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. 2019 Dec 9;9:18622. doi: 10.1038/s41598-019-54738-2

Figure 3.

Figure 3

Cdc7 inhibition reduces HH3 pS28 and CENP-A pS7 in M-phase cells. (a,b,e) 31-8, U2OS derivative carrying shRNA under an inducible promoter, was incubated with or without doxycycline for three days to reduce Cdc7 level. “low” represents Cdc7-depleted cells (with doxycycline), whereas “normal” represents control cells (without doxycycline). (a) HH3 pS28 signal and Hoechst33342 (DNA) during M-phase are shown (upper panels). HH3 pS28 signal on chromatin were calculated by Imaris software and plotted by the Prism software (lower graph). (b) 31-8 cells (normal and low) were either treated with 100 ng/ml nocodazole or DMSO for 20 hrs before analyses by FACS. Western analyses of protein expression are shown in Supplementary Fig. S3a,b. (c) HeLa cells expressing GFP-CENP-A were treated with 10 µM Cdc7 inhibitor (PHA-767491) for 100 min, fixed and stained with anti CENP-A (pSer7) antibody. Cells were observed under LSM780 confocal microscopy. Imaris Spot analyses were performed to determine the Alexa 546 signal (pSer7) intensity on GFP signal (CENP-A) spots. Control, n = 784; Cdc7 inhibitor treated, n = 561 (d) U2OS cells were incubated with 50 ng/ml nocodazole or DMSO (control) for 15 hrs. The IP-kinase assays were conducted using HH3 as a substrate. (e) 31-8 cells (normal and low) were non-treated (−) or treated with 30 ng/ml nocodazole for 45 min (+). The IP-kinase assays were conducted using HH3 as a substrate. (f) Parent and AID-Cdc7 HCT116 cells, treated with or without Auxin for 60 min (cMyc#4) or 90 min (mCl#2 and Parent), were analyzed by FACS. (g) AID-Cdc7 HCT116 (mCl#2) cells were treated with Auxin for 1.5 hrs. The IP-kinase assays were performed using HH3.1 as a substrate. Western blot analyses of the extracts used are shown in Supplementary Fig. S3f. A long exposure of the Aurora B blot is shown in Supplementary Fig. S8. (h) AID-cMyc-Cdc7 (#1, #4 and #6) and parent HCT116 cells arrested with nocodazole were treated with MG132 (−) or Auxin (+) for 1.5 hrs. Western blotting was performed as indicated. (i) The CSK-soluble extracts of #4 clone prepared in (h) were used for IP-kinase assay. Purified GST-Aurora B (rat) was used as a control for the kinase assays (lane 6).