Journal: bioRxiv
Article Title: A ZO-2 scaffolding mechanism regulates the Hippo signalling pathway
doi: 10.1101/2024.10.17.618965
Figure Lengend Snippet: A) ZO-2 is not essential for YAP to enter the nucleus at low cell density. Representative immunostaining of DAPI, ZO-2 and YAP in control and ZO-2 knockdown (KD) under low cell density conditions. B) Quantification of the nuclear to cytoplasmic ratio of YAP intensity at sparse cell density in control and ZO-2 knockdown conditions as shown in (A). Data in box-and-whisker plots show median (midline), 25th to 75th percentiles (box), and minimum and maximum (whiskers) from n>60 cells for each condition, analysed with the Mann-Whitney U test. C) Representative images of DAPI, YAP, and ZO-1 immunostaining of control and ZO-2 depleted cells at confluent cell density 72 hours after seeding. (ZO-1 is at the apical plane, whereas YAP and DAPI are at the lateral plane). D) Quantifying the YAP nuclear to cytoplasmic ratio in cells shown in (C). Data in box-and-whisker plots show median (midline), 25th to 75th percentiles (box), and minimum and maximum (whiskers) from n = 20 cells for each condition, analysed with Student’s t-test. E) Representative cross-sectional views of YAP, DAPI, and gp135 immunostaining in control and ZO-2 depleted cells at confluent cell density 72 hours after seeding. F) Representative immunoblots for ZO-2, YAP, S127-phosphorylated YAP (pYAP S127), MST1, T183/T180-phosphorylated MST1/2(pMST1 T183 / pMST2 T180), and GAPDH from control and ZO-2 depleted confluent monolayer. Data is representative of 5 independent experiments. G) Representative immunoblots for ZO-2 and ZO-1 knockdown in MDCK cells. H) ZO-2 knockdown in confluent monolayer increases the cell number compared to the corresponding controls in the 48 and 72-hour time points. Box-and-whisker plots show median (midline), 25th to 75th percentiles (box), and minimum and maximum (whiskers). All data are from 3 independent experiments, with three technical replicates for each condition (n = 9). Data was analysed with the Mann-Whitney U test. I) ZO-2 knockdown in confluent monolayer increases the fold change of cell proliferation in the 48 and 72-hour time points when compared to the corresponding control. Box-and-whisker plots show median (midline), 25th to 75th percentiles (box), and minimum and maximum (whiskers). All data is from 3 independent experiments, with three technical replicates for each condition (n = 9). Data was analysed with the Mann-Whitney U test. J) ZO-2 knockdown promotes cell proliferation compared to ZO-1 knockdown and control. Proliferation curve of MDCK cells over 72 h. Error bars represent mean ± s.e.m from 3 independent experiments, each with three technical replicates (n=9). Both graphs represent the number of viable cells/ml and fold change relative to seeding cell density. All data are from at least three independent experiments. Scale bar, 25 μm.
Article Snippet: Rabbit polyclonal anti-LATS1 antibody (#9153), rabbit polyclonal anti-phospho-LATS1 (Ser909) antibody (#9157), rabbit polyclonal anti-phospho-YAP (Ser127) antibody (#4911), rabbit polyclonal anti-MST1 antibody (#3682), rabbit polyclonal anti-phospho-MST1 (Thr183) and MST2 (Thr180) antibody (#3681), rabbit polyclonal anti-Histone H3 antibody (#9715), rabbit polyclonal anti-phospho-Histone H3 (Ser10) antibody (#9701) and rabbit monoclonal anti-YAP antibody (#14074) were purchased from Cell Signalling Technology.
Techniques: Immunostaining, Control, Knockdown, Whisker Assay, MANN-WHITNEY, Western Blot
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