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. Author manuscript; available in PMC: 2022 Sep 28.
Published in final edited form as: Eur Respir J. 2020 Dec 17;56(6):1902407. doi: 10.1183/13993003.02407-2019

Fig 5. Time of HDM challenge determines magnitude of contraction of airways to methacholine in lung slice model; muscarinic receptor expression is regulated by time of day and also by HDM challenge and REV-ERBα.

Fig 5

a. Representative images of precision cut lung slice experiment showing contraction of airways to increasing concentrations of methacholine in mice challenged with HDM at ZT11 or ZT23.

b. Concentration-response curves to increasing concentrations of methacholine. There is a significant increase in the maximal effect to methacholine in mice challenged with HDM at ZT11 compared to ZT23 (* P= 0.03). The EC50 for WT mice challenged at ZT11 was 3.2μM, and 6.2μM at ZT23.

c. Concentration-response curves to increasing concentrations of methacholine in Rev-erbα-/- mice challenged with HDM at ZT11 and ZT23. There was no difference in the effects of methacholine by time of day. The EC50 for ZT11 was 9.3μM and for ZT23 was 8.5μM.

Methacholine dose response curves were fitted to a three parameter sigmodal dose-response curve. An extra sum-of-squares F-test was used to test whether one curve could adequately fit the data for ZT11 and ZT23. Black triangles-HDM challenge at ZT11 and grey circles-HDM challenge at ZT23

d. Quantitative PCR for muscarinic receptor Chrm1. Chrm1 expression is significantly increased in PBS challenged mice at ZT11 compared to ZT 23 (* P < 0.05). This time of day difference is not apparent after HDM challenge. There is a significant increase in expression of Chrm1 after HDM challenge at ZT23 compared to control, PBS challenged mice (* P < 0.05). There was no time of challenge difference seen in Rev-erbα-/- mice (n=5-9 per treatment group, in duplicate).

e. Quantitative PCR for muscarinic receptor Chrm2. Chrm2 expression showed no time of challenge differences in WT or Rev-erb-/- mice (n=7-10 per treatment group, in duplicate). There were no time of genotype differences.

f. Quantitative PCR for muscarinic receptor Chrm3. Chrm3 expression is significantly increased after HDM challenge at ZT11 compared to ZT 23 (* P < 0.05). There is no time of day difference in Rev-erbα-/- mice (n=5-9 per treatment group, in duplicate).

All data presented as mean ± SEM and analysed by 1 way ANOVA, followed by Tukey’s multiple comparison test. All QPCR data is compared to expression of the housekeeping gene Gapdh in WT PBS challenged mice at ZT23. Black bars indicate challenge at ZT11 and grey bars indicate challenge at ZT23.