Supplementary Figure 5. (a) KAT6B especifically acetylates lysine 23 of histone H3 in vitro. Recombinant GST-Flag-tagged KAT6B HAT domains (wild type,

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Supplementary Figure 5. (a) KAT6B especifically acetylates lysine 23 of histone H3 in vitro. Recombinant GST-Flag-tagged KAT6B HAT domains (wild type, and K815L mutant) were assayed for the presence of histone acetyltransferase activity (left-top). Core histones used as substrates were stained with Comassie blue (left-midle). Flag-tagged wild-type and catalytically inactive HAT domains were analyzed in western blot (left-bottom). Recombinant GST-Flag-tagged KAT6B HAT were assayed in western blot for the presence of specific histone H3K23 acetyltransferase activity (right-top). Histone H3 from core histones used as substrates was analyzed in western blot (righ-middle). Flag-tag HAT domains were analyzed in western blot (right-bottom). (b) Recombinant GST-Flag-tagged KAT6B HAT domains were assayed for the presence of histone acetyltransferase activity on recombinant histone H3 (top). Recombinant histone H3 was stained with Comassie blue (midle) and GST-Flag-tagged KAT6B HAT domains were analyzed in western blots (bottom). (c) The expression of HDAC1, 2, 3 and 6 was diminished using specific shRNAs targeting each protein in NCI-N417 cells. Western blot analysis of each HDAC and the acetylation of H3K23 revealed that HDAC1 is involved in H3K23 deacetylation. (d) Quantitative reverse-transcription PCR results confirmed the microarray expression data: twenty one genes (in addition to the four genes shown in Fig. 3f) presented significantly less expression in KAT6B shRNA-depleted cells than in scramble cells. Significance of permutation tests: *, p <0.05; **, p < 0.01; ***, p < a b H3K23ac Anti-Flag KAT6B-HAT Total H3 KAT6B- HAT-K815L Coomassie blue [acetyl C]- coenzyme A Anti-Flag KAT6B-HAT KAT6B- HAT-K815L Coomassie blue KAT6B-HAT KAT6B- HAT-K815L [acetyl C]- coenzyme A Anti-Flag NCI-N417 shRNA_3 H3K23ac Lamin B1 HDAC3 NCI-N417 Scramble NCI-N417 shRNA_2a NCI-N417 shRNA_2b H3K23ac Lamin B1 HDAC2 NCI-N417 Scramble H3K23ac Lamin B1 HDAC6 NCI-N417 shRNA_6 NCI-N417 Scramble NCI-N417 shRNA_1a NCI-N417 shRNA_1b H3K23ac Lamin B1 HDAC1 NCI-N417 Scramble c d N417 Scramble N417 shRNA PIAS4 * N417 Scramble N417 shRNA JHDM1D ** N417 Scramble N417 shRNA BNIP3L ** N417 Scramble N417 shRNA ADAMTS15 ** N417 Scramble N417 shRNA ITPRIP ** CPS N417 Scramble N417 shRNA ** N417 Scramble N417 shRNA CNIH3 *** N417 Scramble N417 shRNA CDKL4 ** N417 Scramble N417 shRNA PPAPDC3 ** N417 Scramble N417 shRNA ICK ** N417 Scramble N417 shRNA CACNA1B ** N417 Scramble N417 shRNA CMA1 *** N417 Scramble N417 shRNA MIAT * N417 Scramble N417 shRNA MRPS10 ** N417 Scramble N417 shRNA YRDC ** N417 Scramble N417 shRNA SLC38A10 ** N417 Scramble N417 shRNA PVRL2 *** N417 Scramble N417 shRNA SEC61A2 ** N417 Scramble N417 shRNA ARL8A ** N417 Scramble N417 shRNA GNAS-AS1 *** ADAMTS N417 Scramble N417 shRNA ** Relative expression