Journal: PLoS ONE

Article Title: The Function of Hypoxia-Inducible Factor (HIF) Is Independent of the Endoplasmic Reticulum Protein OS-9

doi: 10.1371/journal.pone.0019151

Figure Lengend Snippet: (A) OS-9 expression in various human cell lines. Equal protein amounts of total cell lysates were used for SDS-PAGE and subsequent Western blotting. For each cell line, two independent samples are shown. Endogenous OS-9 was detected with a polyclonal antibody raised against a peptide corresponding to amino acids 600–667 of isoform 1 of OS-9. (B) Protein stability assay of endogenous OS-9. U2OS cells were treated with the translational inhibitor cycloheximide (100 µM). At indicated time points, whole cell lysates were analysed by immunoblotting. (C) Effect of hypoxia on OS-9 expression. For hypoxia, UT-7 cells were exposed to 1% O 2 for 24 h prior to Western blot analysis. To determine any influence of HIF-1α on OS-9 expression under normoxia, cells were incubated with the prolyl hydroxylase inhibitor DMOG (0.5 mM) for 24 h. (D) Protein interaction between OS-9 and PHD2 in vitro . For co-immunoprecipitation, U2OS cells were transiently co-transfected with the plasmids pOS-9-V5 and pPHD2-His, lysed in NP40 buffer, and subjected to immunoisolation with anti-V5 antibody recognizing OS-9 by its V5-tag. OS-9 and its associated proteins were separated by SDS-PAGE and analyzed by Western blot (lane 2). As controls, samples of untransfected (lane 1) cells or cells transfected with a single plasmid (lanes 3–4) were loaded. Representative Western blots are shown for each subfigure.

Article Snippet: Polyclonal antibodies against OS-9 (NB100-520) and PHD2 (NB100-137) were from Novus Biologicals (Littleton, USA).

Techniques: Expressing, SDS Page, Western Blot, Stability Assay, Incubation, In Vitro, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Function of Hypoxia-Inducible Factor (HIF) Is Independent of the Endoplasmic Reticulum Protein OS-9

doi: 10.1371/journal.pone.0019151

Figure Lengend Snippet: (A) A lectin gel-shift assay was conducted to test for glycosylated proteins. Total cell lysates of U2OS cells were incubated in the presence or absence of the endoglycosidases EndoH and PNGaseF for 6 h at 37°C. Digest products were separated on a reducing 10% SDS-PAGE gel which contained concanavalin A co-polymerized in the top layer of the separating gel to retard mobility of glycosylated proteins . Glycosylated OS-9 is indicated as ‘g’, deglycosylated OS-9 as ‘d’. (B) Detection of OS-9 and PHD2 in the nuclear fraction. HEK293 cells with and without transfection of the plasmid pcDNA3-OS-9 were separated into nuclear fraction (N) and postnuclear supernatant (PS), the latter containing cytoplasm and organelles. Western blot analysis included BiP, GAPDH and lamin A as typical marker proteins for the ER, the cytoplasm and the nucleus, respectively. (C) Detection of OS-9 and PHD2 in the cytoplasm. HEK293 cells were co-transfected with pcDNA3-OS-9 and pPHD2-V5. For hypoxia, cells were exposed to 3% O 2 for 4 h. Cells were treated with 50 µg/ml digitonin and centrifuged to obtain a cytoplasmic (C) and an organelle fraction (O) and subjected to immunoblotting. (D) Isolation of cellular endomembranes. HEK293 cells were lysed mechanically by several passages through a 30½G needle. The postnuclear supernatant was processed further by ultra-centrifugation to separate the organelles (O) from the cytosol (C). High salt treatment (1 M KCl) of the organelle fraction produced a wash fraction (W) that contained dissociated peripheral membrane proteins. For immunoblot analysis of subcellular fractionations, cell aliquots were normalized for cell number prior to loading (B–D). Representative Western blots are shown for each subfigure.

Article Snippet: Polyclonal antibodies against OS-9 (NB100-520) and PHD2 (NB100-137) were from Novus Biologicals (Littleton, USA).

Techniques: Gel Shift, Incubation, SDS Page, Transfection, Plasmid Preparation, Western Blot, Marker, Isolation, Centrifugation, Produced, Membrane

Journal: PLoS Genetics

Article Title: Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila

doi: 10.1371/journal.pgen.1007926

Figure Lengend Snippet: (A) P-p38 after sal E/Pv >rpr induction (n = 14). (B) Inhibition of Ask1 in the anterior compartment ( sal E/Pv >rpr ci>Ask1 RNAi ) stained for P-p38 (n = 7). (C) Mmp1 after sal E/Pv >rpr induction (n = 16). (D) Inhibition of Ask1 in the anterior compartment ( sal E/Pv >rpr ci>Ask1 RNAi ) stained for Mmp1 (n = 14). TP-3: TO-PRO-3 nuclear staining. Purple: sal E/Pv >rpr ablated zone. White dotted line divides the discs into anterior ( Ask1 inhibition; to the left) and posterior (wild-type; to the right) compartments. (E-H) The UAS-Ask1 RNAi was activated in the posterior compartment together with UAS-GFP (green). Two injuries were inflicted with tungsten needles in Schneider’s medium, one in the anterior and one in the posterior compartment. Discs were stained for P-p38 and Mmp1. (E) For P-p38 staining, discs were immediately fixed after physical injury. (F) For Mmp1 staining, discs were first cut and cultured for 5 hours before fixation and processing. White lines indicate the wound site. Scale bar: 50μm. (G) Quantification of P-p38 activation at the wound site (n = 10 discs each genotype). (H) Quantification of Mmp1 at the wound site (n = 8 discs each genotype). Error bars indicate standard deviation. ***P<0.001. (I) Model proposed for the onset of regeneration in the columnar epithelium of the wing imaginal disc.

Article Snippet: Other antibodies used were: Ptc (DSHB, 1:100), P-p38 (Cell Signalling 1:50), Mmp1 (cocktail of three antibodies: DSHB 3A6B4, 5H7B11, 3B8D12 1:100), P-Akt (S473 Cell Signalling 1:100) and P-Histone-H3 (Millipore, 1:1000).

Techniques: Inhibition, Staining, Cell Culture, Activation Assay, Standard Deviation

Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Increased migration behavior of hCSCs after application of human blood serum is dependent on p38-MAPK signaling. ( A ) Exemplary images of hCSCs cultivated without serum directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( B ) Exemplary images of hCSCs cultivated with human blood serum directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( C ) Exemplary images of hCSCs cultivated with human blood serum and p38-MAPK-inhibitor SB239063 directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( D ) Migration distance of hCSCs is significantly increased by the application of human serum. This effect is reversed by the inhibition of p38. ( E ) Migration velocity of hCSCs is significantly increased by the application of human serum. This effect is reversed by inhibition of p38. Data points represent migration dynamics of single hCSCs observed in individual cultivation chambers. Measurements were performed with hCSCs from one donor and blood serum from one donor. Mann-Whitney one-tailed, **** p < 0.0001 was considered significant.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Migration, Inhibition, MANN-WHITNEY, One-tailed Test

Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Immunocytochemistry of Nestin and phospho-Hsp27 in hCSCs treated with human serum and p38-MAPK-inhibitor. ( A ) Untreated cells exhibit a basal level of Hsp27 phosphorylation. ( B ) Phosphorylation of Hsp27 in serum-treated cells is strongly increased. ( C ) Hsp27-phosphorylation is inhibited after the application of the p38-MAPK inhibitor SB239063. ( D ) Quantification of fluorescence intensity across all replicates. Measurements were performed with hCSCs from one donor and blood serum from two donors. Mann-Whitney one-tailed, * p < 0.05; **** p < 0.0001 was considered significant.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Immunocytochemistry, Phospho-proteomics, Fluorescence, MANN-WHITNEY, One-tailed Test

Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Schematic summary of hCSC-migration upon serum-treatment regulated by p38-MAPK and Hsp27.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Migration

Journal: Journal of Microbiology and Biotechnology

Article Title: Establishment and Characterization of Immortalized Human Dermal Papilla Cells Expressing Human Papillomavirus 16 E6/E7

doi: 10.4014/jmb.2310.10035

Figure Lengend Snippet: ( A, B ) The TP53 gene and p53 protein expression level was determined. The relative band intensities were evaluated by ImageJ software. Data are presented as the mean SD ( n = 3). *** p < 0.001 by t -test. ( C ) p53 was labeled with Cy3 (red) in cells, and nuclei were labeled with DAPI (blue). Scale bar = 125 μm. ( D ) The protein level of CDK inhibitor p21 was determined by immunoblotting assay. ( E-G ) The gene (CDK2 and CCNE1) and protein (CDK2 and cyclin E) expression levels were determined. The relative band intensities were evaluated by ImageJ software. Data are presented as the mean SD ( n = 3). * p < 0.05, ** p < 0.01 by t -test.

Article Snippet: Specific primary antibodies against p53 (#NB200-171) (Novus Biologicals, Centennial, USA), cyclin E (#sc-481) (Santa Cruz Biotechnology, USA), p21(#2947T), CDK2 (#2546T), pRb (#9309s), and p-pRb (#8147s) (Cell Signaling Technology, USA) were used.

Techniques: Expressing, Software, Labeling, Western Blot