Active JNK is localized at the base of motile cilia. (A) Immunofluorescence images of DMSO- and SP600125-treated embryos stained with pJNK and acetylated tubulin to mark MCCs. Scale bars, 5 μm. (B) Multiciliated cells stained for total JNK (tJNK) and γ-tubulin that marks the basal foot. (C) Multiciliated cells expressing GFP-JNK stained with an antibody against pJNK. Side view (XZ projection) shows an overlap of the two signals. Scale bars, 10 μm. (D) Confocal images of Xenopus epidermal MCCs injected with GFP displaying a diffuse cytosolic pattern. Scale bars, 10 µm. (E) Color-coded images of the YFP/CFP intensity ratio of the JNK biosensor showing higher ratio at the basal bodies. Graph shows the quantification of the YFP to CFP intensity ratio of the JNK FRET sensor at the basal bodies and cytosol. YFP/CFP ratio at the BBs is higher than in the cytosol. Data represent ± SEM from two experiments (n = 159 BBs, and n = 156 cytosolic regions from 4 embryos), ***, P < 0.001, unpaired two-tailed t test. Scale bars, 5 μm. (F) Representative confocal images of SP600125-treated embryos injected with GFP-JNK and RFP-Centrin. JNK localization is not dependent on its kinase activity. Scale bars, 5 µm. (G) Representative images of human nasal epithelial cells (hNECs) grown at ALI after 21–25 d. Staining with acetylated tubulin and pJNK show apical enrichment of pJNK. Scale bars, 5 µm. (H) Representative confocal images of endogenous localization of acetylated tubulin and pJNK in mouse tracheal epithelial cells (mTECs). Scale bars, 5 µm. (I) Confocal images of MCCs expressing GFP-JNK coinjected with FLAG-MKK7 show partial colocalization with JNK. Expression of FLAG-MKK4 show apical enrichment. Scale bars, 5 µm. (J) Representative images of pJNK staining in Flag-MKK7 and Flag-MKK4 overexpressing MCCs. Quantification of BB-associated fluorescence signal of pJNK shows enhanced phosphorylation in MKK7- and MKK4-overexpressing cells compared to controls (n = 205 controls, n = 179 MKK7-overexpressing cells, n = 192 MKK4-overexpressing cells). Data represent ± SEM from three experiments. ***, P < 0.001, unpaired two-tailed t test. (K) Image sequence of a MCC expressing GFP-JNK/RFP-Centrin upon photobleaching of a region of interest (red square). T0 indicates the time of photobleaching. Normalized fluorescence intensity of GFP-JNK displays a highly dynamic association of JNK with the basal bodies with t1/2 = 0.4 s (n = 11 controls and n = 11 JNK MO from 3 embryos). Scale bars, 10 μm.
Figure S1.

Active JNK is localized at the base of motile cilia. (A) Immunofluorescence images of DMSO- and SP600125-treated embryos stained with pJNK and acetylated tubulin to mark MCCs. Scale bars, 5 μm. (B) Multiciliated cells stained for total JNK (tJNK) and γ-tubulin that marks the basal foot. (C) Multiciliated cells expressing GFP-JNK stained with an antibody against pJNK. Side view (XZ projection) shows an overlap of the two signals. Scale bars, 10 μm. (D) Confocal images of Xenopus epidermal MCCs injected with GFP displaying a diffuse cytosolic pattern. Scale bars, 10 µm. (E) Color-coded images of the YFP/CFP intensity ratio of the JNK biosensor showing higher ratio at the basal bodies. Graph shows the quantification of the YFP to CFP intensity ratio of the JNK FRET sensor at the basal bodies and cytosol. YFP/CFP ratio at the BBs is higher than in the cytosol. Data represent ± SEM from two experiments (n = 159 BBs, and n = 156 cytosolic regions from 4 embryos), ***, P < 0.001, unpaired two-tailed t test. Scale bars, 5 μm. (F) Representative confocal images of SP600125-treated embryos injected with GFP-JNK and RFP-Centrin. JNK localization is not dependent on its kinase activity. Scale bars, 5 µm. (G) Representative images of human nasal epithelial cells (hNECs) grown at ALI after 21–25 d. Staining with acetylated tubulin and pJNK show apical enrichment of pJNK. Scale bars, 5 µm. (H) Representative confocal images of endogenous localization of acetylated tubulin and pJNK in mouse tracheal epithelial cells (mTECs). Scale bars, 5 µm. (I) Confocal images of MCCs expressing GFP-JNK coinjected with FLAG-MKK7 show partial colocalization with JNK. Expression of FLAG-MKK4 show apical enrichment. Scale bars, 5 µm. (J) Representative images of pJNK staining in Flag-MKK7 and Flag-MKK4 overexpressing MCCs. Quantification of BB-associated fluorescence signal of pJNK shows enhanced phosphorylation in MKK7- and MKK4-overexpressing cells compared to controls (n = 205 controls, n = 179 MKK7-overexpressing cells, n = 192 MKK4-overexpressing cells). Data represent ± SEM from three experiments. ***, P < 0.001, unpaired two-tailed t test. (K) Image sequence of a MCC expressing GFP-JNK/RFP-Centrin upon photobleaching of a region of interest (red square). T0 indicates the time of photobleaching. Normalized fluorescence intensity of GFP-JNK displays a highly dynamic association of JNK with the basal bodies with t1/2 = 0.4 s (n = 11 controls and n = 11 JNK MO from 3 embryos). Scale bars, 10 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal