Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: Primers used for real-time PCR.

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Sequencing

Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: ISG15 expression and ISGylation were increased by transfection . HepG2.2.15 cells were transfected with the ISG15 expression plasmid or GFP-expressing plasmid. (a) Levels of ISG15 mRNA were determined by real-time PCR (normalized by GAPDH) 24 hours after transfection. PEI, transfection regent polyethyleneimine (PEI) treatment only; GFP, transfected with 4 μ g GFP-expressing plasmid; ISG15, transfected with 4 μ g ISG15 plasmid. The results are presented as the means ± SD, n ≥ 3; error bars indicate SD. ∗∗∗ P < 0.001. (b) Protein ISGylation was further assessed by western blot with or without ISG15 overexpression. Molecular mass markers are shown on the left (kDa).

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: ISG15 overexpression promotes HBV production in vitro . HepG2.2.15 cells were transfected with the indicated amount of ISG15 plasmid or GFP-expressing plasmid. (a) Real-time PCR showing ISG15 mRNA expression 24 h after transfection of ISG15 plasmid. GFP, transfected with 4 μ g GFP-expressing plasmid; 2 μ g, 4 μ g, 8 μ g, or 16 μ g transfected with 2 μ g, 4 μ g, 8 μ g, or 16 μ g ISG15 plasmid. (b) Supernatant HBV DNA, intracellular, (c) cccDNA, (d) pgRNA, and (e) total HBV DNA were determined by real-time PCR 48 h after transfection, respectively. PEI, transfection regent polyethyleneimine (PEI) treatment only; GFP, transfected with 4 μ g GFP-expressing plasmid; ISG15, transfected with 4 μ g ISG15 plasmid. The results are presented as the means ± SD, n ≥ 3; error bars indicate SD. ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Over Expression, In Vitro, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction

Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: HBV protein expression after ISG15 overexpression . HepG2.2.15 cells were transfected with the ISG15 plasmid or GFP-expressing plasmid. (a) HBsAg and (b) HBeAg in the culture medium were detected by ELISA, respectively. (c) Intracellular HBcAg was assessed by western blot. Relative integrated density was calculated by ImageJ software and normalized by GAPDH expression. PEI, transfection regent polyethyleneimine (PEI) treatment only; GFP, transfected with 4 μ g GFP-expressing plasmid; ISG15, transfected with 4 μ g ISG15 plasmid. The results are presented as the means ± SD, n ≥ 3; error bars indicate SD.

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Software

Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: ISGylation is important in ISG15-promoted HBV production . UBE1L knockdown was performed by RNAi to abrogate ISGylation in HepG2.2.15 cells. (a) Knockdown efficiency was determined by real-time PCR showing UBE1L mRNA expression 24 h after 50 nM negative siRNA, 50 nM UBE1L siRNA, or 100 nM UBE1L siRNA treatment. (b) Comparison of protein ISGylation (western blot for ISG15) after UBE1L knockdown and ISG15 overexpression. Molecular mass markers are shown on the left (kDa). (c) Real-time PCR was used to assess the supernatant HBV DNA after UBE1L knockdown and ISG15 overexpression. Control, untreated control; PEI, ISG15 transfection regent polyethyleneimine (PEI) treatment only; ISG15, ISG15 overexpression by transfection with 1.4 μ g ISG15 plasmid; UBE1L siRNA+ISG15, 50 nM UBE1L siRNA treatment followed by ISG15 overexpression; Mock+ISG15, siRNA transfection regent treatment followed by ISG15 overexpression; NC+ISG15, 50 nM negative siRNA treatment followed by ISG15 overexpression; IFN, 100 IU/mL IFN α 2b treatment. The results are presented as the means ± SD, n ≥ 3; error bars indicate SD. ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Transfection, Plasmid Preparation

Journal: Mediators of Inflammation

Article Title: Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

doi: 10.1155/2016/7417648

Figure Lengend Snippet: Effects of ISG15 overexpression on ISGs and endogenous type I IFNs . HepG2.2.15 cells were transfected with ISG15 plasmid. Real-time PCR was performed to quantify MxA, OAS3, USP18, IFN α , and IFN β 48 h after transfection. PEI, transfection regent polyethyleneimine (PEI) treatment only; ISG15, transfected with 4 μ g ISG15. ∗∗ P < 0.01.

Article Snippet: As previously described [ ], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis

doi: 10.1038/s41388-024-03021-4

Figure Lengend Snippet: A Normalized interaction intensities of JUNI and potential protein interactors, averaged from two incPRINT biological replicates, sorted in increasing order. The horizontal dotted line represents the interaction intensity cutoff used for the classification of JUNI interactors. Red dots are JUNI -interacting proteins; gray dots are proteins that do not bind to JUNI . See the ‘Methods’ section for data normalization. RLU are relative light units. B Intensities of JUNI interactions with DUSP14 and GFP were obtained from two biological replicates of the incPRINT screen. On the left, interaction intensities detected between JUNI and the indicated proteins. On the right, the expression level of each protein was measured using ELISA. C HeLa cells were transfected with DUSP14 or GFP and UV crosslinking immunoprecipitation was performed. The ratio of RNAs coprecipitated with DUSP14 relative to RNAs in the whole cell extract was further normalized to the ratio obtained after GFP precipitation. Enrichment of the indicated lncRNAs is depicted. D HMCB cells were transfected with the indicated siRNAs, irradiated with 30 J/m 2 48 h later and harvested 5 h post irradiation. Levels of the indicated proteins were measured by immunoblotting using specific antibodies as described before. E HMCB cells were transfected with the indicated plasmids and 24 h later irradiated or not with 25 J/m 2 UV. Cells were harvested 4 h later and the levels of the indicated proteins were measured using specific antibodies. DUSP14 was measured using HA antibody. F HMCB and HeLa cells were transfected with the below indicated siRNAs and UV irradiated 40 h later or not. Irradiated cells were harvested 12 h after irradiation and viability was determined using XTT.

Article Snippet: DUSP14-HIS was a kind gift from M. Saleh [ ] pRK5 HA-DUSP14 plasmid (HA tag N-terminal) was obtained by PCR amplification from pOTB7 DUSP14 plasmid (HGMP MRC geneservice, IMAGE cDNA clone 2819474) and subcloning into pRK5 plasmid containing HA sequence. pRK5 HA-DUSP14 C111S was obtained by PCR oligonucleotide site-directed mutagenesis.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Cross-linking Immunoprecipitation, Irradiation, Western Blot

Journal: Oncogene

Article Title: The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis

doi: 10.1038/s41388-024-03021-4

Figure Lengend Snippet:

Article Snippet: DUSP14-HIS was a kind gift from M. Saleh [ ] pRK5 HA-DUSP14 plasmid (HA tag N-terminal) was obtained by PCR amplification from pOTB7 DUSP14 plasmid (HGMP MRC geneservice, IMAGE cDNA clone 2819474) and subcloning into pRK5 plasmid containing HA sequence. pRK5 HA-DUSP14 C111S was obtained by PCR oligonucleotide site-directed mutagenesis.

Techniques:

Journal: Oncogene

Article Title: The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis

doi: 10.1038/s41388-024-03021-4

Figure Lengend Snippet:

Article Snippet: DUSP14-HIS was a kind gift from M. Saleh [ ] pRK5 HA-DUSP14 plasmid (HA tag N-terminal) was obtained by PCR amplification from pOTB7 DUSP14 plasmid (HGMP MRC geneservice, IMAGE cDNA clone 2819474) and subcloning into pRK5 plasmid containing HA sequence. pRK5 HA-DUSP14 C111S was obtained by PCR oligonucleotide site-directed mutagenesis.

Techniques:

Journal: Journal of Molecular Signaling

Article Title: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

doi: 10.1186/1750-2187-2-5

Figure Lengend Snippet: PNRC interacts with a subunit of RNA pol III . A , AD Gal4 -RPC39 212–316 expression plasmid was isolated from a human mammary gland expression library screening using DBD Gal4 -PNRC 270–327 as bait. To confirm the interaction and the specificity of the interaction between PNRC and RPC39, yeast strain Y187 was cotransformed with the expression plasmids for the expression of the fusion proteins as indicated, and transformants containing these plasmids were selected by growth on SD/-Leu/-Trp agar plates. The expression of interacting hybrid proteins in Y187 transformants was analyzed for Lac Z expression. DBD Gal4 (DBD) and DBD Gal4 -human lamin C (hLC) expression plasmids were included as background and negative control, respectively. β-Galactosidase activities in liquid cultures are expressed in Miller units as mean ± s.d. of three independent assays. B , PNRC interacts with the C-terminus, amino acids 212–316, of the human Pol III subunit RPC39. The yeast expression plasmids, pACT2-RPC39 and pACT2-RPC39 1–212 , for the expression of AD Gal4 -RPC39 wild type (WT) or AD Gal4 -RPC 1–212 (1–212) fusion proteins were constructed as described in 'Methods'. The expression plasmids for AD Gal4 -RPC39 212–316 (212–316) and AD Gal4 -RPC39 241–316 (241–316) were isolated from library screening through their interaction with PNRC 270–327 . Y187 cells were cotransformed with the expression plasmids coding for the fusion proteins as indicated. The selection of Y187 transformants and the β-Gal assays on transformants were performed as described in A .

Article Snippet: The coding regions of RPC39 and RPC39 1–211 were amplified by PCR using pOTB7/RPC39 plasmid (Invitrogen) as template.

Techniques: Expressing, Plasmid Preparation, Isolation, Library Screening, Negative Control, Construct, Selection

Journal: Journal of Molecular Signaling

Article Title: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

doi: 10.1186/1750-2187-2-5

Figure Lengend Snippet: PNRC associates with RPC39 in mammalian cells . MCF-7/EGFP or MCF7/EGFP-PNRC stable expression cells were transiently transfected with the RPC39 expression plasmid, pSG5-RPC39, 24 h post-transfection, cells were harvasted and lysed, and 15 mg of total proteins were immunoprecipitated with antibodies against GFP (Clontech) or RPC39 (Santa Cruz Biotechnology). Specifically bound proteins to Protein A agarose beads were separated on a 10% SDS-PAGE and analysed by immunoblotting with either anti-RPC39 ( A , blot: anti-RPC39) or anti-GFP ( B , blot: anti-GFP) antibody, as previously described [2]. An aliquot of cell lysate equal to 100 μg of protein was included in each SDS-PAGE gel for Western blot to examine whether the crude protein extracts used for co-immunoprecipitation contains EGFP-PNRC and RPC39 proteins. The protein bands with molecular weights corresponding to those of RPC39 or EGFP-PNRC were indicated by arrows.

Article Snippet: The coding regions of RPC39 and RPC39 1–211 were amplified by PCR using pOTB7/RPC39 plasmid (Invitrogen) as template.

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Molecular Signaling

Article Title: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

doi: 10.1186/1750-2187-2-5

Figure Lengend Snippet: Co-recruitment of PNRC and RPC39 onto RNA polymerase III-dependent genes . MCF-7/EGFP-PNRC cells were transfected either with pArg maxi reporter plasmid, which contains tRNA arg gene promoter and coding sequence, or with pTZU6 plasmid that carries the U6 RNA gene promoter. Twenty-four hours after transfection, cells were treated with formaldehyde to crosslink endogenous proteins and DNA. Samples of sonicated and purified chromatin were immunoprecipitated with no antibody (No ab), preimmuno IgG (IgG), GFP antibody (anti-GFP), or RPC39 antibody (anti-RPC) as indicated. DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify a 220 bp fragment of the Drosophila tRNA arg gene ( A ) or a 160 bp fragment of the U6 RNA gene ( B ). The amplified PCR products were analyzed on 1.8% agarose gel. The PCR products from the reactions using input DNA, pArg or pTZU6 plasmids as template were included on the gels as positive and size controls.

Article Snippet: The coding regions of RPC39 and RPC39 1–211 were amplified by PCR using pOTB7/RPC39 plasmid (Invitrogen) as template.

Techniques: Transfection, Plasmid Preparation, Sequencing, Sonication, Purification, Immunoprecipitation, Isolation, Amplification, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: (A) RT-PCR results for the ATP5J and uroguanylin genes. (B) Immunohistochemical staining results for adjacent tissue and cancer tissue. (C) Immunohistochemical staining results for adjacent tissue, cancer tissue, and metastatic lymph node tissue.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: Condition of ATP5J expression in colorectal tumor and adjacent tissue.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: Condition of ATP5J expression in colorectal tumor and metastatic lymph nodes.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: Correlation between ATP5J expression and patients’clinical features.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: (A) RT-PCR results for ATP5J mRNA. (B) Western blotting results for ATP5J protein.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: (A) Western blot result for ATP5J protein in different clones of DLD1 cell after stable transfection with the same ATP5J shRNA plasmid. (B) Wound-healing assay for WT, Vector, and ATP5J shRNA/4 DLD1 cells. Data represent one of three similar experiments (original magnification: x100). (C) Migration of WT, Vector, and ATP5J shRNA/4 DLD1 cells was assayed using a 24-Transwell system. The pictures of migrated cells were taken 24 hours after seeding (original magnification: x100). Data represent one of three similar experiments. (D) Quantitative analyses of three cell migration assays. Values represent means ± SD. * P <0.05; (E) Apoptotic ratios of WT, Vector, and ATP5J shRNA/4 DLD1 cells after treatment with 50 µmol/L 5-Fu for 3 days. Data represent one of two similar experiments. Values represent means ± SD. * P <0.05.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Western Blot, Clone Assay, Stable Transfection, shRNA, Plasmid Preparation, Wound Healing Assay, Migration

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: (A) Western blot result for ATP5J protein in different clones of SW620 cell after stable transfection with the same ATP5J shRNA plasmid. (B) Apoptotic ratio of WT, Vector, and ATP5J shRNA/3 SW620 cells after treatment with 50 µmol/L 5-Fu for 3 days. (C) Cell viability assays of WT, Vector, and ATP5J shRNA/3 SW620 cells after treatment with 50 µmol/L 5-Fu for 3 days. Data represent one of two similar experiments. Values represent means ± SD. * P <0.05.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Western Blot, Clone Assay, Stable Transfection, shRNA, Plasmid Preparation

Journal: PLoS ONE

Article Title: Over-Expression of the ATP5J Gene Correlates with Cell Migration and 5-Fluorouracil Sensitivity in Colorectal Cancer

doi: 10.1371/journal.pone.0076846

Figure Lengend Snippet: (A) Western blot result for ATP5J protein in DLD1 cells after stable transfection with pcDNA3.1(+)/ATP5J plasmid. (B) Wound-healing assay for WT, Vector, ATP5J/A2, and ATP5J/A4 cells. Data represent one of three similar experiments (original magnification: x100). (C) Migrations of WT, Vector, ATP5J/A2, and ATP5J/A4 cells were assayed using a 24-Transwell system. The pictures of migrated cells were taken 24 hours after seeding (original magnification: x100). Data represent one of three similar experiments. (D) Quantitative analysis of three cell-migration assays. Values represent means ± SD. * P <0.05. (E) Apoptotic ratios of WT, Vector, ATP5J/A2, and ATP5J/A4 cells after treatment with 50 µmol/L 5-Fu for 3 days. Data represent one of two similar experiments. Values represent means ± SD and * P <0.05.

Article Snippet: A pOTB7 plasmid containing the ATP5J sequence was purchased from Invitrogen (Clone ID 3357779).

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Wound Healing Assay, Migration