Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A–B) Western blot analysis of uPAR and cathepsin B expression in U251 and 5310 cells 72 hrs after transfection with SV, pU, pC and pCU. (C) Quantitative analysis of uPAR and cathepsin B expression by densitometry. Results from three independent experiments are shown as mean ± SD (** p <0.001). GAPDH was used as a loading control. (D) Cell cycle distribution of U251 and 5310 cells. Propidium iodide-stained cells were analyzed for DNA content using flow cytometry. (E) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of three independent experiments. Values are mean ± SD of three different experiments (** p <0.001). (F) Cells were stained for apoptosis using TdT-mediated dUTP nick end-labeling (TUNEL) assay. Data shown are from a representative experiment. (G) Quantification of apoptotic cells expressed as percent of DAPI-stained cells. Bars represent the mean ± SD of three experiments ( *p <0.05; **p <0.001). (H) Analysis of mitochondrial membrane potential using MitoLight kit. Cells were collected, incubated with MitoLight dye for 20 min at room temperature, and observed for fluorescence; red fluorescence indicates healthy cells while green fluorescence indicates apoptotic cells.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Western Blot, Expressing, Transfection, Staining, Flow Cytometry, End Labeling, TUNEL Assay, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A) Expression of pro- and anti-apoptotic molecules in U251 and 5310 cells 72 hrs after transfection with SV or pCU. Human apoptosis antibody arrays were exposed to cell lysates and processed as per the manufacturer's instructions. (B) Densitometric analysis and graphical representation of fold change of pro- and anti-apoptotic molecules. (C) Western blot analysis of Bcl-2 and Bax expression in U251 and 5310 cells 72 hrs transfection. The blots were stripped and re-probed with GAPDH antibody to verify equal loading. The experiments were repeated three times and representative blots are shown. (D) Densitometric analysis showing the Bcl-2/Bax ratio in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; ** p <0.001. (E) Semi-quantitative RT-PCR analysis of Bcl-2 and Bax mRNA expression in pU-, pC- and pCU-transfected U251 and 5310 cells. Total RNA was extracted 72 hrs after transfection, and cDNA was synthesized as described in . PCR was set up using first-strand cDNA as the template for Bcl-2, Bax and GAPDH. (F) Densitometric analysis showing the Bcl-2/Bax mRNA ratio. Columns: mean of triplicate experiments; bars: SE; ** p <0.001, significant difference from untreated control or SV-transfected control.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Synthesized

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A) FACS analysis of pCU-transfected U251 and 5310 cells pretreated with 40 µM broad caspase inhibitor (Z-Asp-2, 6-dichlorobenzoylmethylketone). (B) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of the three independent experiments. Values are mean ± SD of three different experiments (* p <0.05, ** p <0.001). uPAR and cathepsin B downregulation induced activation of caspase-9, caspase-3, ICAD, CAD in pU-, pC- and pCU-treated U251 cells. (C) Western blot analysis for active caspase-9, caspase-3, ICAD and CAD in U251 cells. (D) Expression of cytochrome c in mitochondrial and cytosolic fractions was determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions (MF); GAPDH was used for cytosolic fractions (CF). (E) Immunoprecipitation of Apaf-1 from U251 cell lysates. Total cell lysates were subjected to immunoprecipitation using anti-caspase-9 antibody and then immunoblotted for Apaf-1. Total lysates from SV-, pU-, pC- and pCU-treated U251 cells were separated into mitochondrial and nuclear fractions as per standard protocols and immunoblotted for AIF. (F) Expression levels of AIF in mitochondrial (MF) and nuclear fractions (NF) were determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions; laminin B was used for nuclear fractions. Total lysates from SV-, pU-, pC- and pCU-treated 5310 cells were fractionated into mitochondrial and cytosolic fractions as per standard protocols and immunoblotted for cytochrome c. Immunoblots are representative of three experiments.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Activation Assay, Western Blot, Expressing, Marker, Immunoprecipitation

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: U251 and 5310 cells were transfected with SV, pU, pC or pCU. Untreated cells served as the control. After 72 hrs, cells were collected, and total cell lysates were prepared and western blotted as per standard protocol using normal and phosphorylated forms of PI3K, Akt and PDGFR-β. (A–B) Expression of normal and phosphorylated forms of PI3K, Akt and PDGFR-β in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; * p <005 and ** p <0.001, significant difference from untreated control or SV-transfected control. Effect of PI3K inhibitor (Wortmannin) and PDGFR tyrosine kinase inhibitor (PTKI) on expression of p-PDGFR-β, p-PI3K p85α, Bcl-2 and Bax. U251 and 5310 cells were treated with 20 and 40 µg/mL of PI3K inhibitor (Wortmannin;WN) for 48 hrs, and the expression levels of p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (C–D) Western blot analysis of p-AKT, p-CREB, Bcl-2 and Bax expression levels after treatment with the PI3K inhibitor. Separately, U251 and 5310 cells were treated with 5 nM and 10 nM PDGFR tyrosine kinase inhibitor (PTKI) for 48 hrs. Expression levels of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (E–F) Western blot analysis of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax expression levels in U251 and 5310 cells treated with PDGFR tyrosine kinase inhibitor.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: U251 and 5301 cells were transfected with SV, pU, pC or pCU for 72 hrs. Cell lysates were separated into nuclear (NF) and cytoplasmic fractions (CF) and immunoblotted for p-CREB. Total cell lysates were probed for CREB. (A–B) Western blot analysis of p-CREB in nuclear and cytoplasmic fractions of U251 and 5310 cells. Nuclear extracts were prepared from SV- (control) and pCU-transfected U251 and 5310 cells and supershift analysis of CREB binding activity was carried out using EMSA. For supershift analysis, nuclear extracts were incubated with supershift specific CREB antibody (2.0 µg) or IgG (2.0 µg) prior to incubation with CREB binding buffer. (C–D) Supershift analysis of CREB DNA binding activity in U251 and 5310 cells. The experiments were performed three times and representative blots are shown. Nuclear extracts were prepared from SV-, pU-, pC- and pCU-transfected U251 and 5310 cells, and DNA binding activity of CREB was determined by colorimetric assay using the TransAM ELISA kit. To test specificity, DNA binding activity was tested in the presence of an excess of oligonucleotide containing a wild-type or mutated CREB consensus binding site. (E–F) Inhibition of DNA binding activity of CREB by nuclear extracts of pU-, pC- and pCU-treated U251 and 5310 cells. The bars represent the mean ± SD of three different experiments. *Statistically different compared to controls and pU-, pU- and pCU-treated groups (** p <0.001).

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Western Blot, Binding Assay, Activity Assay, Incubation, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: Intracranial tumors were established in nude mice by injecting U251 and 5310 cells that were treated with SV and pCU as described in . (A–B) The brains were embedded in paraffin, sectioned and stained for apoptosis by TdT-mediated nick end-labeling (TUNEL) followed by DAB staining. Nuclei were counterstained with methyl green. Data shown are representative of five fields. Brown stain around green nuclei indicates apoptotic cells. (C–D) Immunohistochemical analysis of uPAR, cathepsin B, Bcl-2 and Bax was performed in paraffin-embedded U251 and 5310 tumor sections. Appropriate protein-specific antibodies were used. Fields with brown staining as a result of DAB interaction were scored for protein expression. (E–F) uPAR and cathepsin B, Bcl-2 and Bax expression was detected in tumor tissue lysates from intracranial tumors of mice that received SV and pCU. Results are representative of three separate experiments.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Staining, End Labeling, TUNEL Assay, Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Bamboo Lignin Fractions with In Vitro Tyrosinase Inhibition Activity Downregulate Melanogenesis in B16F10 Cells via PKA/CREB Signaling Pathway

doi: 10.3390/ijms23137462

Figure Lengend Snippet: ( a ) Cytotoxicity result (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) of methanol-soluble steam-exploded lignin fraction (SEL-F2). ( b ) Effects of SEL-F2 on melanogenesis and morphological changes in B16F10 cells. ( c ) B16F10 melanoma cells were cultured for 48 h in the presence of 50–100 μg/mL SEL-F2 and 100 μg/mL arbutin as a positive control, or 1 μg/mL α-melanocyte-stimulating hormone (α-MSH). CON: control B16F10 cell line without any treatment. Represents the data of three separate experiments. ** p < 0.01; significant compared with α-MSH.

Article Snippet: B16F10 melanoma cells, forming a murine melanoma cell line, were purchased from ATCC (American Type Culture Collection; Rockville, MD, USA).

Techniques: MTT Assay, Serial Time-encoded Amplified Microscopy, Cell Culture, Positive Control

Journal: Nature Communications

Article Title: Persistent telomere cohesion protects aged cells from premature senescence

doi: 10.1038/s41467-020-17133-4

Figure Lengend Snippet: a FISH analysis of HEK293T wild-type (#23) and TRF1.G18P mutant (#1, #3, #5) mitotic cells with a 16p (triploid in HEK293T cells) telo probe (green). b Quantification of the frequency of mitotic cells with cohered telomeres. Average of three independent experiments ( n ≥ 28 cells each) ± SEM. WT (#23) vs G18P (#1): p < 0.0001, WT (#23) vs G18P (#3): p < 0.0001, WT (#23) vs G18P (#5): p = 0.0026. c FISH analysis of early (PD30) and late (PD54) passage WI38 mitotic cells with a 16p telo probe (green). d Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments ( n ≥ 36 cells each). e Immunoblot analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cell extracts. f Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cells using Myc (red) and TNKS1 (green) antibodies. g FISH analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 mitotic cells using a 16p telo probe (green). h Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n≥33 cells each). i FISH analysis of a TRF1.WT transfected late (PD52) WI38 mitotic cell exhibiting subtelomere copying (arrowhead) using a 16p telo probe (green). j Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n≥33 cells each). ( a , c , f , g , i ) DNA was stained with DAPI (blue). Scale bars represent 2 μm. ** p ≤ 0.01, **** p ≤ 0.0001, Student’s unpaired two-sided t test. Experiments were repeated independently three times (for a ) and twice (for c , e – g , i ) with similar results. Source data are provided as a Source Data file.

Article Snippet: ALT cancer cell lines GM847 and U2OS (ATCC), and the telomerase-positive cancer cell lines HEK293T (ATCC) and HTC75 (obtained from Dr. Titia de Lange) were supplemented with 10% FBS and grown in standard conditions.

Techniques: Mutagenesis, Western Blot, Plasmid Preparation, Transfection, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2

doi: 10.3390/ijms21010072

Figure Lengend Snippet: Association between protein Amuc_1434* and Muc2. ( A ) Determined Muc2 expression in LS174T and HeLa cells using Western blot. ( B ) Effect of Amuc_1434* concentration in the range of 0–16 μg/mL on adhesion of LS174T cells. HeLa cells were used as negative control. LS174T and HeLa cells bound to the wells were photographed. Crystal violet was extracted, and the remaining adherent cells were quantified by spectrophotometric absorbance at 570 nm. Scale bar = 50 μm ( n = 3, *: p < 0.05; **: p < 0.01). The black line indicated which two groups were compared for significant differences in the column chart.

Article Snippet: LS174T colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Negative Control