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. 2017 Sep 19;12(9):e0185089. doi: 10.1371/journal.pone.0185089

Fig 1. Rapamycin and temsirolimus induce Xbp-1 splicing and downstream target gene mRNA levels.

Fig 1

(A) Human osteosarcoma cells (HOS-MNNG) were treated for the indicated times with 2, 5 or 20μM temsirolimus. RT-PCR was conducted using a primer pair flanking the unconventional splice site of the Xbp-1 mRNA. (B) Quantitative RT-PCR was conducted using primer pairs specific for the Xbp-1 target genes, Grp78 and CHOP. Expression values for each transcript were normalized to 18S and vehicle treated control. Experiments were carried out on three independent biological replicates, assayed in triplicate. One-way ANOVA was performed for statistical analysis and asterisk denotes p = <0.05. (C) 143B human osteosarcoma cells were treated with vehicle or 20μM rapamycin and analyzed for Xbp1 splicing (upper panel) and downstream target gene activation of BiP/Grp78 and CHOP at the indicated time points as in (A and B). (D) Cells were treated for the indicated times with either 20μM temsirolimus or an ATP competitive inhibitor of mTOR. Total RNA was harvested and Xbp-1 splicing was analyzed by RT-PCR as in (A).