Microson ultrasonic cell disruptor

The treatment with 25 mg/kg bw PQ did not cause difference in the bw gain, hematological parameters and clinic chemistry. The lung sample from each rat was ground in liquid N₂ using a mortar and pestle. One hundred mg of the powdered lung tissues (wet weight) was extracted with 1.75 mL of a mixture of 1 mL lysis buffer and 0.75 mL protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) under ultrasonication (Microson^™ ultrasonic cell disruptor, Misonix, Farmingdale, NY) at speed level 1 for 15 min at ambient temperature. The lysis buffer contained 40 mM tris-HCl, pH 7.4, 5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride. The homogenate was then centrifuged at 17,000 g for 30 min. The supernatant was filtered through a membrane Econofilter (0.2 mm×25 μm, Agilent, Palo Alto, CA) and then used for protein analysis. Protein concentrations were determined with Coomassie Plus^™ protein assay kit (Pierce, Rockford, IL).

ChIP assays were carried out using EZ ChIP kit (Millipore) according to the manufacturer's protocol with some modifications. 293T cells were treated with Tm or Tg prior to cross-linking. DNA fragments at around 200-1000 bp were achieved by sonication with Microson Ultrasonic Cell Disruptor (Misonix). For IP, the indicated antibodies (i.e. anti-XBP-1 or anti-PCAF antibodies) were added to the sheared chromatin individually and incubated at 4°C overnight. The DNA/protein/antibody complex was then pulled down by protein G agarose and the DNA in the complex was purified using QIAquick PCR purification kit (Qiagen). Quantitative-PCR was performed to determine the relative amount of DNA that was immunoprecipitated by anti-XBP-1 or anti-PCAF antibodies in the presence of Tm or Tg. The primer pairs used to amplify the promoter regions of BiP, CHOP and EDEM genes include: BiP (5′-GATGGGGCGGATGTTATCTA-3′ and 5′-CTCTCACACTCGCGAAACAC-3′), CHOP (5′-GACACTACGTCGACCCCCTA-3′ and 5′-GGTTCCAGCTCTGATTTTGG-3′), and EDEM (Epitect ChIP qPCR primers, Qiagen). Cells treated with DMSO served as a negative control. For overexpression, MCF7 cells were co-transfected with the indicated expression vectors two days prior to cross-linking, followed by ChIP-quantitative-PCR as described earlier.

Tissue was homogenized in ten volumes radioimmunoprecipitation assay (RIPA) buffer (Boston Biotechnologies) with 1X HALT protease inhibitor (Thermo Fisher Scientific). The tissue was sonicated on ice three times for 10 seconds each with one minute between pulses at speed 2 with a Microson Ultrasonic Cell Disruptor (Misonix). The homogenate was incubated on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4 °C and the supernatant transferred to a new tube. If the supernatant still appeared cloudy after centrifugation, the previous step was repeated. Protein concentration was determined with the BioRad Protein Assay. For immature (P7-P30) testes, tissue was homogenized in a glass tissue grinder (Wheaton), then processed the same as above, omitting the sonication step.