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. 2022 Mar 9;28:175–189. doi: 10.1016/j.omtn.2022.03.007

Figure 2.

Figure 2

Silencing circNlgn decreased the effects of doxorubicin on cardiac cell activity and prevented doxorubicin-induced expression of fibrosis-associated molecules

(A) Left, real-time PCR showed that AC16 cells expressed increased circNlgn when cultured in basal medium and treated with doxorubicin for 1 h. ∗p < 0.05, ∗∗p < 0.01 versus doxorubicin 0 μM (n = 4). Right, real-time PCR showed that MCFs expressed increased levels of circNlgn when treated with doxorubicin. ∗p < 0.05, ∗∗p < 0.01 versus doxorubicin 0 μM (n = 4). (B) AC16 cells transfected with circNlgn or the vector were cultured in basal medium treated with doxorubicin for survival assay. circNlgn expression repressed cell survival. ∗∗p < 0.01 versus vector (n = 4). (C) Expression of circNlgn promoted cell apoptosis when treated with doxorubicin, which was dose dependent. ∗p < 0.05, ∗∗p < 0.01 versus vector (n = 4). (D) Silencing circNlgn enhanced AC16 cell survival. ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (E) Silencing circNlgn repressed AC16 apoptosis. ∗∗p < 0.01 versus oligo (n = 4). (F) MCF cells transfected with circNlgn or the vector were cultured in basal medium for the indicated time points. Expression of circNlgn enhanced MCF cell proliferation. ∗∗p < 0.01 versus vector (n = 4). (G) Silencing circNlgn suppressed MCF cell proliferation. ∗∗p < 0.01 versus oligo (n = 4). (H) RNAs were isolated from circNlgn- or vector-transfected MCF cells treated with or without 0.25 μM doxorubicin for 24 h and subjected to RT-PCR. Real-time PCR showed that transfection with circNlgn increased expression of fibrosis markers TGF-β1, CTGF, collagen I, collagen III, fibronectin, and vimentin, which were further enhanced by doxorubicin treatment. ∗∗p < 0.01 (n = 4). (I) Control oligo- and circNlgn siRNA-transfected MCF cells were cultured in basal medium treated with 0.1 μM doxorubicin for 24 h, harvested, and subjected to RT-PCR. MCF cells showed increased expression of fibrosis markers TGF-β1, CTGF, collagen I, collagen III, fibronectin, and vimentin, which could be prevented by silencing circNlgn. ∗∗p < 0.01 versus oligo (n = 4).