Fig. 4: In vivo characterization of Per2-IL1Ra-t2a-Luc chronotherapy circuit.

A Schematic of experiments. B Mouse wheel-running actigraphy (black tick marks) recorded to monitor circadian entrainment to the light cycle. Yellow background is lights on; white background is lights off. IL-1Ra was measured from blood drawn in the day (ZT9, diamonds) and night (ZT 17, triangles) two days before and 15 days after a 12-h shift in the light cycle. Orange lines represent light/dark cycle sampling and blue lines represent dark/light sampling. C Bioluminescence from Per2-IL1Ra:Luc constructs implanted in mice peaked around dusk before and after adjusting to the reversed light cycle (n = 7 mice, 4 constructs/mouse). LD = light/dark cycle. DL = dark/light cycle. D Serum IL-1Ra concentration evaluated 4 h post BLI peak and trough expression (repeated measures t-test, p = 0.0096, n = 6 mice, 4 constructs/mouse). Asterisks denotes significance within each sample, between timepoints. E Serum IL-1Ra concentration evaluated 4 hours post BLI peak and trough expression (repeated measures t-test, p = 0.004, n = 7, 4 constructs/mouse). Asterisks denotes significance within each sample, between timepoints. F Safranin-O staining of a representative engineered cartilage construct 21 days after implantation. Image is from one implant after removal and staining. Scale bar = 100 μm, 6 replicates. G Immunohistochemistry staining for collage type II of a representative engineered cartilage construct 21 days after implantation. Image is from an implant after removal and staining. Scale bar = 200 μm, 6 replicates. H Bioluminescence images of three mice bearing Per2-IL1Ra:Luc flank implants, taken around the peak (ZT13) and trough (ZT5) of Per2 expression. Source data are provided as a Source Data file. Created in BioRender. Created in BioRender. Guilak, F. (2025) https://BioRender.com/m89a345.