Inhibition of JNK activity affects primary and motile ciliogenesis. (A) Western blot analysis of protein extracts from control and JNK morphants. 40 ng of JNK MO was injected into both blastomeres of two-cell stage embryos. Quantification shows the normalized ratio of pJNK/GAPDH intensity. Data represent mean ± SEM from three independent experiments. **, P < 0.01, unpaired two-tailed t test. (B) Representative images of GFP-Centrin expressing embryos injected with high dose MO (80 ng/cell) and stained with acetylated tubulin. Scale bars, 5 µm. (C) Immunofluorescence images of control and JNK MO embryos stained for acetylated tubulin and ZO-1 shows apical enrichment of ZO-1 and absence of signal at the basal region. Scale bars, 10 μm. (D) Representative images of control and JNK MO (low and high dose) embryos injected with GFP-Dishevelled shows an association of Dishevelled with the basal bodies, suggesting that JNK is not involved in the PCP polarity. Scale bars, 5 μm. (E) Western blotting of pJNK protein levels in DMSO- or SP600125-treated epidermal lysates. Quantification shows the normalized ratio of pJNK/GAPDH intensity. Data represent mean ± SEM from three independent experiments, ***, P < 0.001, unpaired two-tailed t test. (F) Immunofluorescence images of DMSO- and BI-78D3 embryos stained for pJNK and acetylated Tubulin. Fluorescence intensity of pJNK in BI-78D3-treated embryos is dramatically decreased compared with DMSO. Scale bars, 5 µm. (G) Immunofluorescence images of DMSO- and BI-78D3 embryos stained for Centrin and acetylated tubulin. Scale bars, 10 µm. Quantification shows decreased acetylated-tubulin fluorescence intensity in BI-78D3-treated MCCs compared to DMSO. Data represent mean ± SEM from three independent experiments, ***, P < 0.001, unpaired two-tailed t test. (H) HeLa cells were serum-starved for 24 h and stained for acetylated α-tubulin in the presence of DMSO or SP600125. DNA was counterstained with Hoechst. Scale bars, 20 µm. Primary cilia were manually counted by scanning through all focal planes. Approximately 700 cells for each condition were scored from three replicates for the presence of a primary cilium. The percentage of primary cilia in SP600125-treated cells is decreased as shown in the graph. Data represent mean ± SEM from three independent experiments, *, P < 0.05, unpaired two-tailed t test. Source data are available for this figure: SourceData FS3.
Figure S3.

Inhibition of JNK activity affects primary and motile ciliogenesis. (A) Western blot analysis of protein extracts from control and JNK morphants. 40 ng of JNK MO was injected into both blastomeres of two-cell stage embryos. Quantification shows the normalized ratio of pJNK/GAPDH intensity. Data represent mean ± SEM from three independent experiments. **, P < 0.01, unpaired two-tailed t test. (B) Representative images of GFP-Centrin expressing embryos injected with high dose MO (80 ng/cell) and stained with acetylated tubulin. Scale bars, 5 µm. (C) Immunofluorescence images of control and JNK MO embryos stained for acetylated tubulin and ZO-1 shows apical enrichment of ZO-1 and absence of signal at the basal region. Scale bars, 10 μm. (D) Representative images of control and JNK MO (low and high dose) embryos injected with GFP-Dishevelled shows an association of Dishevelled with the basal bodies, suggesting that JNK is not involved in the PCP polarity. Scale bars, 5 μm. (E) Western blotting of pJNK protein levels in DMSO- or SP600125-treated epidermal lysates. Quantification shows the normalized ratio of pJNK/GAPDH intensity. Data represent mean ± SEM from three independent experiments, ***, P < 0.001, unpaired two-tailed t test. (F) Immunofluorescence images of DMSO- and BI-78D3 embryos stained for pJNK and acetylated Tubulin. Fluorescence intensity of pJNK in BI-78D3-treated embryos is dramatically decreased compared with DMSO. Scale bars, 5 µm. (G) Immunofluorescence images of DMSO- and BI-78D3 embryos stained for Centrin and acetylated tubulin. Scale bars, 10 µm. Quantification shows decreased acetylated-tubulin fluorescence intensity in BI-78D3-treated MCCs compared to DMSO. Data represent mean ± SEM from three independent experiments, ***, P < 0.001, unpaired two-tailed t test. (H) HeLa cells were serum-starved for 24 h and stained for acetylated α-tubulin in the presence of DMSO or SP600125. DNA was counterstained with Hoechst. Scale bars, 20 µm. Primary cilia were manually counted by scanning through all focal planes. Approximately 700 cells for each condition were scored from three replicates for the presence of a primary cilium. The percentage of primary cilia in SP600125-treated cells is decreased as shown in the graph. Data represent mean ± SEM from three independent experiments, *, P < 0.05, unpaired two-tailed t test. Source data are available for this figure: SourceData FS3.

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