Figure 4. Accumulation of DNA damage in CLN3-KO cells drives c-Abl and nuclear dysmorphism.

(A) Schematic representation of c-Abl activation in response to DNA damage. C-Abl directly phosphorylate YAP at the position Y357 in response to DNA damage. (B) Representative immunoblot image and quantification of p53, γH2AX, p-ATM and ATM protein levels in Wt and CLN3^KO cells. GAPDH was used as loading controls. (C) Confocal fluorescence images of HEK293T Wt and CLN3^KO cells immunostained for p-p53 and γH2AX proteins. Nucleus are outlined by the yellow dashed line using Hoechst staining. Scale bar 10µm. Graphs are the quantification of the mean intensity of p-p53 (upper panel) and γH2AX (bottom panel) at the nucleus from at least 100 cells per condition in each independent experiment, with a total of at least 450 cells. (D) Confocal fluorescence images of HEK293T Wt and CLN3^KO cells immunostained for LaminA/C proteins. Scale bar 10µm. Graphs represent the quantification of nuclear morphology parameters in HEK293T Wt and CLN3^KO cells immunostained for LaminA/C: circularity (up, right), aspect ratio (up, left), roundness (down, left) and solidity (down, right). (E) Transmission electron microscopy of HEK293T Wt and CLN3^KO cells. (F) Confocal fluorescence images of HEK293T CLN3^KO cells transfected with GFP or GFP-CLN3 and immunostained for LaminA/C proteins. Scale bar 10µm. Graphs represent the quantification of nuclear morphology parameters in HEK293T Wt and CLN3^KO cells immunostained for LaminA/C: circularity, aspect ratio, roundness and solidity. (F) Lipid composition of nuclear extracts isolated from HEK293T Wt or CLN3^KO cells. LysoPL= lysophospholipids, PC phosphatidylcholine, PA = phosphatidic acid, DAG= diacylglycerol, Cer= ceramide, SM=sphingomyelin. In the violin plots, yellow dots represent the mean of each individual experiment. All the results are mean±SEM of at least three independent experiments. * p<0.05; ** p<0.01; ***p<0.001. * p<0.05; *** p<0.001; using unpaired t-test.