PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription

Lew, Qiao Jing; Chu, Kai Ling; Lee, Jialing; Koh, Poh Ling; Rajasegaran, Vikneswari; Teo, Jin Yuan; Chao, Sheng-Hao

The UPR inducing compounds, tunicamycin (Tm) (Assay Designs) and thapsigargin (Tg) (Sigma), were dissolved in dimethyl sulfoxide (DMSO) to 10 mg/ml and 3 mM, respectively. All three cell lines, HEK293, 293T and MCF7, exhibited UPR after treating with Tm or Tg. Induction of the UPR genes in the treated cells were confirmed by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) (data not shown). Among the cell lines used in this study, the endogenous XBP-1S target genes in MCF7 cells showed the highest sensitivity to the ectopic expression of XBP-1S (data not shown). Therefore, MCF7 cells were selected for the XBP-1S overexpression experiments followed by the examination of the transcriptional regulation of XBP-1S-dependent genes in vivo. Total RNAs of the transfected MCF7 cells or the Tm (10 mg/ml)/ Tg (300 nM) treated HEK293 cells were isolated using RNeasy mini kit (Qiagen). One microgram of the total RNAs was converted into complementary DNA (cDNA) using ImProm TM -II Reverse Transcription System (Promega). Specific cDNAs were amplified using SYBR Green PCR Master Mix (Applied Biosystems). The primer pairs used in this study include: BiP (5 0 -GGTGAAAGACCCCTGACAAA-3 0 and 5 0 -GTCAGG CGATTCTGGTCATT-3 0 ), CHOP (5 0 -CTTCTCTGG CTTGGCTGACT-3 0 and 5 0 -CCCTTGGTCTTCCTCCT CTT-3 0 ), EDEM (5 0 -AGGTGCTGATAGGAGATG TGG-3 0 and 5 0 -GGATTCTTGGTTGCCTGGTA-3 0 ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5 0 -AACAGCCTCAAGATCATCAGC-3 0 and 5 0 -GGAT GATGTTCTGGAGGACC-3 0 ). GAPDH was used as a control to normalize the cDNA inputs. Amplification and detection of the cDNAs were performed using ABI Prism 7000 Thermal-Cycler (Applied Biosystems).