American Society of Hematology

Fig. 3.

$Fig. 3. Histone acetylation by HDACIs in HL60 cells. / (A) HL60 cells were incubated with or without 1.0 mmol/L SB for 6 hours. Each histone fraction was extracted and analyzed on an AUT gel. The gel was stained with Coomassie blue. (B) Histone hyperacetylation by HDACIs was detected in situ using antiacetylated histone H4 antibody. HL60 cells were incubated for 3 hours with either 1.0 mmol/L SB or 100 ng/mL TSA and subjected to immunostaining. In comparison with the control, almost all the cells were stained positively after 3 hours of treatment with both SB and TSA. (C) Soluble chromatin preparations from cell cultures treated with or without SB were immunoprecipitated with the antibody against acetylated histone H3 or H4. Precipitated DNA was quantitated by real-time PCR using SYBR Green Master Mix. On the other hand, aliquots of chromatin solution were directly subjected to DNA extraction followed by real-time PCR, and these were used as input DNA. The ratio of the DNA amount at the indicated time relative to the corresponding input DNA amount was calculated. Finally, the relative amount of each precipitated DNA normalized by input DNA was shown. (D) The PCR products with 27 cycles of conventional PCR using the same primers and DNA template were visualized with ethidium bromide staining. No-Ab indicates that antibodies were not added for ChIP.$

Histone acetylation by HDACIs in HL60 cells.

(A) HL60 cells were incubated with or without 1.0 mmol/L SB for 6 hours. Each histone fraction was extracted and analyzed on an AUT gel. The gel was stained with Coomassie blue. (B) Histone hyperacetylation by HDACIs was detected in situ using antiacetylated histone H4 antibody. HL60 cells were incubated for 3 hours with either 1.0 mmol/L SB or 100 ng/mL TSA and subjected to immunostaining. In comparison with the control, almost all the cells were stained positively after 3 hours of treatment with both SB and TSA. (C) Soluble chromatin preparations from cell cultures treated with or without SB were immunoprecipitated with the antibody against acetylated histone H3 or H4. Precipitated DNA was quantitated by real-time PCR using SYBR Green Master Mix. On the other hand, aliquots of chromatin solution were directly subjected to DNA extraction followed by real-time PCR, and these were used as input DNA. The ratio of the DNA amount at the indicated time relative to the corresponding input DNA amount was calculated. Finally, the relative amount of each precipitated DNA normalized by input DNA was shown. (D) The PCR products with 27 cycles of conventional PCR using the same primers and DNA template were visualized with ethidium bromide staining. No-Ab indicates that antibodies were not added for ChIP.

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