Fig. 2
(A-E): Circadian difference in gene expression profiles in primary microglia
(A) Western blot as proof of HO-1 knockout efficiency in LyzM-Cre-Hmox1fl/fl compared to Hmox1fl/fl cultured primary microglia cells. The lower panel shows corresponding loading control using total protein staining. (B) microglial Per-2 mRNA level in the late phase of the circadian cycle demonstrated as fold change vs. early phase in Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl cells. Early and late phases in the circadian cycle were induced by different timing of the medium change. (Hmox1fl/fl late vs. early phase p = 0.0003, LyzM-Cre-Hmox1fl/fl late vs. early phase p = 0.0359, n = 6 per group). (C) microglial Cry-1 mRNA levels demonstrated as fold change vs. early phase in Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl cells (Hmox1fl/fl late vs. early phase p = 0.009, n = 5–6 per group). (D) The circadian difference in microglial Per-2 mRNA expression demonstrated as fold change with carbon monoxide (CO) treatment (250ppm, 1 h) vs. control in Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl cells (Hmox1fl/fl late phase CO vs. control p = 0.0498, n = 6). (E) circadian difference in microglial Cry-1 mRNA expression demonstrated as fold change with CO treatment (250ppm, 1 h) vs. control in Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl cells (Hmox1fl/fl early phase CO vs. control p = 0.0188, n = 6) Used statistical analysis: one sample t-test; results presented as box blot with median, minimum and maximum. Statistically significant values were defined as p ≤ 0.05 (* p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)