Extended Data Figure 3: Testing fidelity of FabLEM for detecting RNAP2 and histone modifications.
From: Regulation of RNA polymerase II activation by histone acetylation in single living cells
a–e, Representative FabLEM experiments in single living cells that were either untreated (a, b) or treated with inhibitors (c–e). Scale bars 5 μm. c, Cells were treated with 1 μM flavopiridol for 1 h and then activated with hormone (time roughly corresponds to post-activation). Flavopiridol inhibits P-TEFb, which phosphorylates the RNAP2 CTD at Ser 2, preventing elongation at the gene array (marked by yellow arrow). FabLEM experiments confirm this, showing no array decondensation and no accumulation of Ser 2ph Fab (red) at the array upon hormone treatment even though Ser 5ph (purple) does accumulate, indicating RNAP2 initiation (although to a lesser extent than in untreated cells). d, The same experiment as above, but now examining histone acetylation levels at the array (blue, H3K27ac), which no longer go down post-activation, as in untreated cells. e, Cells were treated with 100 nM of the histone deacetylase inhibitor trichostatin A (TSA) for 1 h. As with flavopiridol treatment, the array no longer decondenses, H3K27ac levels remain high (and in fact the total nuclear intensity is higher, indicating global increases in H3K27ac levels) and levels of RNAP2 initiation are low, indicating little or no RNAP2.
