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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Mol Cancer Ther. 2018 Feb 26;17(6):1280–1290. doi: 10.1158/1535-7163.MCT-17-0603

Figure 1. BRAFMT CRC cells are dependent on the UPR for survival.

Figure 1

A. Results of the siRNA screen. BRAFMT cells were reverse transfected with all stars’ non-targeted siRNA sequence and sequence specific siRNA. Cell viability was calculated following 72h transfection using the CellTitre-Glo™ assay. Data for siHSPA5 are shown. Western blotting (WB) for GRP78 and PARP following siHSPA5 (HA) for 48h. Full siRNA screen data can be found in Table S4. B. Left: PARP, pEIF2αS51, EIF2α, ATF4, CHOP, ATF6, GRP78, pIRE1αS724 and IRE1α expression in VACO432 and VT1 cells, following treatment with HA15 for the indicated time. Right upper: Caspase-3/7 activity levels in VACO432 cells, following treatment with HA15. Right lower: VACO432 cells were transfected with scrambled control (SC), caspase-8 (siC8) or caspase-9 siRNA (siC9) for 24h and thereafter treated with HA15 for 24h. Cleaved-PARP, caspase-3, pro-caspase-8 and caspase-9 were determined by WB. C. Upper: PARP, BCL-2, BCL-XL, MCL-1, PUMA, NOXA, BID and BIM levels in BRAFMT VACO432, HT-29 and BRAFWT VT1 cells following treatment with HA15 for the indicated time. Lower left: CRC cells were treated with HA15 for 24h and DR5 cell membrane expression assessed by flow cytometry using receptor-specific phycoerythrin-conjugated mAbs. Expression was compared with a nonspecific isotype-matched control antibody. Lower right: MTT cell viability of BRAFMT cells following 72h treatment with HA15. D. Upper: VACO432 cells were transfected with SC or DDIT3 (siCH) siRNA for 24h and thereafter treated with HA15 for 24h. Cleaved-PARP, caspase-3, CHOP, PUMA, BCL-2 and MCL-1 expression were determined by WB. Lower: MTT cell viability of BRAFMT cells transfected with siCH and co-treated with HA15 for 72h. SE=short exposure; LE=long exposure.