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1.
FIG. 5

FIG. 5. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

Induction of hTERT expression by E2. LEA (a, b, e, and f) and LLO (c, d, g, and h) cells were grown in the absence (a to d) or presence (e to h) of E2 and stained with anti-hTERT antibody K-370 (a, c, e, and g) or with Hoechst 33258 (b, d, f, and h). Uninduced cells (a and c) did not express hTERT, while treatment with the hormone for 6 h resulted in abundant nuclear accumulation of the protein (e and g). Magnification, ×85.

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.
2.
FIG. 4

FIG. 4. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

Expression of hTERT mRNA in HOSE cells upon estrogen treatment. Total RNA was extracted from LEA (lanes 1 and 2) and LLO (lanes 3 and 4) cells grown in the presence (+) or absence (−) of 10−7 M E2 for 6 h. RT-PCR analysis was performed using primers specific for the hTERT and housekeeping aldolase genes and the conditions described in Materials and Methods. Lane 5, HeLa cell RNA as a control; lane 6, no cDNA template. Positions of molecular size markers are indicated.

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.
3.
FIG. 6

FIG. 6. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

Telomerase activity in response to E2 treatment. Telomerase activity was assayed by TRAP in extracts from GRO (lanes 3 and 4), LEA (lanes 7 to 10), LLO (lanes 11 to 14), and WOO (lanes 15 to 18) cells. Assays shown were performed with 5 μg of protein, except in the case of WOO cells (10 μg of protein; similar results were obtained with 1 μg of protein). Cells were grown in the presence of E2 at 10−7 M for the indicated times. As positive and negative controls, 0.1 μg of protein from telomerase-positive HeLa cells was assayed before and after heat inactivation (no E2) (lanes 2 and 1, respectively).

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.
4.
FIG. 3

FIG. 3. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

Effects of E2 and ERs on hTERT promoter activity. (a and b) NIH 3T3 or WOO cells, grown in the presence or absence of 10−7 M E2, were cotransfected with 5 μg of the hTERT promoter-luciferase reporter plasmids (P-1009, P-1009Mut, and P-330) or the control vector pGL2-Enhancer (pGL2), 5 μg of the ERα expression vector, and 250 ng of pCMV-βgal. Cells were assayed for luciferase and β-galactosidase activities after 48 to 72 h. Data are expressed as light units/β-galactosidase units in the presence (+) or absence (−) of hormone. Results represent the average (± standard error [SE]) of a minimum of three independent experiments, each performed in duplicate. (c) NIH 3T3 cells were transfected with P-1009, alone (− ER) or in combination with expression vectors for ERα (+ ERα) or ERβ (+ ERβ), in the presence of E2 or TAM. The VIT promoter (nucleotides −596 to +8), containing a perfect ERE, was used as a control reporter (VIT). Results represent the average (± SE) of three independent experiments, each performed in duplicate, and values are expressed as fold induction (ratio with and without ligand). (d) NIH 3T3 cells were cotransfected with (+ ERα) or without (− ERα) the expression vector for ERα and the hTERT-ERE-TK and TK reporters as indicated and cultured in the absence or presence of E2. Results of a representative experiment out of two, each performed in triplicate, are expressed as fold induction as described for panel c.

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.
5.
FIG. 2

FIG. 2. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

(a) Expression of endogenous ERα by Western blot analysis. ERα-negative (HeLa and MDA-MB231) and ERα-positive (OVCA-433 and MCF-7) cells were lysed directly in protein sample buffer, and equal amounts of protein were separated on a denaturing 12% polyacrylamide gel. Immunostaining was performed with anti-ERα antibody HC-20. As a loading control, proteins were stained with Ponceau S (data not shown). Treatment with E2 did not affect the levels of ERα in the ER-positive cells (data not shown). (b to e) DMS genomic footprinting of the hTERT promoter. Cells were treated with the DNA-alkylating reagent DMS, and their DNA was cleaved with piperidine and analyzed by LM PCR with primers specific for the region of the hTERT promoter from bp −1025 to −917 relative to the ATG (Fig. 1a). (b) Breast cancer MCF-7 cells. (c) Breast cancer MDA-MB231 cells. (d) Ovarian cancer OVCA-433 cells. (e) Cervical cancer HeLa cells. Length (in hours) of treatment with E2 (lanes 2, 3, 5 to 9, and 11 to 13) is indicated. In vitro-methylated DNA from each cell line is shown in lanes 1, 4, 10, and 14. Protected guanine residues over the ERE region are indicated by open arrows, while relevant hypermethylated guanine residues are indicated by filled arrows (b and d). Corresponding G residues unmodified in ERα-negative cells are indicated by arrowheads (c and e). The asterisks (in panel d) indicate two protected guanine residues, of unknown significance, downstream of the ERE region.

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.
6.
FIG. 1

FIG. 1. From: Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells.

(a) Schematic diagram and nucleotide sequence of the hTERT gene 5′-flanking sequences. The region extending to bp 1009 upstream of the hTERT ATG (+1) and the locations of the two ERE half-sites and of an additional downstream half-site (black triangles) are indicated. The hTERT promoter sequence between bp −1009 and −755 is shown below the diagram. The boxes define the composite regulatory unit comprising an imperfect palindromic ERE at positions −949 to −935, a partially overlapping AP1 binding site, an adjacent SP1 motif, and the single ERE half-site at positions −794 to −789. The asterisks indicate G residues altered in the genomic footprints shown in Fig. 2. (b) ERα binding to the hTERT ERE. A 32P-labeled double-stranded oligonucleotide containing the hTERT ERE sequence was incubated with extracts of Sf9 cells infected with wild-type (wt) baculovirus (lane 2) or recombinant baculovirus expressing human ERα (lanes 3 to 10). Lane 1, probe alone; lane 3, recombinant ERα alone; lanes 4 to 9, like lane 3 but with 25-, 100-, and 250-fold molar excesses of unlabeled oligonucleotides containing the hTERT (lanes 4 to 6) or FXII (lanes 7 to 9) ERE sequences; lane 10, like lane 3 but with a 250-fold molar excess of an unrelated unlabeled oligonucleotide (NS). (c) ERβ binding to EREs. 32P-labeled double-stranded oligonucleotides containing the VIT ERE (lanes 1 to 4) or the hTERT ERE (lanes 5 to 8) were incubated with extracts of Sf9 cells infected with recombinant baculovirus expressing human ERβ (lanes 2 to 4 and 6 to 8) in the presence of (E2) (lanes 2, 4, 6, and 8) or of TAM (lanes 3 and 7). Anti-ERβ antibodies (lanes 4 and 8) were used for supershifting ERβ-ERE complexes. Lane 1, VIT ERE probe alone; lane 5, hTERT ERE probe alone.

Silvia Misiti, et al. Mol Cell Biol. 2000 June;20(11):3764-3771.

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