Fig. 4: circDDX21 cooperates with PABPC1 to increase PGAM1 mRNA stability.

A Real-time RT-PCR analysis of PGAM1 mRNA levels in HepG2 cells transduced with lentiviruses control shRNA, circDDX21 shRNA#1, or circDDX21 shRNA#2. Data shown are mean ± SD (n = 3), **p < 0.01, ***p < 0.001. B Real-time RT-PCR analysis of PGAM1 mRNA levels in HepG2 cells transduced with lentiviruses empty vector (EV) or circDDX21. Data shown are mean ± SD (n = 3), *p < 0.05. C HepG2 cells transduced with lentiviruses control shRNA, circDDX21 shRNA#1, or circDDX21 shRNA#2 were incubated with actinomycin D (2 μg/mL) for the indicated periods of time, followed by real-time RT-PCR analysis to examine PGAM1 mRNA stability. Data shown are mean ± SD (n = 3), ***p < 0.001. D Lysates from HepG2 cells were subjected to a biotin pull-down assay using sense or antisense biotin-labeled DNA oligomers corresponding to circDDX21. The pull-down complexes were analyzed by real-time RT-PCR and western blotting. E Lysates from HepG2 cells were subjected to RNA immunoprecipitation using an anti-PABPC1 antibody or a control IgG. The input and immunoprecipitates were analyzed by RT-PCR and western blotting. F Purified recombinant Flag-PABPC1 bound with anti-Flag M2 beads was incubated with in vitro-synthesized circDDX21 or its antisense RNA. The bead-bound RNAs were then analyzed by RT-PCR. G HepG2 cells transduced with lentiviruses expressing control, circDDX21 shRNA, PABPC1, or both circDDX21 shRNA and PABPC1 were incubated with actinomycin D (2 μg/mL) for the indicated periods of time, followed by real-time RT-PCR analysis to examine PGAM1 mRNA stability. Data shown are mean ± SD (n = 3), **p < 0.01, ***p < 0.001, ns., no significance. H Real-time RT-PCR analysis of PGAM1 mRNA levels in HepG2 cells transduced with lentiviruses expressing control, circDDX21 shRNA, PABPC1, or both circDDX21 shRNA and PABPC1. Data shown are mean ± SD (n = 3), **p < 0.01, ***p < 0.001. I Western blot analysis of PGAM1 protein levels in HepG2 cells transduced with lentiviruses expressing control, circDDX21 shRNA, Flag-PABPC1, or both circDDX21 shRNA and Flag-PABPC1. J Real-time RT-PCR analysis of PGAM1 mRNA levels in HepG2 cells transduced with lentiviruses expressing control, circDDX21, PABPC1 shRNA, or both circDDX21 and PABPC1 shRNA. Data shown are mean ± SD (n = 3), **p < 0.01, ns., no significance. K Western blot analysis of PGAM1 protein levels in HepG2 cells transduced with lentiviruses expressing control, circDDX21, PABPC1 shRNA, or both circDDX21 and PABPC1 shRNA. L Real-time RT-PCR analysis of PGAM1 mRNA levels in HepG2 cells with stable expression of circDDX21 or the circDDX21 mutant (ΔPABPC1 BS). Data shown are mean ± SD (n = 3). ***p < 0.001; ns., no significance. M Western blot analysis of PGAM1 protein levels in HepG2 cells with stable expression of circDDX21 or the circDDX21 mutant (ΔPABPC1 BS). N HepG2 cells expressing control, circDDX21 shRNA, Flag-PABPC1, or both circDDX21 shRNA and Flag-PABPC1 were transfected with psiCHECK2-PGAM1-3′UTR. Twenty-four hours later, reporter activity was measured. Data shown are mean ± SD (n = 3). *p < 0.05, ***p < 0.001. O HepG2 cells expressing control, circDDX21, PABPC1 shRNA, or both circDDX21 and PABPC1 shRNA were transfected with psiCHECK2-PGAM1-3′UTR. Twenty-four hours later, reporter activity was measured. Data shown are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ns, no significance. P HepG2 cells transduced with lentiviruses expressing circDDX21 shRNA and Flag-PABPC1 in the indicated combination, followed by an RNA immunoprecipitation assay using anti-Flag antibody. The input and immunoprecipitates were analyzed by RT-PCR and western blotting. Q HepG2 cells transduced with lentiviruses expressing circDDX21 and Flag-PABPC1 in the indicated combination, followed by an RNA immunoprecipitation assay using anti-Flag antibody. The input and immunoprecipitates were analyzed by RT-PCR and western blotting.