ER stress in CLL cells. (A) Confocal fluorescence microscopy for ATF6. Z stacks were collected (0.4 μm per slice), and images were chosen from the middle of nuclei. Side views (across bottom and side of figures) are also shown to depict ATF6 nuclear localization in CLL cells treated with flavopiridol (2μM) or thapsigargin (1μM) for 4 hours. Results shown are representative of 4 experiments. (B) ChIP assay data showing enrichment of ATF6 at the GRP78 promoter region with flavopiridol and thapsigargin treatment in 697 cells. Values of enrichment at promoter are presented relative to the IgG negative control. (C) ChIP assay data showing enrichment of ATF6 at GRP78 promoter region in cells from CLL patients undergoing flavopiridol treatment, collected before or at the end of flavopiridol infusion (4.5 hours). (D-E) Coimmunoprecipitation assay for IRE1-ASK1-TRAF2 complex formation 697 cells (D) or samples from flavopiridol-treated CLL patients (E) were immunoprecipitated with TRAF2 and immunoblotted for ASK1 or IRE1. (F) Immunoblots for total and phosphorylated JNK1 and p38MAPK in CLL cells treated in vitro with flavopiridol (2μM), thapsigargin (1μM), F-ara-A (5μM), and rapamycin (5μM). Results shown are from one representative patient sample. (G) Immunoblots for total and phosphorylated JNK1 and p38MAPK in samples from 6 CLL patients treated with flavopiridol; samples were collected before treatment and at 4.5 hours into flavopiridol infusion.