Fig. 2

CLOCK overexpression inhibits GCs proliferation. A The overexpression efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 4), ^*P < 0.05. B Western blotting reveals the expression levels of CLOCK. C Quantitative statistics of CLOCK. Data are expressed as mean ± SEM (n = 3), ^**P < 0.01. D Flow cytometry determines cell percentages in different cell-cycle phases. E Cell-cycle analysis statistical results. Data are expressed as mean ± SEM (n = 3), ^**P < 0.01, ^***P < 0.001. F EdU staining was used to quantify the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 3), ^*P < 0.05. G CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 4), ^**P < 0.01. H RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 3), ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. I Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCND1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. J Quantifying the Western blot analysis of CLOCK, CCNB1, CCND1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), ^*P < 0.05, ^**P < 0.01