Journal: Cell Proliferation

Article Title: Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression

doi: 10.1111/j.1365-2184.2007.00462.x

Figure Lengend Snippet: hTERT‐immortalized cell lines show prolonged lifespan with activated telomerase function. (a) Growth curve of hTERT‐immortalized OSE lines (IOSE‐C9, C10, C21) and parental normal OSE cells (NOSE21R) in 180 days of culture. IOSE‐C9, C10 and C21 cells show stable population growth rates 30 days after subcloning. (b) Serum dependence and growth factor dependence analysis. After culturing the hTERT immortal line IOSE‐C21 in complete medium (A), serum‐free medium (B) and growth factor‐free medium (C) for 14 days, cells underwent population growth arrest in serum‐free medium, and slowed their expansion in growth factor‐free medium, but with a change to elongated morphology. Compared to cultures in complete medium, there were significant decreases of live cell populations in both serum and growth factor withdrawn cultures (P < 0.001). (c) TRAP assay show positive telomerase activity in all three immortal lines. 1: Positive control; 2: IOSE‐C21; 3: IOSE‐C9; the missing control band indicates the existence of PCR inhibitor in reaction mix. 4: IOSE‐C10; 5: NOSE21R (parental cells). The lowest two bands of each lane present the PCR endogenous control. Lanes 1–4 show the telomere ladder with 6 base pair (T2AG3) difference between each band, which is a positive indication of telomerase activity. (d) Telomere length assays showed relatively longer telomere length in the three immortal cell lines compared to the parental cells. 1: Telomere ‘high‐level’ control (supplied by the commercial kit); 2: Telomere ‘low‐level’ control (supplied by the commercial kit); 3: NOSE21R (parental cells); 4: IOSE‐C21 cells at passage 9; 5: IOSE‐C21 cells at passage 14; 6: IOSE‐C21 cells at passage 18; 7: IOSE‐C10 cells at passage 4; 8: IOSE‐C10 cells at passage 5; 9: IOSE‐C9 cells at passage 4; 10: IOSE‐C9 cells at passage 5.

Article Snippet: Telomerase activity was measured using the TRAPeze telomerase detection kit (Oncor Inc., Gaithersburg, MD, USA), following the manufacturer's protocol, for the radioactive assay (TRAP assay, telomere repeat amplification protocol).

Techniques: Subcloning, TRAP Assay, Activity Assay, Positive Control, Control

Journal: Cells

Article Title: hTERT Downregulation Attenuates Resistance to DOX, Impairs FAK-Mediated Adhesion, and Leads to Autophagy Induction in Breast Cancer Cells

doi: 10.3390/cells10040867

Figure Lengend Snippet: Analysis of human telomerase reverse transcriptase (hTERT) downregulation effect in MCF7 and MDA-MB-231 cells. ( A ) Representative images of GFP expression in the MCF7 and MDA-MB-231 cells transduced with pLV-THEM-shTERT (shRNA hTERT) and pLV-THEM-shRNA (shRNA Control); magnification 40×, scale bars 100 μm; ( B ) hTERT gene expression at the protein level, semi-quantitative analysis of Western blot results using Image Studio Lite software. GAPDH was used as loading control. ( C ) Relative hTERT gene expression at the transcriptional level measured by qPCR. ( D ) Relative telomerase activity determined by telomeric repeat amplification protocol (TRAP) assay. ( E ) Relative telomere length measured by qPCR. shRNA Control, cells transduced with lentiviral vectors containing scramble shRNA; shRNA hTERT, cells transduced with lentiviral vectors containing shRNA against hTERT. 0.1 μM doxorubicin (DOX); 8h treatment. Data are presented as the mean ± standard deviation. (*) the symbol for p < 0.05; (**) for p < 0.01, with comparison to shRNA Control.

Article Snippet: The effect of hTERT downregulation on telomerase activity was assessed using the quantitative TRAPEZE ® RT Telomerase Detection Kit (Merck Millipore, Darmstadt , Germany), according to the manufacturer’s instructions.

Techniques: Expressing, Transduction, shRNA, Western Blot, Software, Activity Assay, Amplification, TRAP Assay, Standard Deviation

Journal: Cells

Article Title: hTERT Downregulation Attenuates Resistance to DOX, Impairs FAK-Mediated Adhesion, and Leads to Autophagy Induction in Breast Cancer Cells

doi: 10.3390/cells10040867

Figure Lengend Snippet: Telomerase downregulation decreases the proliferation of breast cancer cells. ( A ) The cumulative population doublings was calculated for each 96 h ((log (total number of cells counted at the day of passage) − log (number of cells initially seeded at the previous passage))/log2). The monitoring period of proliferation potential is 9 weeks after transduction. The experiment was repeated three times. ( B ) Immunodetection of Ki-67, p53, p-p53, and p21 was performed using Western blot in MCF7 and MDA-MB-231 cells on the 21st-day. 0.1 μM DOX; 8h treatment, followed by densitometry analysis ( C ). (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 relative to shRNA Control. Densitometry analysis was performed out of three scanned membranes from 3 independent experiments. ( D ) Analysis of the biochemical-aging marker, the enzyme β-galactosidase (SA-β-Gal). Cells were subjected to hTERT downregulation, and the effect was assessed after 21 days from transduction. A typical result out of 2 replicates was demonstrated (arrows indicate green/β-galactosidase signal), magnification 100×, scale bars 100 μm.

Article Snippet: The effect of hTERT downregulation on telomerase activity was assessed using the quantitative TRAPEZE ® RT Telomerase Detection Kit (Merck Millipore, Darmstadt , Germany), according to the manufacturer’s instructions.

Techniques: Transduction, Immunodetection, Western Blot, shRNA, Marker

Journal: Human Molecular Genetics

Article Title: FANCC suppresses short telomere-initiated telomere sister chromatid exchange

doi: 10.1093/hmg/ddp556

Figure Lengend Snippet: Telomerase activity and Tert and Terc RNA levels in wild-type and Fancc−/− mouse bone marrow cells derived from C57BL/6 mice. qT-PCR analysis indicates a comparable telomerase activity (A), and Tert and Terc RNA levels (B) in wild-type and Fancc−/− mouse bone marrow cells (n = 6). Error bars represent the standard error obtained from different mice of each genotype. P-values are not significant between wild-type and Fancc−/− mouse bone marrow cells.

Article Snippet: Telomerase activity was measured by using Biomax Telomerase detection kit (Biomax, Inc., MD, USA) according to the manufacturer's recommendations.

Techniques: Activity Assay, Derivative Assay

Journal: Human Molecular Genetics

Article Title: FANCC suppresses short telomere-initiated telomere sister chromatid exchange

doi: 10.1093/hmg/ddp556

Figure Lengend Snippet: Telomerase activity in wild-type and Fancc−/− bone marrow cells after two serial bone marrow transplantations. qT-PCR analysis indicates a comparable telomerase activity in serially transplanted wild-type and Fancc−/− mouse bone marrow cells (n = 4). Error bars represent the standard error from different mice of each genotype.

Article Snippet: Telomerase activity was measured by using Biomax Telomerase detection kit (Biomax, Inc., MD, USA) according to the manufacturer's recommendations.

Techniques: Activity Assay

Journal: Cell Proliferation

Article Title: Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation

doi: 10.1111/j.1365-2184.2009.00589.x

Figure Lengend Snippet: Characteristics of the adult spermatogonial stem cell (SSC) line. (a) Characteristic example of Oct4 expression in an SSC clump. Green indicates Oct4 immunofluorescence, blue indicates DAPI immunofluorescence. (b) Most SSCs were negative for c‐kit (99 ± 1%), the red channel represents c‐kit immunofluorescence and the blue channel represents DAPI immunofluorescence. (c, d) Cytogenetic analysis by DAPI staining showed that these SSCs had a normal karyotype (40, XY). (e) Flow cytometry analysis of SSCs, DNA content was measured to show cell ploidy. Adult mouse testes were used as a reference (data not shown). (f) TRAP telomerase activity assay showed that the cells had high telomerase activity. M, DNA marker; lane 1, F9 cells (positive control); lane 2, SSCs; lane 3, STO (negative control). Data shown are representative of four experiments. Scale bars: 10 µm in (a), 25 µm in (b) and 6 µm in (c).

Article Snippet: Telomerase activity was tested using the TRAP‐cyber green stain Telomerase Detection Kit (GENMED, Boston, MA, USA) following the manufacturer's instructions ( 24 ).

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Telomerase Activity Assay, Activity Assay, Marker, Positive Control, Negative Control

Journal: Frontiers in Veterinary Science

Article Title: Telomerase Reverse Transcriptase (TERT) Expression, Telomerase Activity, and Expression of Matrix Metalloproteinases (MMP)-1/-2/-9 in Feline Oral Squamous Cell Carcinoma Cell Lines Associated With Felis catus Papillomavirus Type-2 Infection

doi: 10.3389/fvets.2020.00148

Figure Lengend Snippet: Expression of TERT, cMyc, and telomerase activity in SCCF2 and SCCF3 (A,B) TERT and cMyc gene expression in SCCF2 and SCCF3. Data are normalized for β2-microglobulin expression as housekeeping gene and expressed as relative quantization by the 2 −ΔΔCt method. Relative mRNA levels of TERT were lower while cMyc gene expression was higher in SCCF3 with respect to SCCF2 in at least three repeated, independent experiments. (C) A representative Western blotting (WB) gel showing lower TERT protein levels but higher cMyc amounts in SCCF3 vs. SCCF2 is illustrated. Hela whole cell lysate run along with feline samples confirmed the identity of the band. The blot was stripped and reprobed for tubulin to ensure comparable protein loading and allow normalization. Boxes are cut from the same gel at the same exposure time and properly aligned according to molecular standards loaded onto the gel. Full scans from original gels are shown in . (D,E) Mean densitometric values ± SD from at least three repeated, independent WB experiments, normalized for tubulin expression (statistically significant, * P < 0.05 and ** P < 0.01). (F) Telomerase activity was detected in SCCF2 and SCCF3 by telomeric repeat amplification protocol (TRAP) assay. Hela cell lysate was run along with feline samples as positive control. A representative gel out of four independent experiments, showing lower telomerase activity in SCCF3 vs. SCCF2 is illustrated (C–: negative control, sample with no lysate; L100bp: 100 base pairs DNA ladder, the first band from the bottom is 100 bp). (G) Quantification of telomerase activity in SCCF2 and SCCF3 by densitometric analysis expressed in % relative to SCCF2. Data were calculated as the ratio between TERT products ladder and 36 bp internal standard and represent the mean ± standard deviations (SD) of four repeated, independent experiments (statistically significant, ** P < 0.01).

Article Snippet: Cells were plated in six-well-plates at 1 × 10 5 density and harvested by trypsinization after 48 h. Telomerase activity in cells was assessed by a TRAP assay by using TRAPeze® Telomerase Detection Kit (Merck #S7700) following the manufacturer's protocol.

Techniques: Expressing, Activity Assay, Western Blot, Amplification, TRAP Assay, Positive Control, Negative Control