Fig. 4: DARS-AS1 blocks PACT-mediated PKR activation.

a The colocalization of PACT and PKR in control cells or DARS-AS1-overxpression cells were observed by fluorescence confocal microscope. Cell nucleus was stained by DAPI. The statistic results were obtained by 16 photos. b Co-immunoprecipitation (co-IP) was performed using anti-PACT antibody in cell lysates of SW620 control cells or DARS-AS1-overexpressing cells. c Flag-tagged PACT, purified PKR, and in vitro transcribed DARS-AS1 or mock RNA were incubated for in vitro protein binding assay. Immunoprecipitation used anti-flag antibody. d Immunoblot using indicated antibodies was performed in SW620 and HCT116 cells transfected with control shRNAs or DARS-AS1-shRNAs, followed by serum starvation. e The expression level of DARS-AS1 alters cell sensitivity of thapsigargin. SW620 cells were transfected with DARS-AS1 shRNAs, DARS-AS1-overexpression plasmids or control plasmids. Cells were treated by thapsigargin for 48 h and detected cell viability by MTS reagents. f In vitro transcribed DARS-AS1 or mock RNA and purified PACT were used for in vitro activation assay and immunoblot detection. g Immunoblot using indicated antibodies were performed in SW620-ctrl cells (left) or cells overexpressing PKR mutant (right). These cells were then transfected with control shRNAs or DARS-AS1-shRNAs, followed by serum starvation. h Inactive mutant PKR compensated SW620 cell apoptosis induced by DARS-AS1, as revealed by flow cytometry. i Immunoblot using indicated antibodies were performed in SW620 (left) or HCT116 cells (right). Cells transfected with control shRNAs or DARS-AS1-shRNAs were treated by serum starvation, suppling with 100 nM PKR inhibiter C16 or DMSO. Scale bars =5 µm. Data shown are means ± SD in triplicates experiments. *p ≤ 0.05, by two-tailed Student’s t test.