(A) Six genes from the hMitChip3 cDNA microarray results were chosen to be re-evaluated using qRT-PCR and the resultant fold changes compared to those from the microarray. Genes were selected to represent a range of fold changes from within the significance limits, 3 up-regulated (RHOT2, PCK2, and GLUD1) and 3 down-regulated (LDHA, OGDH and MDH2). The hMitChip3 cDNA microarray fold change is shown for each gene/transcript (up-regulated in red and down-regulated in green) and the graphs show the average real time RT-PCR fold change (delta CT) of UOK268 relative to HK-2. Student t-tests were used to produce p-values.
(B) Western Blot analysis of the levels of PCK2, HK1, HADHA and FH protein present in the lysates of UOK268, HK-2, UOK262 and 262FH+ (re-expressing wild-type fumarate hydrase) cells. The expression levels of β–ACTIN were used as a loading control.
(C) Quantative RT-PCR results comparing the mRNA levels of PCK2 and HK1 genes between UOK268, HK-2, UOK262 and 262FH+.