Fig 1.
Colon tissue Per2::Luc mice Per2 protein displays typical interaction with Cry2 and Bmal1 circadian proteins.
We compared interactions of Per2::Luc fusion protein vs. WTBL6 Per2 protein with well validated circadian protein binding partners for Per2, namely Cry2 and Bmal1 proteins using immunoprecipitation with Per2 Ab of colon tissue lysates. Data for Cry2 (left) and Bmal1 (right) show no significant difference in binding of either Cry2 or Bmal1 with the Per2::Luc protein vs. the normal BG proteins. The relative density of Cry2 and Bmal1 are represented for BL6 and Per2::Luc mice. There was no difference in colon tissue levels of Cry2 or Bmal1 levels at a single time point (p = 1.0 and 0.7, respectively). Blots are representative of n = 4 mice.
Fig 2.
Circular variance of circadian period and phase measures is increased in LD shifted vs. non-shifted in Per2::Luc mice colon tissue and organoids.
Circular variance quantifies the variability in circadian data. The top section depicts the circadian period of the colon tissue and organoids from shifted and non-shifted mice. The period of the shifted mice for both the organoids and tissue are much more variable than the periods for the non-shifted organoids and tissue. Similarly, the acrophase (phase) for the colon organoids and tissue from shifted mice has much more variability than the colon organoids and tissue from the non-shifted mice. This demonstrates that LD shifting mice creates more variability (disruption) in their colon tissue circadian clock and that colon organoids maintain the circadian rhythm disruption of the disrupted host colon tissue. Data are for n = 8-10/group.
Fig 3.
Circadian LD shifting increases colonic permeability in Per2::Luc mice.
Intestinal permeability was determined by gavage of a sugar cocktail and 5h urine analysis (Methods). (A) The mean percent excretion of the oral dose of sucralose (permeability) for non-shifted Per2::Luc (1.769±0.271) is statistically significantly lower than that for LD shifted Per2::Luc mice (2.921±0.318, p = 0.0089). (B) Similarly, the mean ratio of percent excretion of sucralose to lactulose ratio (permeability) for non-shifted mice (0.3368±0.044) was significantly lower than that for the shifted mice (0.4783±0.068, p = 0.0355). *p<0.05. n = 8-10/group.
Fig 4.
Colon tissue and organoid AJC protein expression is decreased with LD shifting in Per2::Luc mice.
(A) Immunofluorescent staining of E-cadherin (red) in the colon tissue and organoids of shifted and non-shifted Per2::Luc mice. Both demonstrated a significant decrease in E-cadherin in the shifted mice (p<0.05). (B) Western blot protein quantification demonstrated a similar trend in E cadherin levels. While the difference was non-significant in the tissue, the protein levels of E-cadherin were significantly lower in the colon organoids of the shifted Per2::Luc mice. (C) Immunofluorescent staining of ZO-1 (green) in the colon tissue and organoids of the shifted and non-shifted Per2::LUC mice. In both organoids and tissue, the shifted mice again demonstrated a lower amount of tight junction ZO-1 in the colons of the shifted mice (p<0.05). (D) Immunofluorescent staining of cytoskeleton protein Krt-20 (green) in the colon tissue and organoids of shifted and non-shifted Per2::Luc mice. Both tissue and the organoids demonstrated a significant decrease in Krt-20 after shifting (p = 0.0086, 0.020). (E) Western blot protein quantification of Krt-20 levels in colon tissue and organoids of shifted and non-shifted Per2::Luc mice. No difference was seen between the non-shifted and shifted colon tissue levels of Krt-20, however, a lower amount was seen in the colon organoids of the shifted mice compared to those of the non-shifted mice (p<0.05). This could be due to the same dilution effect seen in section B.
Fig 5.
Colon tissue of a night shift worker exhibits loss of AJC permeability protein E-cadherin and Krt-20 proteins vs. a day worker’s colon tissue.
Colonic biopsy tissues from a Night shift (shifted) worker and a day worker nurse were assessed for AJC protein E-cadherin and cytoskeletal protein Cytokeratin 20 (Krt-20). with IF microscopy as described in Methods. (A) E-cadherin levels were lower in the night shift worker compared to the day worker (p = 0.001). (B) Krt-20 levels were lower in the night shift worker compared to the day worker as well (p = 0.011). Data points are random field values for each sample.