Journal: PLoS ONE

Article Title: High-mobility group box 1 fragment suppresses adverse post-infarction remodeling by recruiting PDGFRα-positive bone marrow cells

doi: 10.1371/journal.pone.0230392

Figure Lengend Snippet: The second examination was performed using the same MI rats prior to HMGB1 treatment to assess the expression of SDF1, a representative homing factor of MSCs. A: Study protocol of second examination. B: Histological analysis revealed SDF1 expression along the peri-infarction area in MI model and normal rats (40×, scale bar = 200 μm). C: RT-PCR analysis indicated that SDF1 expression increased significantly in MI rats (n = 6) compared with normal rats (n = 10). P -values were calculated using the Welch’s t-test. P < 0.05*, P < 0.01**.

Article Snippet: In the second examination, the frozen sections in MI and normal rats were stained with rabbit anti-SDF1 polyclonal antibody (1:50; Abcam, Cambridge, UK), and were evaluated using the confocal laser microscopy.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: High-mobility group box 1 fragment suppresses adverse post-infarction remodeling by recruiting PDGFRα-positive bone marrow cells

doi: 10.1371/journal.pone.0230392

Figure Lengend Snippet: Intravital imaging analysis was performed using the GFP-BMT rat MI model to visualize HMGB1-induced GFP + -cells mobilization to the damaged heart tissue in real time. A: Details of the study protocol using intravital imaging. B: Histological findings in GFP-BMT rat MI model 12 h after HMGB1 treatment. 1, 2) More GFP + /PDGFRα + cells were visualized in the HMGB1 group compared with the control (100×, 600×; scale bar = 100, 200 μm, respectively). C: In the HMGB1 group, GFP + -cells were recruited along the peri-infarction area with SDF1 over-expression (100×, scale bar = 200 μm). Conversely, the recruitment of GFP + -cells was not enhanced at the remote area, where significant expression of SDF1 was not observed. P -values were calculated using the Welch’s t-test. P < 0.01**.

Article Snippet: In the second examination, the frozen sections in MI and normal rats were stained with rabbit anti-SDF1 polyclonal antibody (1:50; Abcam, Cambridge, UK), and were evaluated using the confocal laser microscopy.

Techniques: Imaging, Over Expression, Expressing

Journal: PLoS ONE

Article Title: High-mobility group box 1 fragment suppresses adverse post-infarction remodeling by recruiting PDGFRα-positive bone marrow cells

doi: 10.1371/journal.pone.0230392

Figure Lengend Snippet: Systemic administration of HMGB1 fragment can mobilize BM mesenchymal cells, including BM-MSCs, to blood circulation. Consequently, these BM-derived mesenchymal cells accumulate in the damaged myocardium through the SDF1/CXCR4 signaling complex, leading to functional recovery by paracrine activity of the BM-MSCs or to differentiation of some vascular constituent cells.

Article Snippet: In the second examination, the frozen sections in MI and normal rats were stained with rabbit anti-SDF1 polyclonal antibody (1:50; Abcam, Cambridge, UK), and were evaluated using the confocal laser microscopy.

Techniques: Derivative Assay, Functional Assay, Activity Assay

Journal: Antiviral Research

Article Title: Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells

doi: 10.1016/j.antiviral.2016.04.008

Figure Lengend Snippet: VEEV-nsP3 interacts with components of the host translational machinery . A) Working model for VEEV-nsP3 interaction with eukaryotic initiation factors. During shut off of cellular transcription and translation pre-existing DDX3, eIF4A, eIF4G and PABP complexes are stalled in cytoplasmic SGs. During VEEV infection, VEEV-nsP3 interacts with these pre-existing complexes to initiate translation of viral mRNA. eIF4E initially recognizes 5′ methylguanosine caps on VEEV RNA and then complexes with eIF4A (helicase) and eIF4G (scaffold protein) to form the eIF4F complex, which then recruits the 40S ribosome. PABP binds to the poly(A) tail present at the 3′-end of VEEV RNA circularizing VEEV RNA. B) U87MG cells were infected with rTC-83-nsP3-HA (MOI:20) for 1 h. At 6 hpi cell lysates were collected, quantified and 2 mg of total protein was immunoprecipitated with an isotype IgG control or an HA antibody. The immunoprecipitates were resolved by SDS-PAGE and the subsequent immunoblot probed with antibodies to eIF4A, eIF4G, PABP, DDX3 or HA. The western blot is a representative image from 2 independent experiments.

Article Snippet: Primary antibodies to HA, DDX1 (Abcam, ab31963), DDX3 (Cell Signaling, 2635), VEEV-capsid (BEI Resources, NR-9403), HRP conjugated actin, PABP (Santa Cruz Biotechnology, sc-28834), TIA-1 (Santa Cruz Biotechnology, sc-1751), eIF4G (Cell Signaling, C45A4) and eIF4A (Cell Signaling, C32B4) were used according to the manufacturer's instructions.

Techniques: Infection, Immunoprecipitation, Control, SDS Page, Western Blot

Journal: Antiviral Research

Article Title: Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells

doi: 10.1016/j.antiviral.2016.04.008

Figure Lengend Snippet: RK-33 disrupts VEEV-nsP3 interactions with host translational machinery . U87MG cells were seeded at 10,000 cells per well in a 96-well plate and treated with varying concentrations of RK-33. At 24 h post treatment cell viability was measured using the Cell Titer Glo Luminescent Cell Viability Assay according to the manufacturer's instructions (A). The graph is representative of 2 independent experiments performed in triplicate and is depicted as a percentage of cell viability. B) U87MG cells seeded in a 96-well plate at a density of 10,000 cells per well were pre-treated with RK-33 for 2 h and infected with rTC-83 (MOI.0.1) for 1 h. Supernatants were collected at 4, 8, 16 and 24 hpi and analyzed by plaque assay. The graph is representative of an independent experiment performed in triplicate. C) U87MG cells were infected with rTC-83-nsP3-HA (MOI:20) for 1 h. At 6 hpi cell lysates were collected, quantified and 2 mg of total protein was immunoprecipitated with an isotype IgG control or an HA antibody. The immunoprecipitates were resolved by SDS-PAGE and the subsequent immunoblot probed with antibodies to eIF4A, eIF4G, PABP, DDX3 or HA. Densitometric counts for eIF4A, eIF4G, PABP and DDX3 were obtained using the Quantity One Analysis Software. The normalized data are represented graphically. The western blot is a representative image from 2 independent experiments. * = p ≤ 0.05.

Article Snippet: Primary antibodies to HA, DDX1 (Abcam, ab31963), DDX3 (Cell Signaling, 2635), VEEV-capsid (BEI Resources, NR-9403), HRP conjugated actin, PABP (Santa Cruz Biotechnology, sc-28834), TIA-1 (Santa Cruz Biotechnology, sc-1751), eIF4G (Cell Signaling, C45A4) and eIF4A (Cell Signaling, C32B4) were used according to the manufacturer's instructions.

Techniques: Cell Viability Assay, Infection, Plaque Assay, Immunoprecipitation, Control, SDS Page, Western Blot, Software

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Sequences of the integrin β1, β1A, β1C, hnRNP E1-GFP and β-actin PCR primers.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques:

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Activity of Fyn regulates the phosphorylation and nuclear localization of hnRNP E1 via PAK1. (A–C) Western blot analysis revealed the relative expression levels of hnRNP E1 and PAK1 protein among BxPC3, BxPC3GFP and BxPC3KdFyn cells. β-actin blotting was used as a loading control. (D and E) Co-immunoprecipitation analysis showed the threonine-phosphorylation of hnRNP E1 and the interaction of hnRNP E1 with PAK1 among BxPC3, BxPC3GFP and BxPC3KdFyn cells. (F and G) Confocal microscopy analysis showed that the nuclear distribution of hnRNP E1 was increased in BxPC3KdFyn cells compared with BxPC3GFP cells. * P<0.05. Data are presented as the mean ± standard deviation from ≥3 independent experiments.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Confocal Microscopy, Standard Deviation

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Fyn regulates the alternative splicing of integrin β1 via hnRNP E1. (A) Co-immunoprecipitation analysis showed the expression of GFP-hnRNP E1 fusion protein in BxPC3 cells. (B, F and G) Splicing of integrin β1 was analyzed under different Fyn activity levels or hnRNP E1 expression levels in BxPC3 cells. (C–E) Knockdown of hnRNP E1 expression decreased the phosphorylation of hnRNP E1 in BxPC3 cells. (H and I) Splicing of integrin β1 was analyzed under different Fyn activity levels or hnRNP E1 expression levels in Panc1 cells. (J) RNA immunoprecipitation showed the interaction between hnRNP E1 and the pre-mRNA of integrin β1. * P<0.05. Data are presented as the mean ± standard deviation from ≥3 independent experiments. Sequences of the PCR primers for integrin β1 pre-mRNA, which cover the exon and intron sequences of integrin β1C are listed in .

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Immunoprecipitation, Expressing, Activity Assay, Standard Deviation

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Proteins identified by mass spectrometry in the hnRNP E1 spliceosome of BxPC3KdFyn cells.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Mass Spectrometry, Protein Binding

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: hnRNP E1 spliceosome regulates integrin β1 splicing by hnRNP A1 and SF2/ASF. (A) Schematic representation of the genomic sequence of integrin β1. Exon sequences are shown as boxes and introns as solid lines. The splicing pattern used to generate β1A and C transcripts are depicted, and the potential SR protein-binding site is indicated with a vertical arrow. The positions of the stop codons for the various splice variants are also indicated by arrows. The horizontal arrows indicate the primers used in PCR experiments . (B–D) Western blot analysis and co-immunoprecipitation were used to analyze the expression levels and abundance of hnRNP E1-specific spliceosome components among BxPC3, BxPC3GFP and BxPC3KdFyn cells. β-actin blotting was used as a loading control. (E and F) Co-immunoprecipitation and splicing analysis were used to explore the functions of hnRNP A1 and SF2/ASF on the construction of the hnRNP E1 spliceosome complex and the splicing of integrin β1. (G) RNA immunoprecipitation showed that hnRNP A1 and SF2/ASF, but not hnRNP E1, were directly bound to the pre-mRNA of integrin β1. * P<0.05. Data are presented as the mean ± standard deviation from ≥3 independent experiments.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Sequencing, Protein Binding, Western Blot, Immunoprecipitation, Expressing, Standard Deviation

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Effect of Fyn and hnRNP E1 on the invasion and metastasis of pancreatic cancer cells. (A–C) Transwell and wound-healing assays revealed that the activity of Fyn and/or the expression of hnRNP E1 significantly affected the invasion and migration abilities of BxPC3 pancreatic cancer cells in vitro . (D–F) An assay of the subcutaneous tumor mass and liver metastatic nodules revealed that the activity of Fyn and/or the expression of hnRNP E1 significantly affected the tumorigenesis and metastasis of BxPC3 pancreatic cancer cells in vivo . Each group of xenograft experiments included 5 mice. * P<0.05. Data are presented as the mean ± standard deviation from ≥3 independent experiments.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Activity Assay, Expressing, Migration, In Vitro, In Vivo, Standard Deviation

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Associations between the expression of integrin β1A, hnRNP E1 and the categorical clinicopathological parameters of pancreatic carcinoma.

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin β1

doi: 10.3892/ijo.2017.4018

Figure Lengend Snippet: Correlation between the expressions of β1A (mRNA) and hnRNP E1 (protein).

Article Snippet: Briefly, 5 mg extracted proteins were pre-cleared with 50 µ l protein A/G Plus agarose and then incubated with 10 µ g hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) overnight at 4°C with constant rotation.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms

doi: 10.3892/mmr.2015.4364

Figure Lengend Snippet: Effect of SAHA on levels of GSH-associated enzyme and GSH in CLL cells co-cultured with stromal cells. (A) SAHA increased the expression of Nrf2 in CLL cells. CLL cells were treated with 2 µ M SAHA for 20 h, and cell lysates were assayed for Nrf2 using western blot analysis. Representative western blot results from three samples from patients with CLL are shown. The right panel shows the quantification of Nrf2 band density of eight CLL samples, with β-actin expression as an internal control (mean ± standard deviation; * P<0.05; Ctrl, control cells without treatment; S, SAHA treatment). (B) SAHA induced the translocation of Nrf2 between the cytosol and nucleus. SAHA (2 µ M) was added to the CLL cells for 20 h, and the cells were cytospun and immunostained with Nrf2 antibodies, and observed using a confocal laser scanning microscope. The nuclei were stained with 4,6-diamidino-2-phenylindole. (C) Upregulation of mRNA expression of GCLC following SAHA treatment. CLL cells were treated with 2 µ M SAHA for 22 h and the GCLC mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. (D) SAHA increases the expression of GCLC in CLL cells. CLL cells were treated with 2 µ M SAHA for 24 h, and the cell lysates were then assayed for the expression levels of GCLC by western blot analysis. The upper panel shows the representative western blot results from samples of four patients with CLL. The lower panel shows the quantification of GCLC band density of eight CLL samples, with β-actin as the internal control (mean ± standard deviation; ** P<0.01. (E) Treatment with SAHA enhanced stromal-mediated GSH upregulation in CLL cells. The CLL cells were treated with 2 µ M SAHA for 48 h in the presence or absence of HS5 cells. In another treatment group, CLL and HS5 cells in co-culture were incubated with cystine transporter inhibitor S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Values are presented as the mean ± standard deviation of three independent experiments using three CLL samples. (F) Sensitization of CLL cells to SAHA by inhibiting the cystine transporter with S-4-CPG. CLL and HS5 cells in co-culture were incubated with S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Cell viability was analyzed using an Annexin V/PI assay. Representative dot plots are shown. CLL, chronic lymphocytic leukemia; SAHA, suberoylanilide hydroxamic acid; GSH, glutathione; PI, propidium iodide; Nrf2, nuclear factor-E2-related factor 2; CPG, carboxyphenylglycine Ctrl, untreated control; S, SAHA treatment.

Article Snippet: The membranes were then probed with the following primary antibodies: Rabbit anti-human GCLC polyclonal antibody (cat. no. sc-28965, Santa Cruz Biotechnology, Inc.) at a 1:1,000 dilution; rabbit anti-human Nrf2 polyclonal antibody cat. no. sc-13032; Santa Cruz Biotechnology, Inc.) at a 1:1,000 dilution; rabbit anti-human Mcl1 polyclonal antibody (cat. no. sc-819; Santa Cruz Biotechnology, Inc.) at a 1:1,000 dilution;and mouse anti-human actin monoclonal antibody (cat. no. MA5-11869; Pierce Biotechnology) at a 1:10,000 dilution.

Techniques: Cell Culture, Expressing, Western Blot, Control, Standard Deviation, Translocation Assay, Laser-Scanning Microscopy, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Co-Culture Assay, Incubation