DKK1, Dickkopf WNT signaling pathway inhibitor 1.

Article Snippet: For transient transfection, MC3T3-E1 cells were cultured in 12-well plates at a density of 5×10 4 cells per ml overnight, and co-transfected with 900 ng pMIR-REPORT-DKK1 UTR and 100 ng pRL-TK Vector (Promega Corporation) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Plasmid Preparation, Luciferase, Negative Control

Journal: eLife

Article Title: Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells

doi: 10.7554/eLife.61630

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , RPE-1 tet on Cas9 TP53 KO , , PMID: 32084359 , Originally derived from hTERT RPE-1 (ATCC Cat# CRL-4000, RRID: CVCL_4388 ).

Techniques: Derivative Assay, Knock-Out, CRISPR, Mutagenesis, Knockdown, Control, Transfection, Construct, Over Expression, Plasmid Preparation, Sequencing, Modification, Mass Spectrometry, Immunofluorescence, Expressing, Isolation, cDNA Synthesis, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Software, Microscopy

Journal: Immunity

Article Title: Programmed death-1 receptor signaling downregulates asparaginyl endopeptidase to maintain Foxp3 stability in induced Regulatory T cells

doi: 10.1016/j.immuni.2018.05.006

Figure Lengend Snippet: Murine CD4+CD25− T cells were transduced with WT human Foxp3, Mutant (N154) human Foxp3, WT murine Foxp3 or mutant murine Foxp3 (N153). Transduction efficiency at day 4 was measured by flow cytometry (A). Host BALB/c mice were subjected to lethal total body irradiation (TBI; 950cGy) and then reconstituted with B6 T depleted bone marrow (BM, 5×106 cells) alone, or with CD4+CD25− T cells (CD45.1 marked, 0.1×106). Certain cohorts were treated with BM plus CD4+CD25− T plus non-transduced T cells (NT, 0.1×106) or T cells transduced with WT human Foxp3 (WT hFoxp3, 0.1×106), or human mutant Foxp3 (Mu hFoxp3, N154; 0.1×106) or murine WT Foxp3 (WT mFoxp3, 0.1×106) or murine mutant Foxp3 (N153; Mu mFoxp3, 0.1×106). GvHD lethality was monitored (n=6 per cohort) (B). Host BALB/c mice were subjected to TBI and then reconstituted with bone marrow (BM, 107 cells), CD4+CD25− T effector cells (CD45.1 marked; 1×106 cells) and either Tbet+iTreg cells (1×106 cells) that were expanded with AEP inhibitor or control Tbet+iTreg cells (1×106 cells). At day 14 post-transplant, splenocytes were harvested and the Foxp3 was measured in the Tbet+iTreg cell populations (marked with CD45.2). Representative flow plots showing Foxp3 expression in the different cell populations (C); frequency of Foxp3 expression (D). A survival curve experiment was set up to test the efficacy of blocking AEP in preventing GvHD. Animals were conditioned with TBI and then reconstituted with BM alone or plus CD4+CD25− T cells. Cohorts were then treated with Tbet+iTreg cells, Tbet+iTreg cells expanded with AEP inhibitor (AEPi), Tbet+iTregPDL1 cells or Tbet+iTregPDL1 cells overexpressing AEP (E). Experiments were repeated with CD45.2+ Tbet+iTreg cells that were expanded with AEP shRNA or scramble shRNA and then tested in GvHD. Frequency and absolute numbers of CD45.2+Foxp3+ cells in the different cohorts (F–H) and absolute numbers of Tbet+ cells at day 14 post-transplant in Tbet+ iTreg cells (I). Experiments were repeated twice with n=4-6 mice for immunological studies and n=6-10 mice for survival curve. Representative data from one experiment is shown as Mean±SEM. Please also refer to Figure S5.

Article Snippet: For NLS experiments AEP NLS site was mutated whereby KRK was replaced to AAA at site 318-320. pMIGR-mFoxp3 was a gift from Prof. Dan Littman (Addgene plasmid # 24067, unpublished).

Techniques: Transduction, Mutagenesis, Flow Cytometry, Irradiation, Expressing, Blocking Assay, shRNA