Fig. 3: Cell cycle regulation by circTshz2-2.

A The distribution of cell cycle phase following circTshz2-2 knockdown in undifferentiated (Undiff.) Neuro-2A cells at day 3 and differentiated (Diff.) Neuro-2A cells at day 5. The graph in the left panel depicts the representative results from three experiments (n = 3), and the data were averaged to produce the histogram in the right panel (mean ± SEM). Percentages of cells in each phase were calculated using FlowJo software and siCtr indicates the negative control siRNA. PE-A indicates phycoerythrin-area, while 2N, 2N–4N, 4N, <2N, and >4N denote G1, S, G2/M, sub-G1, and any phase greater than G2/M, respectively. B The changes in nuclear morphology and neurite length following circTshz2-2 knockdown in differentiated Neuro-2A cells at day 5. These data are depicted using representative cells from three independent cultures (n = 3) and the beta-tubulin and nuclei (arrow) are indicated by red and blue (DAPI), respectively. The rectangular box is a magnified field of view. In A and B, the data from the sicircTshz2-2 cells represent the outcomes of mixing two different siRNAs against circTshz2-2 (#1 and #2). C The changes in the protein levels of Cyclin B2, CDK1, and p-CDK1-Y15 following circTshz2-2 knockdown in differentiated Neuro-2A cells at day 5. These expression changes are depicted as the mean ± SEM (n = 3). p-CDK1-Y15 indicates the CDK1 protein phosphorylated at tyrosine 15. The level of p-CDK1-Y15 was normalized against CDK1. D The changes in Bdnf transcription following circTshz2-2 knockdown in neuronal cells were measured and are described as the mean ± SEM (n = 3). PCN indicates mouse primary cortical neurons. DIV indicates the days in vitro. E The changes in proBDNF and mBDNF protein levels following circTshz2-2 knockdown in neuronal cells were evaluated and are depicted as the mean ± SEM (n = 3). In A, and C–E, statistical significance was analyzed using an unpaired two-tailed t-test with Welch’s correction; ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001.