Inhibition of JNK in mature MCCs leads to defects in CBF and apical actin without affecting rotational polarity. (A) Western blot analysis of pJNK protein levels in tadpoles’ skin lysate from DMSO- or SP600125-treated embryos. *, P < 0.05, data represent mean ± SEM from three independent experiments, unpaired two-tailed t test. (B) Ciliary beat frequency in hertz (beats per second) of motile cilia in DMSO- or SP600125-treated Xenopus epidermal MCCs. Error bars indicate ± SEM, ***, P < 0.001 (n = 5 embryos for each condition), unpaired two-tailed t test. (C) Immunofluorescence images of DMSO- and BI-78D3 embryos labeled for F-actin and acetylated tubulin. Scale bars, 10 µm. Quantification shows decreased acetylated tubulin intensity (n = 23 DMSO-treated MCCs and n = 28 BI-78D3-treated MCCs) and apical cell surface (n = 33 DMSO-treated MCCs, n = 57 BI-78D3-treated MCCs) in BI-78D3-treated embryos compared to DMSO. Error bars indicate ± SEM from three experiments, ***, P < 0.001, unpaired two-tailed t test. (D) Representative higher-magnification immunofluorescence images of MCC treated with DMSO or BI-78D3 for 2 h and stained with Phalloidin. Fluorescence intensity profiles along the lines show an impaired apical actin network in BI-78D3-treated MCCs. Scale bars, 10 µm. (E) Immunofluorescence images of MCCs treated with SP600125 for 2 h at stage 30 followed by drug washout and subsequent imaging of the apical actin network 0, 1, 2, and 3 h after washout. Scale bars, 10 μm. (F) Representative images of DMSO- and SP600125-treated embryos injected with GFP-Clamp as a marker of rootlets and RFP-Centrin to monitor cilia orientation. Circular diagrams of mean cilia orientation from polarized embryos treated at stage 30 with either DMSO or SP600125 for 2 h (n = 212 BB from four DMSO-treated embryos, n = 243 BBs from 4 SP600125-treated embryos). Rotational polarity is not affected in SP600125-treated embryos as reflected by the similar circular standard deviation (CSD). Scale bars, 10 µm. (G) Quantification of the number of basal bodies in DMSO- (n = 63 MCCs from three embryos) and SP60125-treated stage 30 embryos (n = 61 from 3 embryos). Error bars indicate ± SEM from two experiments, unpaired two-tailed t test. (H) Confocal images of stage 30 embryos coinjected with GFP-IFT52 and RFP-Centrin. The association of IFT52 with the basal bodies is not affected in 2 h SP600125-treated embryos. Scale bars, 10 µm. Source data are available for this figure: SourceData FS5.
Figure S5.

Inhibition of JNK in mature MCCs leads to defects in CBF and apical actin without affecting rotational polarity. (A) Western blot analysis of pJNK protein levels in tadpoles’ skin lysate from DMSO- or SP600125-treated embryos. *, P < 0.05, data represent mean ± SEM from three independent experiments, unpaired two-tailed t test. (B) Ciliary beat frequency in hertz (beats per second) of motile cilia in DMSO- or SP600125-treated Xenopus epidermal MCCs. Error bars indicate ± SEM, ***, P < 0.001 (n = 5 embryos for each condition), unpaired two-tailed t test. (C) Immunofluorescence images of DMSO- and BI-78D3 embryos labeled for F-actin and acetylated tubulin. Scale bars, 10 µm. Quantification shows decreased acetylated tubulin intensity (n = 23 DMSO-treated MCCs and n = 28 BI-78D3-treated MCCs) and apical cell surface (n = 33 DMSO-treated MCCs, n = 57 BI-78D3-treated MCCs) in BI-78D3-treated embryos compared to DMSO. Error bars indicate ± SEM from three experiments, ***, P < 0.001, unpaired two-tailed t test. (D) Representative higher-magnification immunofluorescence images of MCC treated with DMSO or BI-78D3 for 2 h and stained with Phalloidin. Fluorescence intensity profiles along the lines show an impaired apical actin network in BI-78D3-treated MCCs. Scale bars, 10 µm. (E) Immunofluorescence images of MCCs treated with SP600125 for 2 h at stage 30 followed by drug washout and subsequent imaging of the apical actin network 0, 1, 2, and 3 h after washout. Scale bars, 10 μm. (F) Representative images of DMSO- and SP600125-treated embryos injected with GFP-Clamp as a marker of rootlets and RFP-Centrin to monitor cilia orientation. Circular diagrams of mean cilia orientation from polarized embryos treated at stage 30 with either DMSO or SP600125 for 2 h (n = 212 BB from four DMSO-treated embryos, n = 243 BBs from 4 SP600125-treated embryos). Rotational polarity is not affected in SP600125-treated embryos as reflected by the similar circular standard deviation (CSD). Scale bars, 10 µm. (G) Quantification of the number of basal bodies in DMSO- (n = 63 MCCs from three embryos) and SP60125-treated stage 30 embryos (n = 61 from 3 embryos). Error bars indicate ± SEM from two experiments, unpaired two-tailed t test. (H) Confocal images of stage 30 embryos coinjected with GFP-IFT52 and RFP-Centrin. The association of IFT52 with the basal bodies is not affected in 2 h SP600125-treated embryos. Scale bars, 10 µm. Source data are available for this figure: SourceData FS5.

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