Fig. 6
NSUN2 and YBX1 regulate QSOX1 expression via manipulation of mRNA translation. a H1650 and H1975 cells were transfected with siNSUN2 or siCtrl for 72 h and the whole cell lysates were detected by western blotting with indicated antibodies. b Representative images of QSOX1 IHC staining and the quantitative H-scores of tumors obtained from the H1650-shCtrl and H1650-shNSUN2 xenografts. Image magnification: 200 × (upper panel) and 400 × (lower panel). c H1650 cells pre-treated with siNSUN2 were transfected with pcDNA-NSUN2-WT or pcDNA-NSUN2-Mut (C271A&C321A) plasmid and western blotting analysis of QSOX1 expression. d H1650 or H1975 cells transfected with siCtrl or siNSUN2 were treated with 200 nM puromycin for the indicated time and the whole cell lysates was detected by western blotting with indicated antibodies. e QSOX1 CDS containing either wild type or mutant (C-to-T/A mutation) m5C sites was cloned into luciferase reporter vector. f Relative luciferase activity of the wild-type and mutant form of QSOX1 CDS reporter vectors in H1650 cells transfected with shCtrl or shNSUN2, respectively. g The expression of QSOX1 in H1650 or H1975 cells transfected with siCtrl or siYBX1 was determined by western blotting analysis. h RNA immunoprecipitation of YBX1 and QSOX1 mRNA was carried out in H1650 cells transfected with siCtrl or siNSUN2 using anti-YBX1 antibody, with IgG as the control. i H1650 cells pre-treated with shYBX1 were transfected with 3FLAG-YBX1-WT or 3FLAG-YBX1-Mut (W65A) plasmid and western blotting analysis of QSOX1 expression. j H1650 or H1975 cells transfected with siCtrl or siYBX1 were treated with 200 nM puromycin for the indicated time and the whole cell lysates were detected by western blotting with indicated antibodies. k Relative luciferase activity of the wild-type and mutant form of QSOX1 CDS reporter vectors in H1650 cells transfected with shCtrl or shYBX1, respectively. l H1650 cells were transfected with siNSUN2 or siYBX1 alone or in combination for 72 h and the whole cell lysates was detected by western blotting with indicated antibodies. Error bars are means ± SD of three independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.