Journal: Cell Death Discovery

Article Title: Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species

doi: 10.1038/cddiscovery.2017.60

Figure Lengend Snippet: Aging/aged and GL TM cells and TM cells exposed to oxidative-stress displayed elevated cell senescence markers, p16, p21 and SA- β -gal activity with reduced hTERT expression and activity. ( A ) Western analysis showing increased expression of senescence markers, p16 and p21 in old normal and GL cells compared with younger normal TM cells. ( B ) Western analysis revealed that normal TM cells facing oxidative-stress showed further increases in levels of p16 and p21 in an age-dependent manner. ( C ) Lysate samples containing equal amounts of protein from normal TM cells of different ages and GL TM cells were incubated with SA- β -gal substrate, and SA- β -gal activities were measured and presented in the form of histograms. Values are mean±S.D. of three independent experiments. A significant age-dependent increase in the SA- β -gal activity was observed in aging/aged, and GL TM cells (* P <0.001). ( D ) Oxidative-stress increased levels of SA- β -gal activity in TM cells. Lysate was prepared from normal TM cells of different ages after exposure to H 2 O 2 , and was processed for SA- β -gal activity assay. Histogram values are mean±S.D. of three independent experiments, each with triplicate wells. An age-dependent significant increase in levels of SA- β -Gal activity was observed (* P <0.001), suggesting that oxidative-stress promotes cell senescence. ( E ) Expression analyses showing reduced expression of Prdx6 was related to reduced expression of hTERT (telomerase) expression in GL cells ( E a) Cellular extracts from normal (4M old subject) and glaucomatous (56Y old subject) TM cells having equal amounts of protein were immunoblotted with Prdx6 or hTERT antibodies. β -actin was used as loading control. ( E b) Total RNA was isolated from the same subjects’ TM cells, and processed for qPCR for hTERT mRNA expression by using specific probe. Histogram values are mean±S.D. of three independent experiments (* P <0.001). ( F ) Relative telomerase activity was evaluated using quantitative telomerase detection kit (QTD kit, Allied Biotech, Inc.,) in normal aging/aged and GL TM cells as indicated. Expression of telomerase activity was found to be age-dependent, and was highly reduced in GL TM cells as indicated. Histogram values represent mean±S.D. of three independent experiments (* P <0.001).

Article Snippet: Telomerase activity was evaluated using quantitative telomerase detection kit (QTD kit, Allied Biotech, Inc., Vallejo, CA, USA).

Techniques: Activity Assay, Expressing, Western Blot, Incubation, Control, Isolation

Journal: Cell Death Discovery

Article Title: Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species

doi: 10.1038/cddiscovery.2017.60

Figure Lengend Snippet: Blocking of increased ROS by means of Prdx6 delivery rescued senescence processes in aging/aged/glaucomatous TM cells. ( A ) Normal TM cells of different ages as indicated, GL TM cells overexpressing Prdx6 or TM cell transfectants with vector were seeded in 96 well plate and processed for assessing ROS levels by CellRox assay. The values (mean±S.D.) from two experiments are presented as histograms (* P <0.001). ( B ) The increased generation of bioactive TGF β was attenuated by Prdx6 overexpression. Transfectants expressing Prdx6 or vector were cultured, and supernatants were collected at 72 h and processed for bioactive TGF β assay. Values derived from two experiments are presented as histograms (mean±S.D.) as indicated. ( C ) Accumulation of LPO contents was blunted by Prdx6 overexpression. The same batch of transfectants overexpressing Prdx6 or empty vector were seeded for 72 h, and thereafter processed for LPO assay as described earlier. Histograms represent values (means±S.D.) of two independent experiments as indicated. * P <0.001, statistically significant difference. ( D ) Prdx6 overexpression increased telomerase activity in aging/aged and GL TM cells. The transfectants overexpressing Prdx6 or vector only were cultured. After 48 h transfectants were subjected to assessment of telomerase activity. The values (means±S.D.) showing telomerase activity in cells of different ages and GL cells of three independent experiments are presented as histograms. (* P <0.001). ( E ) Prdx6 overexpression released restriction on cell proliferation, and slowed CDK inhibitors. Transfectants overexpressing pGFP-Prdx6 or pGFP-Vector derived from variable ages/aged/GL TM cells were seeded and 24 h later processed for BrdU assay ( E a) according to the manufacturer’s protocol. Histogram values are means±S.D. of three independent experiments. (* P <0.001), statistically significant difference. ( E b) An aliquot of collected transfectants was tested for levels of p16 and p21, CDK inhibitors and markers for senescence. Cell extracts from transfectants were immunoblotted with antibodies specific to p16 and p21. The levels of p16 and p21 were reduced in GL TM cells overexpressing Prdx6 compared with the control vehicle, transfectants containing vector only. ( F ) Cell senescence assay showing increased SA- β -gal activity in aging TM cells. Aging TM cells overexpressing Prdx6 show reduced SA- β -gal activity as indicated. (* P <0.001).

Article Snippet: Telomerase activity was evaluated using quantitative telomerase detection kit (QTD kit, Allied Biotech, Inc., Vallejo, CA, USA).

Techniques: Blocking Assay, Plasmid Preparation, Over Expression, Expressing, Cell Culture, Derivative Assay, Activity Assay, BrdU Staining, Control

Journal:

Article Title: Significance of Immunological Detection of Human Telomerase Reverse Transcriptase

doi:

Figure Lengend Snippet: A: Telomerase activity in nuclear and cytoplasmic extracts from each cell line. B: Assay to examine whether composition of lysis buffers affect levels of the TRAP activity. Buffer A and C mean lysis buffers with which cytoplasmic and nuclear extracts were extracted, respectively. Nuclear extracts of C33A were diluted with an equal amounts of cytoplasmic lysis buffer (buffer A), and cytoplasmic extracts of C33A were diluted with an equal amounts of nuclear lysis buffer (buffer C). This manipulation equalizes the composition of each extraction buffer. IC, internal control to verify amplification efficiency of PCR.

Article Snippet: TRAP Assay TRAP assays were performed using the TRAPeze telomerase detection kit (Serologicals Corporation, Norcross, GA), according to the manufacturer’s protocols.

Techniques: Activity Assay, Lysis, Extraction, Control, Amplification

Journal:

Article Title: Significance of Immunological Detection of Human Telomerase Reverse Transcriptase

doi:

Figure Lengend Snippet: Western blot analysis of hTERT expression in cell lines. Nuclear or cytoplasmic extracts (100 μg) from each cell line were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to membranes, and probed with specific antibodies against hTERT (KM2604) or Sp1. Immunoreactive bands were visualized by the ECL detection system. Specific signals of the expected size (127 kd) were observed in both nucleic and cytoplasmic extracts from cancer cell lines. In contrast, this signal was faint or absent in telomerase-negative SaOs2, SUSUMU-1, HRCEs, and NHFs; additional bands of lower molecular weight (∼110 kd) were observed in these cells. N, nuclear extract; C, cytoplasmic extract.

Article Snippet: TRAP Assay TRAP assays were performed using the TRAPeze telomerase detection kit (Serologicals Corporation, Norcross, GA), according to the manufacturer’s protocols.

Techniques: Western Blot, Expressing, Polyacrylamide Gel Electrophoresis, Molecular Weight

Journal: BMC Cell Biology

Article Title: Age-related changes in rat bone-marrow mesenchymal stem cell plasticity

doi: 10.1186/1471-2121-12-44

Figure Lengend Snippet: Telomerase activity, growth and immunocytochemical assessment of DNA double strand break in young and old BM-MSCs: (A) Representative micrographs of anti- γ-H2AX staining in cultured BM-MSCs from each experimental group. (B) Columns show comparison of telomerase activity between young and old BM-MSCs by qPCR. C q values for young BM-MSCs are significantly lower indicating higher enzymatically active telomerase. Data represent Mean ± SEM for three biologically independent experiments (n = 3). The '‡' sign indicates a significant difference between the indicated group and any passage 3 groups from older donors ( p < 0.05). (C) Passage 0 BM-MSCs from individual young and old rats were trypsinized, and enumerated with a Cedex HiRes non-flow imaging cytometer over 15 days.

Article Snippet: Telomerase activities from young and old rat MSCs were determined using the Quantitative Telomerase Detection Kit (MT3010; Allied Biotech, Inc., Germantown, MD) according to the manufacturer's protocol.

Techniques: Activity Assay, Staining, Cell Culture, Comparison, Imaging, Cytometry