Research Paper Volume 11, Issue 22 pp 10220—10241

Circular RNA CircCCNB1 sponges micro RNA-449a to inhibit cellular senescence by targeting CCNE2

Figure 4. CircCCNB1 suppresses cellular senescence in human diploid fibroblasts. (A) Model showing shRNA specifically targeting the back-splicing sites of CircCCNB1. (B) qRT-PCR measuring the knock down efficiency by sh-CircCCNB1 in 2BS cells after transfection with lentiviruses expressing sh-ctr, sh-scramble, sh-circCCNB1-1, sh-circCCNB1-2, **P<0.01. (C) qRT-PCR detects CCNB1 mRNA level in 2BS cells transfected with lentiviral expressing sh-ctr, sh-scramble, sh-CircCCNB1-1and sh-CircCCNB1-2, N.S, nonsignificant. (D) Immunoblot analysis of CCNB1 protein level in 2BS cells transduced with lentiviruses expressing sh-ctr, sh-scramble, sh-CircCCNB1-1and sh-CircCCNB1-2. (E) CCK-8 measurement of the proliferative ability of 2BS cells after transfection with lentiviruses expressing sh-ctr, sh-scramble, sh-CircCCNB1-1, sh-CircCCNB1-2, **P<0.01. (F) SA-β gal staining of 2BS cells transfected with lentiviruses expressing sh-ctr, sh-scramble, sh-CircCCNB1-1and sh-CircCCNB1-2, bar, 100 μm, **P<0.01. (G) Clonogenicity assay of 2BS cells transfected with lentiviruses expressing sh-ctr, sh-scramble, sh-CircCCNB1-1and sh-CircCCNB1-2, *P<0.05. (H) Immunoblots analysis of P53, P21 and p16 levles in 2BS cells transfected with lentiviruses expressing sh-ctr, sh-scramble, sh-CircCCNB1-1and sh-CircCCNB1-2.