Figure 6. SAHA upregulates mRNA transgene level by inhibiting
histone deacetylation.
SAHA’s effect on mRNA transgene levels and histone acetylation levels
were verified by RT-qPCR and western-blot respectively. Flask liquid
cultures of clones 16, 17, 18, 20, 21 and WT treated with 5 µM of SAHA
and grown for 12 days were all tested at day 6. A. RT-qPCR bar graph
result of relative mVenus mRNA expression normalized on histoneh3 in clones 16, 17, 18, 20, 21 and WT treated with SAHA and DMSO
(negative control). B. Immunoblot analyses to determine the levels of
mVenus (27 kDa upper panel), histone H3 Lysine 9 acetylated (H3K9ac,
15.4 kDa, below), histone 3 (H3, middle panel and actin (40 kDa, lower
panel) in clones 16, 17, 18, 20, 21 and WT treated with SAHA and DMSO
(negative control). Total soluble protein was separated by denaturing
12% SDS-PAGE, blotted and probed with specific antibodies as indicated.
For mVenus and H3K9ac detection, 50 µg and 25 µg of total soluble
proteins were loaded respectively. Purified mVenus was used as control.
The blot, from the upper and lower panels, was detected with
anti-mVenus, following by anti-actin detection. Middle panels blot was
first incubated with anti-H3K9ac and then anti-H3. Molecular weights
were deduced from co-migrating protein markers.