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Fig. 1 | BMC Cancer

Fig. 1

From: PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment

Fig. 1

The ectopic expression of hPARG in 3 T3 cells does not affect their viability in vitro. A Schematic of the 13.5 kb lentiviral cassette pLVX-TetONE-hPARG-IRES-mClover3-NLS-Puro co-expressing hPARG and nucleus-targeted fluorescent protein mClover3 under control of the TetON promoter. The cassette was used to generate a 3 T3 cell line which stably expresses hPARG in the presence of doxycycline (3 T3-hPARG). The Puromycin-resistant gene (Puro) was used as a selection marker. The cells were split into two test groups, one of which received 500 ng/ml doxycycline (+) and the other receiving no doxycycline (−). These cells were then subjected to the assays indicated. B Rabbit monoclonal anti-hPARG and mouse anti-pADPr antibodies were used to detect the hPARG polypeptide and pADPr moieties, respectively, in 3 T3-hPARG cells that were cultured either in the absence (−) or in the presence (+) of doxycycline. Staining with anti-tubulin antibodies was used as a loading control (Tubulin). The uniform expression of hPARG in cultured cells and the corresponding reduction in pADPr was confirmed by immunofluorescence (Supplemental Fig. S4). Uncropped version of Western Blot is presented at Supplemental Fig. S5. C Doubling time assay comparing 3 T3-hPARG cell cultures grown either in the presence (‘With Dox’; red line) or in the absence (‘No Dox’; blue line) of doxycycline with regard to the number of cells produced over the indicated time periods. The two treatment conditions showed a similar doubling time (26 ± 7 hours). D Clonogenic assay comparing # of clones (≥50 cells/each) formed by single 3 T3-hPARG cells over 14 days either after doxycycline induction (+DOX) or in the absence of doxycycline (−DOX) (n = 3 for each group; error bars represent SEM). E Wound healing assay shows the ability of cultured 3 T3-hPARG cells to restore the damaged monolayer in the presence (DOX) and in the absence of doxycycline (No DOX). F 3 T3-hPARG wound healing assay quantified as % of wound closure (n = 5; the difference is not statistically significant (t-test p value = 0.2076). Error bars represent SEM

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