Fig 1: MYBL2-related complex contributed to RRM2 transcription during S-phase in CRC cells.A DLD1 cells were synchronized by 40uM lovastatin treatment for 36 h, and then released for analyses at the indicated time points. Left, FACS analyses of cell cycle phases (NT nontreatment, T treatment with lovastatin, RT remove treatment); Middle, qPCR for RRM2 and MYBL2; Right, western blotting for RRM2, RRM2B, MYBL2, E2F1, Cyclin E, Cyclin D1, and TUBULIN (as loading control). B DLD1 cells were transfected with empty vector (EV) or Flag-MYBL2 expression plasmid for 48 h, the cell lysates were co-immunoprecipitated with anti-Flag antibody, and MYBL2-interacting proteins were identified by LC-MS/MS. C The above MYBL2-interacting proteins were validated by western blotting with antibodies against RBBP4, LIN9, TAF15, FLAG, and GAPDH (as loading control). D The effects of knockdown of RBBP4, LIN9, or TAF15 on the activity of RRM2 promoter reporter (pRRM2-S) upregulated by MYBL2 transfection in DLD1 cells. E, F DLD1 cells were transfected with the siRNAs of negative control, TAF15, RBBP4, and LIN9, respectively, and then transfected with empty vector (EV) or MYBL2 expression plasmid for 48 h. qRT-PCR and western blotting were performed for analyses as indicated. G, H The effects of overexpression or knockdown of TAF15 on the mRNA and protein levels of RRM2 were analyzed by qRT-PCR (normalized by actin) and western blotting (GAPDH as loading control), respectively, in HCT116 cells. I Relative luciferase reporter activity of the truncated or mutated RRM2 promoter while co-transfected with TAF15 expression plasmid for 48 h in HCT116 cells, pRL-SV40 as an internal control reporter. J Nuclear proteins obtained from HCT116 cells were pulled down by a non-biotin-labeled (cold) or biotin-labeled (hot) DNA probe of RRM2 promoter (-2465/+23), western blotting was used to detect the biding of TAF15 and Ku80 (as binding control). K Chromatin extracted from HCT116 cells was immunoprecipitated with the Flag or IgG antibodies, qRT-PCR was carried out on immunoprecipitated DNAs using the primer pair2 as shown in Fig. 3A. L The whole lysates of HCT116 cells transfected with Flag-EV or Flag-MYBL2 were pulled down by GST-TAF15 or GST and then analyzed by immunoblotting. M HCT116 cells were plated into coverslip in six-well plate and then immunofluorescent staining was performed with antibody against TAF15 (red) or MYBL2 (green), scale bars: 10 µm.