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. 2013 Feb 1;41(6):3734–3747. doi: 10.1093/nar/gkt047

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Effect of overexpressed ψPPM1K on cell growth and clonogenic activity in transfected Huh-7 cell clones. (a) HCC line Huh-7 and HepG2 cells were transfected with ψPPM1K-expressing recombinant plasmid to isolate stably transfected cell clones (TPG1, TPG2 and TPG7 for Huh-7; TPG for HepG2) by G418 selection, or with empty vector plasmid for G418-selected control mock2 cells (Vector). Equivalent amounts of total RNA from each cell clone were used for RT–PCR/gel electrophoresis analysis of ψPPM1K and GAPDH mRNA. (b) All three ψPPM1K-expressing cell lines have a slower proliferation rate than the vector control cell line (TPG7 cells: P = 0.036 for Day 2, P = 0.018 for Day 4; TPG1 cells: P = 0.045 for Day 4). (c) Serial photographs of the same colonies at Day 5, Day 7 and Day 9 showing the 2D growth of mock2, TPG1, TPG2 and TPG7 transfected Huh-7 clones on plastic culture dishes. (d) Clonogenic activity of mock2, TPG1, TPG2 and TPG7 was determined in soft-agar cultures incubated under normoxic 19% O2 and hypoxic 3% O2 conditions in 5% CO2 incubators for 12 days. Numbers of colonies formed per 1000 cells were counted for mock2, TPG1, TPG2 and TPG7 cell clones. Decreased clonogenicity compared with the mock2 control was calculated for TPG1 (P = 1.54E-06), for TPG2 (P = 0.0001) and for TPG7 (P = 0.013) in normoxic conditions; for TPG1 (P = 2.22E-05), for TPG2 (P = 5.71E-05) and for TPG7 (P = 0.0011) in hypoxic conditions. (*P < 0.05).