Hypoxic induction of LDH in P19 cells. (A) LDH activity; (B) LDHA mRNA. In (A), P19 cells and Hep3B cells were cultured in 21% and 2% oxygen for 24 hours and cellular LDH activity was determined. LDH activity (Wro units of the LDH assay kit) per milligram of cellular protein is shown. Hypoxic inducibility (activity ratios in 2% and 21% oxygen) is indicated. Each value is the mean ± SD of triplicate experiments. In (B), P19 cells were cultured in 21%, 5%, and 2% oxygen. LDHA mRNA in total RNA was amplified by RT-PCR, varying the PCR cycle number (22, 23, and 24 cycles). LDHA mRNA-derived product of 353 bp is shown. β-Actin mRNA was also amplified to use for normalization of efficiency in RT (see the Materials and Methods). Normalized band intensities relative to that of the product amplified (22 cycle) from LDHA mRNA of the cells cultured in 21% oxygen were plotted against PCR cycle number. (•) 21% oxygen; (▪) 5% oxygen; (▴) 2% oxygen.