hTERT overexpression in HeLa and U2OS cell lines.

<p>hTERT is overexpressed in U2OS and HeLa cell lines. (A & D) mRNA level of hTERT in U2OS & HeLa cell lines was determined by quantitative real-time PCR. (B & E) Western blotting confirms overexpression of hTERT in U2OS and HeLa cell line. (C & F) Histograms depict densitometric quantification of the hTERT overexpression of three corresponding independent Western blot experiments in U2OS and HeLa cell lines.</p

List of proteins differentially expressed following hTERT overexpression in HeLa cells.

<p>List of proteins differentially expressed following hTERT overexpression in HeLa cells.</p

Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

<div><p>Reverse transcriptase activity of telomerase adds telomeric repeat sequences at extreme ends of the newly replicated chromosome in actively dividing cells. Telomerase expression is not detected in terminally differentiated cells but is noticeable in 90% of the cancer cells. hTERT (human telomerase reverse transcriptase) expression seems to promote invasiveness of cancer cells. We here present proteomic profiles of cells overexpressing or knocked down for hTERT. This study also attempts to find out the potential interacting partners of hTERT in cancer cell lines. Two-dimensional gel electrophoresis (2-DE) of two different cell lines U2OS (a naturally hTERT negative cell line) and HeLa revealed differential expression of proteins in hTERT over-expressing cells. In U2OS cell line 28 spots were picked among which 23 spots represented upregulated and 5 represented down regulated proteins. In HeLa cells 21 were upregulated and 2 were down regulated out of 23 selected spots under otherwise identical experimental conditions. Some heat shock proteins viz. Hsp60 and Hsp70 and GAPDH, which is a housekeeping gene, were found similarly upregulated in both the cell lines. The upregulation of these proteins were further confirmed at RNA and protein level by real-time PCR and western blotting respectively.</p></div

Primers used for PCR-based assays.

<p>Primers used for PCR-based assays.</p

List of proteins differentially expressed following hTERT overexpression in U2OS cells.

<p>List of proteins differentially expressed following hTERT overexpression in U2OS cells.</p

hTERT overexpression enhances the migration of cancer cells.

<p>A) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in U2OS cells. B) Histogram represents quantification of the effect of hTERT overexpression on cell mobility (% migration) in U2OS cell line. C) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in HeLa cells. D) Histogram representing quantification of the effect of hTERT overexpression on cell mobility (% migration) in HeLa cell line.</p

Two dimensional gel electrophoresis of hTERT overexpressing (A) U2OS and (B) HeLa cell line.

<p>Total proteins were extracted from hTERT overexpressing U2OS and HeLa cells and separated non-linearly on IPG strip of PH 3–10, followed by electrophoresis through 12% polyacrylamide gels. The gels were further stained and analyzed by image master 2D platinum software.</p

Validation of upregulation of heat shock proteins in U2OS and HeLa cell lines.

<p>The hTERT induced upregulation of Hsp60 and hsp70 were confirmed by qRT-PCR in (A) U2OS and (B) HeLa cell lines respectively. C) Western blotting showing upregulation of Hsp60 and Hsp70 at protein level. D) Histogram shows the results by applying ImageJ software. D) To confirm the upregulation of Hsp90 in U2OS cell line E) QRT-PCR and F) western blotting is performed.</p

Two-dimensional gel electrophoresis of rat kidney lysate; Coomassie blue-stained gels from control (a) and MSG-treated rats (b).

<p>Proteins were resolved on 7 cm pH 3–11 IEF strips (NL) followed by SDS-PAGE (12%). The differentially expressed spots detected by the image Master 2D Platinum 7.0 software are circled. The gels shown are representative of three independent experiments.</p

Proposed model of MSG-induced ROS production in rat kidney.

<p>The higher level of glutamate on chronic MSG intake accelerates TCA cycle. The increased level of α-KGDH may stimulate ROS production hence oxidative stress occurs in the kidney of the MSG-treated rats.</p