Fig. 8

HEK293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 h and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 h. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysates from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analyzed by SDS–PAGE, and subjected to Western blot using antibodies against Flag and S6. Upper and lower rows of Flag- and S6 blots were Endouc- and EndoucK242A- expressing cells, respectively. B, C. The lysates from control (pCS2), Endouc-expressing (Endouc), and EndoucK242A-expressing (EndoucK242A) cells were applied to PPA. The resulting fractions from 1-10 collected from sucrose gradient were subsequently subjected to RT–qPCR assay to quantify (B) exogenous huORFchop-tagged luc (luc mRNA) and (C) endogenous CHOP transcripts (CHOP mRNA). The distribution of relative abundance of luc and CHOP transcripts contained in each fraction was determined. D, E. HEK293T cells were co-transfected with pCS2-Endouc and phuORFchop-luc for 24 h and then subjected to DMSO or TH treatment for 2 h. The lysates of transfected cells were then applied to PPA. Total RNAs were isolated from heavy polysome fractions followed by performing 5′RACE to characterize the nucleotide sequences of truncated (D) exogenous huORFchop-luc and (E) endogenous huORFchop-CHOP transcripts. The first sequence at N-terminus of RNA samples from the truncated forms derived from exogenous huORFchop-luc (n = 14) and endogenous huORFchop-CHOP transcripts (n = 11) was determined; the percentages of positive colonies containing various 5′UTR lengths (truncated forms) of luc and CHOP transcripts among total colonies examined were calculated.