Fig 1.
TIMELESS is overexpressed in cancer.
(A) TIMELESS mRNA expression (RNASeq) data from TCGA for unpaired primary tumors and normal solid tissue samples. The results published here are in whole or part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/. Data are shown as mean ± SEM. (B) RT-qPCR and (C) representative western blot of TIMELESS levels in a panel of colon tumor cell lines and immortalized, non-transformed HCECs. RT-qPCR data are shown as mean ± SD and represent three independent experiments. *** p < 0.001, **** p < 0.0001. Western blot in (C) is representative of five independent experiments comparing the expression of TIMELESS in HCECs to eight colon cancer cell lines as depicted.
Fig 2.
Activated ERK promotes TIMELESS expression.
(A) Western blot of TIMELESS in HCECs, HCECs that stably express H-RasG12V, and HCT116 colon cancer cells with DMSO or 1 μM SCH772984 (ERK inhibitor) treatment for 48 hours. (B) Western blot of TIMELESS following RNAi-mediated ERK depletion for 72 hours in HCT116 cells. Western blots shown in (A) and (B) are representative of three or more independent experiments.
Fig 3.
TIMELESS depletion is detrimental to colon cancer cells, but does not induce cell death.
(A) Cell state was assessed in HCT116 colon cancer cells following RNAi-mediated depletion of TIMELESS that were replated on polyHEMA-coated plates 48 hours following transfection to simulate anchorage-independent conditions. Cell state was measured using CellTiter-Glo 0 and 24 hours after replating the cells. (B) Metabolic capacity of HCECs and HCT116 cells measured using alamarBlue following RNAi-mediated TIMELESS depletion for 72 hours in normal culture conditions. (C) Metabolic capacity was measured in a panel of colon cancer cells following RNAi-mediated depletion of TIMELESS using alamarBlue 96 hours after transfection. (D) Western blot of TIMELESS and PARP following RNAi-mediated TIMELESS depletion for 72 hours in HCT116, HCT15, SW480, SW620, and RKO colon cancer cells. Data are shown as mean ± SD and represent four independent experiments in (A), three independent experiments in (B), and five independent experiments in (C). **** p < 0.0001.
Fig 4.
TIMELESS depletion induces G2/M arrest and decreases cell proliferation in colon cancer cells.
(A) Quantification of the percent of cells in each phase of the cell cycle following RNAi-mediated TIMELESS depletion for 72 hours in HCT116, RKO, SW620, SW480 and HCT15 colon cancer cells from three independent experiments. Apoptosis (% of cells in the sub-G1 peak) and cell cycle were evaluated using propidium iodide staining followed by flow cytometry analysis. (B) Representative cell cycle histograms from (A). (C) Overlay histogram for three independent experiments of flow cytometry analysis of CFSE staining following RNAi-mediated TIMELESS depletion in CFSE-stained HCT116 cells for 96 hours. Control replicates are shown in gray, and TIMELESS-depleted replicates are shown in black. (D) Quantification of mean CFSE staining from (C). Data are shown as mean ± SD and represent three independent experiments in all cases. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 5.
TIMELESS depletion induces G2/M arrest via CHK1 phosphorylation, which leads to CDK1 phosphorylation and inactivation.
Western blot of phospho- and total-H2AX, phospho- and total-CHK1, and phospho- and total-CDK1 following RNAi-mediated TIMELESS depletion for 72 hours in a panel of colon cancer cells. The western blot shown is representative of three or more independent experiments in all five cell lines.
Fig 6.
TIMELESS depletion sensitizes colon cancer cells to Wee1 and CHK1 inhibition.
Cell metabolic capacity was measured in a panel of colon cancer cells following RNAi-mediated depletion of TIMELESS with Wee1 inhibition or CHK1 inhibition using alamarBlue assays 96 hours after transfection. 300 nM of Wee1 (MK-1775) or CHK1 (AZD7762) inhibitor was added 48 hours after transfection. Data are normalized to the DMSO treated control transfection (far left bar). Data are shown as mean ± SD and are representative of four or five independent experiments for each condition and cell line. The lower case letters denote a statistical significance (one-way ANOVA with Bonferroni’s Multiple Comparison test for the specified comparisons) with a p value less than 0.001 for the following comparisons: a—DMSO-treated siCont vs DMSO-treated siTIM; b—MK-1775-treated siCont vs MK-1775-treated siTIM; c—AZD7762-treated siCont vs AZD7762-treated siTIM; d—DMSO-treated siTIM vs MK-1775-treated siTIM; e—DMSO-treated siTIM vs AZD7762-treated siTIM.