Custom sirnas

Transfection of siRNAs was performed as previously described (32 (link)). Briefly, the cells were transfected with siRNAs targeting GRP78 (si78) or control scrambled (siCtrl) using Lipofectamine RNAiMAX Tranfection Reagent (ThermoFisher Scientific, Waltham, MA). Cell lysates were harvested 48 hr after the transfection for Western blot analysis. Custom siRNAs were purchased from GE Healthcare Dharmacon, Inc. (Chicago, IL). The sequences of the siRNAs are as follow: siCtrl: 5’-GAGAUCGUAUAGCAACGGU-3’; si78 (CDS): 5’-GGAGCGCAUUGAUACUAGA-3’; si78 (3’UTR): 5’-CUUAAGUCUCGAAUGUAAU-3’.

Custom siRNAs targeting 5’-CACCAUGAGUGGCAGUCAG-3’^{95 (link)} for FRA1 and 5′-GUUCACUGCUGGCCUAUAA-3’^{96 (link)} for FLI1 were ordered from GE Healthcare Dharmacon, Inc. As negative control, MISSION® siRNA Universal Negative Control SIC001 was used (MERCK). In each knockdown assay, siRNAs were transfected using Thermo Fisher Lipofectamine 3000 Transfection Reagent (Catalog Number: L3000001) according to transfection reagent’s protocol. 75 nM siRNA was transfected to T24 cells (65% confluent) for FLI1 and FLI1 unique knockdowns and 50 nM siRNAs targeting FLI1 and FRA1 were used for co-knockdown of FLI1 and FRA1. T24 cells were incubated in transfection medium for 24 h. Then, the transfection medium was removed and transfected cells were passaged further to be collected for RNA and protein isolation.