Figure 2. Loss of CLN3 promotes the interaction between YAP and p73.

(A) Representative immunoblot image and quantification of YAP protein levels phosphorylated at the tyrosine residue 357 (pTyr357-YAP) in Wt and CLN3^KO cells. GAPDH and Ponceau were used as loading controls. (B) Confocal fluorescence images of Wt and CLN3^KO cells immunostained for pTyr357-YAP protein. Nucleus are outlined by the yellow dashed line using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of YAP at the nucleus of at least 500 cells per each condition. The yellow dots represent the mean of each independent experiment. (C) Immunohistochemistry analysis of pTyr357-YAP staining of control (Wt) and Cln3^Δex7/8 animals. On the right, quantification of the number of positive nucleus to pTyr357-YAP per the same area of hippocampus (first) and thalamus (second). (D) Representative immunoblot image and quantification of pTyr357-YAP in Wt and Cln3^Δex7/8 animals. GAPDH and Ponceau were used as loading controls. (E) Confocal fluorescence images of Wt and CLN3^Kd cells immunostained for p73 protein. Nuclei are outlined by the yellow dashed lines using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of p73 at the nucleus of at least 500 cells per each condition. (F) Correlation between the intensity levels of nuclear YAP (x axis) and p73 (y axis) proteins. At least 150 cells were analysed. (G) Confocal fluorescence images and quantification of HEK293T (upper panels and graph) and ARPE19 (bottom panels and graph) parental cell lines treated with scramble or CLN3-siRNA and immunostained for p73 protein. Nuclei are outlined by the yellow dashed line using Hoechst staining. Scale bar 10µm. All the results are mean±SEM of at least three independent experiments. (H) Confocal fluorescence images of CLN3^KO cells transfected with a control or two siRNA against YAP protein and immunostained for p73 protein. Nuclei are outlined by the yellow dashed lines using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of p73 at the nucleus of at least 450 cells per each condition. (I) Representative immunoblot image and quantification of p73 co-immunoprecipitated with YAP protein in Wt and CLN3^Kd cells. The results are mean±SEM of four independent experiments. In the violin plots, yellow dots represent the mean of each individual experiment. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparisons test (G and H), and Student’s t-tests when comparing two experimental groups. * p<0.05; ** p<0.01; ***p<0.001. * p<0.05; *** p<0.001; **** p<0.0001.