Fig 2: A model depicting that PER3 regulates stemness of PCSCs via WNT/ß-catenin pathway. Our results suggest that in PCa TME, PCa DN cells (non-PCSCs) bear high levels of PER3, which downregulates the expression of BMAL1 and prevents the translocation of ß-catenin from cytoplasm into nucleus to activate WNT/ß-catenin pathway. On the other hand, PCa DP cells (PCSCs) have low levels of PER3, which upregulates BMAL1 expression, leading to the translocation of ß-catenin from cytoplasm into nucleus to eventually activate WNT/ß-catenin signaling pathway.

Fig 3: PER3 regulates stemness of PC3 DP cells in vitro. (A) RT-qPCR analysis was used to evaluate efficiency of PER3 overexpression (OE) and knockdown (KD, or sh-PER3) in PC3 DP and PC3 DN cells, respectively. (B) Western blotting was performed to examine PER3 levels of PER3 OE in PC3 DP cells and PER3 KD in PC3 DN cells, respectively. (C,D) In sphere formation assays, PER3 OE in PC3 cells impairs their sphere-forming abilities whereas PER3 KD in PC3 DN cells improved their sphere formation. A total of 1,000 cells for each well were seeded in ULA plates. Spheres were imaged (C) and counted (D) for sphere efficiency 1–2 weeks later. (E,F) In colony formation assays, PER3 OE in PC3 DP cells abrogates their colony-forming abilities whereas PER3 KD in PC3 DN cells improved their clonogenicity. A total of 1,000 cells for each well were mixed with Matrigel and plated in 24-well culture plates. Colonies were imaged (E) and counted (F) for colonies efficiency 1–2 weeks later. Data are presented from three independent experiments. Scale bars, 100 µm. **P < 0.01, ***P < 0.001.

Fig 4: Time kinetic of TNF effects on Twist1 and Dbp expression, and suppression of Dbp by overexpressiom of Twist1.(A) Expression of Dbp and (B) of Twist1 mRNA in NIH-3T3, which were treated with TNF (10 ng/ml) for 3 to 24 hr or transfected with the pCMV6::Twist1 expression vector. The control consisted of transfection with the empty vector (pCMV6). (C) Expression of clock gene mRNA after transfection with pCMV6::Twist1 expression vector or empty vector control in NIH-3T3 cells. Overexpression of Twist1 was controlled by RT-qPCR and the effect on Twist2 assessed at 4hr. Data of RT-qPCR assays of Dbp, Per1, Per2, Per3, and Twist1 mRNA expression show the mean +/- SEM (error bars) of triplicates from three independent experiments. Data are analyzed with one way-ANOVA and Bonferroni post-hoc test; * p<0.01, ** p<0.01, *** p<0.001

Fig 5: Insulin Selectively Increases PER Expression, Related to Figure 4(A) No significant increase in luciferase expression is observed following insulin application to fibroblasts expressing luciferase constitutively under the control of the SV40 promoter (n = 3, mean ± SEM, 2-way ANOVA, Sidak’s multiple comparisons test). (B) Addition of insulin to cells expressing Cry1:LUC produces a phase shift but no acute induction (n = 4, mean ± SEM). (C) Western blotting on whole cell lysate from PER2::LUC fibroblasts shows a significant increase in the abundance of PER1 and PER3 following insulin treatment but no significant increase in the abundance of CRY1 or CRY2 (n = 3). All samples were harvested 3 h after insulin addition. (D) Quantification of western blotting, normalized against relevant loading control (n = 3, mean ± SEM, Welch’s t test). E, F Western blotting on mouse livers harvested 1 h following IP injection with glucose (3 g/kg) and insulin (2.25 IU/kg) shows a significant increase in both PER1 and PER2 abundance (n = 3, mean ± SEM, Welch’s t test), samples were normalized to histone H3 levels. Positive control (OX) in left-hand lane is extract from HEK cells transiently transfected with PER expression constructs. G,H Insulin addition to Bmal1-/- PER2::LUC fibroblasts elicits a modest but significant PER2::LUC induction (n = 3, mean ± SEM, 2-way ANOVA, Tukey’s multiple comparisons test). WT traces repeated from Figure 4A. I,J Combined addition of phosphodiesterase inhibitor (IBMX) and adenylyl cyclase activator (forskolin) to Bmal1-/- PER2::LUC fibroblasts increases basal PER2::LUC transcription (first arrow), allowing the effect of acute insulin treatment (second arrow) to be readily observed (n = 4, mean ± SEM, 2-way ANOVA, Sidak’s multiple comparisons test).