Fig. 4

CHRM3 ciliary localization is associated with cilia length. (a) In wildtype cells, immunostaining using antibodies against acetylated α-tubulin and CHRM3 was used to examine (i.) cilia length in response to treatment with cevimeline (500 μM) in cells where CHRM3 was localized to the ciliary base, ciliary axoneme, or to “Other” non-ciliary compartments. N = 651 cells, 1-Way ANOVA with Bonferroni’s posthoc test, F(5,646) = 10.75, P < 0.0001 denotes significant difference in cilia length in all groups compared to cilia length where CHRM3 was localized to the ciliary base in vehicle-treated cells; #P < 0.05, ##P < 0.01, and ###P < 0.001denotes significant difference between cilia length in cevimeline-treated compared to all other groups. Individual data points represent cilia length measurements. Means are ± SEM, (ii.) CHRM3 localization frequency (%) to ciliary base, ciliary axoneme, or to “Other” non-ciliary compartments was quantified in response to cevimeline treatment (500 μM). N = 986 cells, unpaired two-tailed t-test, **P < 0.0081, t = 4.890, df = 4 significant difference in CHRM3 localization between cevimeline- and vehicle-treated cells, and (iii.) Immunostaining using antibodies against acetylated α-tubulin (red) and CHRM3 (green) was used to show representative images of CHRM3 localization to the ciliary base and axoneme. (b) In HBEC cells, immunostaining using antibodies against (i.) acetylated α-tubulin and CHRM3, and (ii.) PIP2 and CHRM3 was used to examine CHRM3 and PIP2 localization frequency (%) to ciliary base, axoneme, or to “Other” non-ciliary compartments in response to cevimeline treatment (500 μM). N = 658 cells, unpaired two-tailed t-test, NS non-significant difference in CHRM3 and PIP2 localization between cevimeline- and vehicle-treated (control) cells. None denotes no CHRM3 localization was found. Means are ± SEM.