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. 2021 Mar 26;296:100601. doi: 10.1016/j.jbc.2021.100601

Figure 2.

Figure 2

ADAR1 is involved in circadian regulation of P-gp expression in human RPTECs.A, circadian oscillation of PERIOD2 and BMAL1 mRNA (left), and ADAR1 and P-gp protein (right) in the kidney of cynomolgus monkeys. The mRNA levels were assessed by quantitative real-time RT-PCR analysis and their expression levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 3). The protein levels of β-ACTIN are shown as loading controls. B, temporal expression profiles of PERIOD2 and BMAL1 mRNA in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX for 2 h. The mRNA levels were assessed by quantitative real-time RT-PCR analysis and their expression levels were normalized to that of 18S rRNA. Values are the mean with S.D. (n = 3). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There were significant time-dependent variations in PERIOD2 and BMAL1 mRNA expression (F13,28 = 56.917, p < 0.001 and F13,28 = 46.243, p < 0.001 for PERIOD2 in mock-transduced and ADAR1-KD RPTECs, respectively; F13,28 = 14.565, p < 0.001 and F13,28 = 5.157, p < 0.001 for BMAL1 in mock-transduced and ADAR1-KD RPTECs, respectively; ANOVA). ∗∗p < 0.01; significant difference between the two groups (t4 = 38.454, p < 0.001; unpaired t-test, two-sided). C, temporal protein expression profiles of ADAR1 in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX. The protein levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 4). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There were significant time-dependent variations (F5,18 = 10.414, p < 0.001 for mock-transduced; F5,18 = 8.214, p < 0.001 for ADAR1-KD; ANOVA). ∗∗p < 0.01; significant difference between the two groups (t6 = 4.641, p = 0.004; unpaired t-test, two-sided). D, temporal expression profiles of P-gp in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX. The protein levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 4). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There was a significant time-dependent variation in mock-transduced RPTECs (F5,18 = 2.899, p = 0.043 for mock-transduced; ANOVA). ∗p < 0.05; significant difference between the two groups (t6 = 2.617, p = 0.040; unpaired t-test, two-sided). In panel C and D, β-ACTIN was reused as loading control because the same protein samples were used for Western blotting for detection of ADAR1 and P-gp. E, intracellular accumulation of digoxin in mock-transduced and ADAR1-KD RPTECs 32 and 44 h after DEX treatment. Concentrations of digoxin were measured by LC-MS/MS analysis 1 h after incubation with the drug. Values are the mean with S.D. (n = 4). ∗p < 0.05; significant difference between the two groups (F3,12 = 7.487, p = 0.004; ANOVA with Tukey–Kramer’s post hoc test).