Figure 7. HDAC8 and p300/CBP are involved in determining susceptibility to LeTx in BMDMs as in RAW264.7 cells.

BMDMs from 129/S1/Svlmj mice were treated with a sub-lethal dose of LeTx (100 ng/ml LF and 100 ng/ml PA) for 24 h. Surviving cells were then re-plated onto new dishes or 96-well plates with fresh media and used as TIR BMDMs. (A) Wild-type and TIR BMDMs were harvested for Western blot analysis for MEK1 and p38 (upper panel), or treated with a lethal dose of LeTx (250 ng/ml LF and 500 ng/ml PA) for 5 h. (B) TIR BMDMs were transfected with scramble (si-scramble) or Caspase-1 targeting (si-Casp-1) siRNAs for 16 h, followed by PN or none for another 9 h. These cells were then treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 5 h. (C) TIR BMDMs were treated with none or panobinostat (PN; 50 nM) for 9 h and expression of BNIP3 and BNIP3L was analyzed by real-time qPCR. (D) TIR BMDMs were treated with scramble (si-scramble) or HDAC8 targeting (si-HDAC8) with or without caspase-1 (si-Casp-1) siRNAs for 24 h. These cells or wild-type BMDMs treated with si-scrample were exposed to LeTx (250 ng/ml LF and 500 ng/ml PA) for 5 h. (E) BMDMs were pre-exposed to LeTx with a sublethal dose of LeTx (100 ng/ml LF and 100 ng/ml PA) for 24 h, and surviving TIR cells were then transfected with scramble (si-scramble) or HDAC8 targeting (si-HDAC8) siRNAs for 24 h. Expression of BNIP3, BNIP3L and MLN64 was analyzed by RT-qPCR, and levels of mRNAs were expressed as fold of change relative to those of wild-type BMDMs (dotted line). (F) Wild-type BMDMs were treated with si-scramble or p300 (si-p300), CBP (si-CBP) or PCAF (si-PCAF) targeting siRNAs for 24 h, and then exposed to LeTx (250 ng/ml LF and 500 ng/ml PA) for 5 h. Cell death was measured by MTT assays and the extent of cell death was expressed as % of cell death relative to that of non-treated (A, B) or wild-type BMDMs treated with si-scramble but without LeTx treatments (D, F). Data are expressed as means ± SD (n=3), * P< 0.05; ** P < 0.01 (Student’s t-test).