Journal: OncoTargets and therapy

Article Title: Tumor heterogeneity as a rationale for a multi-epitope approach in an autologous renal cell cancer tumor vaccine

doi: 10.2147/OTT.S92182

Figure Lengend Snippet: List of assays used and the respective sample dilutions

Article Snippet: hTERT , Quantitative Telomerase Detection Kit (QTD Kit)/US Biomax, Rockville, MD, USA , 1:1–1:2.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Oncotarget

Article Title: DNA hypermethyation and silencing of PITX1 correlated with advanced stage and poor postoperative prognosis of esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21375

Figure Lengend Snippet: (A) Growth curves of PITX1 -transfected and mock-transfected KYSE30 clones and KYSE150 stable transfectants as determined by MTT assay (left). HEEpiC cells were transiently transfected with control or PITX1-shRNA. Forty-eight hours after transfection, cell growth was assessed by MTT assay (right). Data are shown as mean ± SD of assays performed in triplicate. (B) PITX1 (left) and TERT (right) mRNA levels in 2 mock-transfected and 2 PITX1 -transfected clones of KYSE30 cells. Numbers in parentheses indicate clone names. Data are shown as mean ± SD of assays performed in triplicate. (C) Increased expression of TERT in ESCC. TERT transcript levels were measured in paired samples from 32 ESCC samples by RT-PCR. Data are reported as expression relative to the mean level in normal tissues. (D) Telomerase activity in PITX1 -transfected and mock-transfected clones of KYSE30 cells. Data are shown as mean ± SD of assays performed in triplicate. (E) Inhibition of telomerase activity in KYSE30 cells after treatment with 5 μM of 5-aza-dC. Data are shown as mean ± SD, assays were repeated five times. (F) PITX1 (left) and TERT (right) mRNA levels in stably mock-transfected and PITX1 -transfected KYSE150 cells. Data are shown as mean ± SD of assays performed in triplicate. (G) Colony formation of stably PITX1 -transfected and mock-transfected KYSE30 and KYSE150 cells. Cells were cultured for 2 weeks. Colonies were visualized by crystal violet staining. (H) Representative images of subcutaneous xenografts (left). Nude mice were subcutaneously inoculated with PITX1 -transfected KYSE30 cells in the right flank and mock-transfected KYSE30 cells in the left flank. Three weeks after inoculation, tumor volumes were measured.

Article Snippet: The lysates of PITX1-transfected KYSE30 cells were subjected to telomerase assay using Quantitative Telomerase Detection (QTD) Kit (Allied Biotech Inc., Vallejo, CA) according to the manufacturer's instructions.

Techniques: Transfection, Clone Assay, MTT Assay, Control, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Inhibition, Stable Transfection, Cell Culture, Staining

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