Journal: Cell reports
Article Title: Multicilia dynamically transduce Sonic Hedgehog signaling to regulate choroid plexus functions.
doi: 10.1016/j.celrep.2025.115383
Figure Lengend Snippet: Figure 2. Choroid plexus cilia mediate Shh signaling to regulate Aqp1 and Atp1a2 expression (A and B) Immunostaining of Smo demonstrated its translocation into cilia in explant culture upon Shh treatment (E14.5 and P0) (A, n = 3) or in whole-mount tissue (E14.5) (B, n = 3). Scale bar, 5 mm. (C) In real-time PCR analysis, E14.5 WT choroid plexus explant induces Gli1 upon Shh treatment (10 nM), and this induction was abolished by Smo inhibitor Cyclopmaine-KAAD (KAAD). DMSO vs. Shh, n = 3, **p = 0.00178; Shh vs. Shh + KAAD, n = 3, **p = 0.0085; error bars, SD; unpaired two-tailed Student’s t test. (D) Immunostaining demonstrates an inhibitory effect of Shh signaling (10 nM) on the apical expression of Aqp1 (top) and Atp1a2 (bottom) in WT choroid plexus explants, which was blunted by Smo inhibition using KAAD. Apical Aqp1 expression (n = 6), DMSO vs. Shh, **p = 0.001; Shh vs. Shh + KAAD, ****p < 0.0001. Apical Atp1a2 expression (n = 3), DMSO vs. Shh, *p = 0.0497; Shh vs. Shh + KAAD, *p = 0.0257. (E) Shh (10 nM) downregulates apical Aqp1 expression in E14.5 WT choroid plexus explants but not littermate-controlled FoxJ1/ samples. WT PBS vs. WT Shh (n = 6), **p = 0.01; FoxJ1/ PBS vs. FoxJ1/ Shh (n = 3), n.s. (F–I) Choroid plexus cilia mediate Shh signaling to reduce cAMP level. Error bars, SD. (F) Top, a diagram illustrates the Epac-SH187 (exchange protein directly activated by cAMP) cAMP sensor, which decreases FRET upon cAMP binding. Bottom, E14.5 WT choroid plexus explants were treated with increasing concentrations of Shh, followed by ISO, and, finally, with forskolin (FSK; an activator of adenylyl cyclase) to achieve the maximal cAMP production. The dosage-dependent effect of Shh to reduce cAMP was measured by its inhibition on ISO-induced cAMP
Article Snippet: Primary antibodies used in this experiment include mouse monoclonal antibody against mouse b-actin (Proteintech Cat. 66009-1-Ig, 1:2000), rabbit polyclonal antibody against Aqp1 (Proteintech, Cat. #20333-1-AP, 1: 1000), rabbit polyclonal antibody Atp1a2 (Proteintech, Cat. #16836-1-AP, 1: 1000).
Techniques: Expressing, Immunostaining, Translocation Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Inhibition, Binding Assay