Fig. 6: PEAK1 deletion triggers ZO-1 degradation by exposing an LC3-interacting region on ZO-1.

a–b Representative western blot analysis of wild-type (WT) and PEAK1 knockout (KO) Caco-2 cells treated with 100 nM Bafilomycin A1 (BafA1, autophagy inhibitor) (a) or 100 μM Rapamycin (Rapa, autophagy activator) (b) for 12 h. c Co-immunoprecipitation of Caco-2 cell lysates using anti-ZO-1 antibodies, showing LC-3B binding to ZO-1. d Immunofluorescence images showing colocalization of ZO-1 and LC-3B in autophagosomes of Caco-2 cells treated with 100 nM BafA1. Scale bars, 10 µm. e In vitro pull-down assays using different FC-tagged ZO-1 mutants and GFP-tagged PEAK1. FC-tagged ZO-1 deletions are visualized by Ponceau staining. f Predicted interaction motifs between ZO-1 and LC-3B, identified using an online tool (https://ilir.warwick.ac.uk/). PSSM: Position Specific Scoring Matrix (PSSM). High PSSM scores are assigned to the most frequent residues. g, h Co-immunoprecipitation analysis of HEK293T cells transfected with GFP-tagged ZO-1 (WT) or GFP-tagged mutant ZO-1 with a 1212-1217 amino acid deletion (Δ1212-1217), confirming this amino acid region regulates ZO-1 binding to LC-3B (g) and PEAK1 (h). (i) Co-immunoprecipitation using an anti-ZO-1 antibody in wild-type (WT) and PEAK1 knockout (KO) Caco-2 cells treated with 100 nM BafA1 for 12 h. j Representative Immunofluorescence images (left panel) and quantification of the ratio of ZO-1 and LC-3B co-localization to total ZO-1 puncta per cell (right panel) in WT and PEAK1 KO Caco-2 cells treated with 100 nM BafA1. Scale bars, 10 µm. KO = PEAK1 KO. Data are presented as mean ± SD. 79 cells per group were analyzed. Unpaired two-tailed Student’s t-test. k Co-immunoprecipitation using anti-ZO1 antibodies in Caco-2 cells treated with 100 nM BafA1 for 12 h, followed by an additional treatment with 10 uM PP2 inhibitor or vehicle (DMSO) for 2 h. (l) Left panel: immunofluorescence microscopy images detecting ZO-1 (red) and LC-3B (green) in Caco-2 cells treated with 100 nM BafA1 for 12 h, followed by treatment with 10 uM PP2 inhibitor or vehicle (DMSO) for 2 h. Scale bars, 10 µm. Right panel: quantification of the ratios of ZO-1 and LC-3B co-localization to total ZO-1 puncta per cell. Data are presented as mean ± SD. WT, n = 79 cells per group. Unpaired two-tailed Student’s t-test. m Co-immunoprecipitation analysis in the protein lysates from WT and Y724F mutant Caco-2 cells expressing CSK (sgNC controls) or lacking CSK (CSK KO) and treated with 100 nM BafA1 for 12 h. n Representative confocal images showing ZO-1 (red) and LC-3B (green) in WT and Y724F mutant Caco-2 cells expressing CSK (sgNC controls) or lacking CSK (CSK KO) and treated with 100 nM BafA1 for 12 h. Scale bars, 10 µm. Right panel: quantification of the ratios of ZO-1 and LC-3B co-localization to total ZO-1 puncta per cell. Data are presented as mean ± SD. n = 79 cells per group. Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. The quantified relative expression levels shown in western blots are indicated in red, and represents the results of at least three repeated experiments.