Journal: Nanoscale

Article Title: Ultrasmall Prussian blue nanoparticles attenuate UVA-induced cellular senescence in human dermal fibroblasts via inhibiting the ERK/AP-1 pathway.

doi: 10.1039/d1nr04268h

Figure Lengend Snippet: Fig. 3 USPBNPs decreased SA-β-gal activity, attenuated cell cycle arrest and reduced the expression of p16, p21 and p53 in UVA- radiated HDFs. (A) SA-β-gal staining of HDFs at 24 h after UVA radiation with or without USPBNPs pretreatment; scale bar: 100 μm. (B) Percentage of SA-β-gal positive stained cells calculated by Image J. (C) Cell cycle analysis of HDFs at 24 h after UVA irradiation with or without USPBNPs pretreatment by flow cytometry. (D) Cell cycle distribution in each group. (E) The protein level of p16, p21 and p53 in HDFs at 24 h after UVA irradiation with or without USPBNPs pretreatment detected by western blot. (F) Quantification of the western blot band signals of p16, p21 and p53 by Image J. *P < 0.05, **P < 0.01, ***P < 0.001, versus control group; #P < 0.05, ##P < 0.01 and ###P < 0.001, versus UVA group.

Article Snippet: The following primary antibodies were used, including anti-p16 (CST), antip21(CST), anti-p53 (CST), anti-IL-6 (Abcam), anti-TNF-α (Proteintech), anti-MMP-1 (Proteintech), anti-MMP-3 (Proteintech), anti-MMP-9 (Proteintech), anti-γH2AX (CST), anti-p-ERK (CST), anti-ERK (CST), anti-c-Jun (CST), anti-c-Fos (CST) and anti-GAPDH (CST).

Techniques: Activity Assay, Expressing, Staining, Cell Cycle Assay, Irradiation, Flow Cytometry, Western Blot, Control

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Transfection

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Oncogene

Article Title: Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

doi: 10.1038/onc.2012.324

Figure Lengend Snippet: Figure 1. WEE1 is downregulated in melanoma metastases as compared with primary melanomas. (a) WEE1 protein expression was analysed in tissue sections of paraffin-embedded primary melanomas (PM; n ¼ 10) and distant cutaneous metastases (META; n ¼ 10) by immunohistochemistry. Representative melanoma sections show positively stained nuclei for WEE1 at different magnification (upper panel, 10; lower panel 40) (b) Percentage of WEE1-positive cells in primary melanomas and distant cutaneous metastases (n ¼ 10). Results are expressed as mean±s.e.m. (Mann–Whitney U-Test). (c) Primary melanomas (n ¼ 8) and cutaneous metastases samples (n ¼ 10) were tested for WEE1 expression by qRT–PCR. Relative WEE1 expression was represented as 2 DDCt values where one of the primary melanoma samples was used as normalizing control to calculate DDCt. GAPDH expression was used for normalization (Mann–Whitney U-Test). (d) WEE1 protein expression was analysed in melanoma cell lines of different aggressiveness, namely non-metastatic BRO, A-375, 1F6 and RPM-MC cells (Cell Line Panel-1) and metastatic SK-Mel-147, SK-Mel-28 and SK-Mel-29 cells (Cell Line Panel-2) using immunoblotting. Normalized WEE1 intensities were quantitated and compared with the mean WEE1 intensities across all cell lines. Results are expressed as mean±s.e.m. of relative WEE1 expression (unpaired t-test).

Article Snippet: Immunoblotting Immunoblotting was performed as described.40 The following primary antibodies were used (all purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA): anti-WEE1 mouse monoclonal Ab (sc-5285), antipCdk1 rabbit polyclonal Ab (sc-7989).

Techniques: Expressing, Immunohistochemistry, Staining, MANN-WHITNEY, Quantitative RT-PCR, Control, Western Blot

Journal: Oncogene

Article Title: Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

doi: 10.1038/onc.2012.324

Figure Lengend Snippet: Figure 3. miR-195 regulates WEE1 by directly targeting its 30UTR in melanoma. (a) The miR-195 seed sequence binding region is conserved across different organisms, including humans as shown by sequence alignments (http://www.targetscan.org). (b) WEE1 mRNA expression was analysed by qRT–PCR after transfection of SK-Mel-28 cells with WEE1 siRNA, miR-195 and miR-195 antagomir, respectively. Fold change in WEE1 mRNA expression is shown relative to non-targeting miRNA-transfected control cells. Data are given as mean±s.e.m. (Mann–Whitney U-Test). (c, d) Immunoblot analysis was performed for WEE1 expression after transfecting the indicated oligomers into SK-Mel-28 (c) and SK-Mel-147 (d) cells, respectively. Fold changes in WEE1 expression are indicated above the blots. (e–g) Luciferase reporter gene analyses using indicated WEE1 30UTR normal and mutant reporter constructs cotransfected with miR-195 in SK-Mel-28 (e, f) and SK-Mel-147 (g) melanoma cells, respectively. Data were normalized by calculating the ratio of firefly luciferase activity with that of Renilla luciferase and expressed as percentage of relative luciferase activity. Data are given as mean±s.e.m. for the percentage of relative luciferase activity (unpaired t-test). antag, antagomir.

Article Snippet: Immunoblotting Immunoblotting was performed as described.40 The following primary antibodies were used (all purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA): anti-WEE1 mouse monoclonal Ab (sc-5285), antipCdk1 rabbit polyclonal Ab (sc-7989).

Techniques: Sequencing, Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Control, MANN-WHITNEY, Western Blot, Luciferase, Mutagenesis, Construct, Activity Assay

Journal: Oncogene

Article Title: Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

doi: 10.1038/onc.2012.324

Figure Lengend Snippet: Figure 4. miR-195 abrogates genotoxic stress-induced G2-M arrest in melanoma cells. (a, b) SK-Mel-28 cells were either left untreated or pulse- treated with 100 nM doxorubicin for 2 h. At 24 h after treatment cells were either fixed and stained with propidium iodide for cell cycle analysis (a) or lysed to test WEE1 expression and Cdc2-Y15 phosphorylation by immunoblotting (b). (c–e) SK-Mel-28 cells were individually transfected with the indicated oligomers. At 48 h after transfection, cells were either left untreated or pulse-treated with doxorubicin for 2 h. Another 24 h later the cells were lysed to either check for WEE1 expression or phosphorylation of Cdc2-Y15 by immunoblotting (c) or fixed and stained with propidium iodide for cell cycle analyses of WEE1 siRNA transfected (d) and miR-195 transfected SK-Mel-28 cells (e). The data for cell cycle analysis are given as mean±s.e.m. for the percentage of cells in the indicated phases of cell cycle (unpaired t-test).

Article Snippet: Immunoblotting Immunoblotting was performed as described.40 The following primary antibodies were used (all purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA): anti-WEE1 mouse monoclonal Ab (sc-5285), antipCdk1 rabbit polyclonal Ab (sc-7989).

Techniques: Staining, Cell Cycle Assay, Expressing, Phospho-proteomics, Western Blot, Transfection

Journal: Oncogene

Article Title: Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

doi: 10.1038/onc.2012.324

Figure Lengend Snippet: Figure 5. WEE1 overexpression reverses miR-195-mediated G2-M abrogation and reduces clonogenicity. A stably WEE1 overexpressing SK-Mel- 28-WEE1 cell line was generated by transduction of SK-Mel-28 cells with a lentiviral construct. (a) qRT–PCR and immunoblot analyses for WEE1 mRNA or protein expression in SK-Mel-28-control (untransduced) cells and SK-Mel-28-WEE1 cells. The qRT–PCR data are given as mean±s.e.m. of fold change in WEE1 mRNA expression in SK-Mel-28-WEE1 cells as compared with SK-Mel-28-control cells. (b) Colony formation assay of SK-Mel-28-control and SK-Mel-28-WEE1 cells. The graph is representative of three independent experiments and the data are given as mean±s.e.m. of the average colony number. (c–e) SK-Mel-28-WEE1 and SK-Mel-28-control cells were transfected with miR-195. At 48 h after transfection, cells were treated with doxorubicin and another 24 h later cells were harvested for cell cycle analysis by flow cytometry (c, d) or lysed to test WEE1 expression and Cdc2-Y15 phosphorylation by immunoblotting (e). Cell cycle data show mean±s.e.m. of the percentage of cells in the indicated cell cycle phases. Fold changes in WEE1 expression and Cdc2-Y15 phosphorylation are indicated above the blots.

Article Snippet: Immunoblotting Immunoblotting was performed as described.40 The following primary antibodies were used (all purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA): anti-WEE1 mouse monoclonal Ab (sc-5285), antipCdk1 rabbit polyclonal Ab (sc-7989).

Techniques: Over Expression, Stable Transfection, Generated, Transduction, Construct, Quantitative RT-PCR, Western Blot, Expressing, Control, Colony Assay, Transfection, Cell Cycle Assay, Cytometry, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

doi: 10.1074/jbc.M112.425066

Figure Lengend Snippet: G93A SOD1 astrocytes released a lower amount of proteins in the media. A , media were collected from similar numbers of cells expressing WT or G93A SOD1, and secreted proteins were prepared as described for slot blot experiments and quantified by BCA protein assay. Protein quantification was performed in at least seven samples per group. Each sample was a 3-well pool. Values are the amounts of secreted proteins normalized to total cell proteins (secreted proteins/total cell proteins) and are means ± S.E. ( error bars ). An asterisk indicates statistical significance ( p < 0.05), Student's t test. B , for two-dimensional gel electrophoresis maps ( n = 3 for each genotype), the same amount of secreted proteins (30 μg) were loaded on IPG strips (pI 4–7). The gel maps were stained with Sypro Ruby and compared by computerized image analysis. Six proteins were differentially secreted by astrocytes expressing mutant SOD1 and controls. Spot numbers refer to proteins listed in . C and D , slot blot and relative quantification of SOD1 ( C ) and VCP/p97 ( D ) released by the astrocytes confirmed the proteomic analysis and showed that the total level of the extracellular proteins were higher than in control conditions. The astrocytes were plated in 6-well dishes, and equal volumes of conditioned media from 3-well pools were used for the slot blot analysis. Values are immunoreactivities and are means ± S.E. ( n = 3) normalized to WT, set as 100. An asterisk indicates statistical significance ( p < 0.05), Student's t test.

Article Snippet: The antibodies anti-human SOD1 (1:500; Millipore), anti-CypA (1:1,000; Millipore), and rabbit monoclonal anti-valosin-containing protein (VCP)/p97 (1:50,000; Epitomics) were incubated overnight at 4 °C to reveal the immunoreactivity in secreted proteins and exosome-enriched fractions.

Techniques: Expressing, Dot Blot, Bicinchoninic Acid Protein Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining, Mutagenesis, Quantitative Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

doi: 10.1074/jbc.M112.425066

Figure Lengend Snippet: Differentially secreted proteins in primary astrocytes derived from G93A SOD1 mice Proteins were identified by MALDI-TOF mass spectrometry as reported under “Experimental Procedures” and in supplemental Table S1 .

Article Snippet: The antibodies anti-human SOD1 (1:500; Millipore), anti-CypA (1:1,000; Millipore), and rabbit monoclonal anti-valosin-containing protein (VCP)/p97 (1:50,000; Epitomics) were incubated overnight at 4 °C to reveal the immunoreactivity in secreted proteins and exosome-enriched fractions.

Techniques: Derivative Assay, Mass Spectrometry, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

doi: 10.1074/jbc.M112.425066

Figure Lengend Snippet: WT and G93A SOD1 are released by the astrocytes, and different amounts are present in the exosomes. A , electron microscopy analysis of purified membrane vesicles with typical exosomal shape and dimension (diameter ranging from 80 to 140 nm; scale bar , 200 nm). B , slot blot of astrocyte protein lysate, secreted proteins, and ultracentrifuged proteins (supernatant and exosomal fractions). An exosomal marker, flotillin-1, is present in whole lysate, in unpurified secreted proteins, and in the exosomal fraction but not in the supernatant. C and E , G93A SOD1 expression increases exosomal proteins. Protein quantification of the supernatant and exosomal fractions was done by BCA protein assay in at least seven samples per genotype. Each sample was a 3-well pool. Values are the amounts of non-exosomal or exosomal proteins normalized to total astrocytic proteins (non-exosomal or exosomal proteins/total astrocytic proteins) and are means ± S.E. ( error bars ) Asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01), Student's t test. D , flotillin-1 was measured by slot blot in WT and G93A SOD1 astrocyte-conditioned media. Anti-flotillin-1 immunoreactivity was normalized to the total protein loaded, as measured after Sypro Ruby blot staining. Values are means ± S.E. ( n = 3). The asterisk indicates a G93A sample mean significantly higher ( p < 0.05) than the WT sample mean, Student's t test. F and G , slot blot for SOD1 and VCP/p97 in the exosomal and non-exosomal (supernatant) fractions. Equal volumes of exosomal or non-exosomal fractions were used to measure protein levels in the G93A SOD1 and WT SOD1 conditions. Each sample was taken from a 3-well pool. Values are immunoreactivities and are means ± S.E. ( n = 4) normalized to WT, set as 100. An asterisk indicates statistical significance ( p < 0.05), Student's t test.

Article Snippet: The antibodies anti-human SOD1 (1:500; Millipore), anti-CypA (1:1,000; Millipore), and rabbit monoclonal anti-valosin-containing protein (VCP)/p97 (1:50,000; Epitomics) were incubated overnight at 4 °C to reveal the immunoreactivity in secreted proteins and exosome-enriched fractions.

Techniques: Electron Microscopy, Purification, Membrane, Dot Blot, Marker, Expressing, Bicinchoninic Acid Protein Assay, Staining