![Identification of E- and N-cadherin interactors, roles of NuMA and its interactors LGN and Band 4.1 in BC elongation, and E-cadherin function in spindle orientation, related to Fig. 4. (A) BioID analysis of E- and N-cadherin interactors. Streptavidin blot of Can 10 cells expressing TurboID-tagged E- or N-cadherin. MWs of marker proteins are indicated in kDa. (B) Analysis scheme for the E- and N-cadherin BioID data. Raw iBAQ values of proteins with ≥2 unique peptides were log2-normalized and filtered using two sequential criteria. Proteins were retained if, compared to both negative controls [biotin(−) and untransfected], they showed (1) significant differential expression [adjusted P < 0.05, fold change (FC) >2] or (2) detection in both replicates with complete absence in controls. (C) Venn diagram of filtered proteins showing overlap between E-cadherin and N-cadherin, as well as those specific to each. Numbers in the circles indicate overlapping proteins (620), E-cadherin–specific proteins (133), and N-cadherin–specific proteins (81). (D) Depletion of NuMA or RhoA does not affect cell proliferation. Can 10 cells transfected with NuMA or RhoA siRNAs were cultured for 36 h or 72 h, stained with DAPI and WGA, and analyzed for cell proliferation. Data represent means ± SD from three independent experiments. (E) Knockdown efficiencies of LGN, 4.1R, and 4.1G in Can 10 cells. Lysates from siRNA-transfected cells were analyzed by immunoblotting using antibodies against LGN, 4.1R, 4.1G, and α-tubulin. The asterisk marks the AGS3 isoform of LGN. MWs of marker proteins are indicated in kDa. (F) LGN, 4.1R, and 4.1G function in the same pathway to promote BC elongation. Shown are representative confocal images of Can 10 cells transfected with LGN siRNA alone or in combination with both 4.1R and 4.1G siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and 4.1G. (G) Quantification of polarity and BC structures in single and double knockdown cells. Data represent means ± SD from four independent experiments (≥742 cells per condition). (H) E-cadherin depletion alters mitotic spindle orientation. Rose diagrams (left) and combined scatter and box-and-whisker plots (right) show spindle angle distributions relative to the BC. Values are from two independent experiments (≥84 cells per condition) and were analyzed using the Wilcoxon rank-sum test with Holm’s correction. Scale bars, 20 µm. P values are indicated at the top of each graph; n.s., not significant. iBAQ, intensity-based absolute quantification; MWs, molecular weights. Source data are available for this figure: SourceData FS3. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jcb/225/8/10.1083_jcb.202509170/4/jcb_202509170_figs3.png?Expires=1783123264&Signature=a1B0ylzCD~E9ItyLO3dF7qLaUGedHD4012mRmhW7TNmOabKZI4Kc7ra2~fiGqao6EOBMX~kRMvJj6YUrmIiug0wt0ZsZ7hSavoovNFArzDMREzoFA~jnyf5sZL-i99SQTpgy5GfzE0Hj0EclfFTCWcH0NKRulWAMv7RG8W0AjGDNZejGLawQ5B0jmumgvfhCg7xbFo6AHtdEi8mN-nJFDnFVjsvEq4Hx0kDOO9srEGN131d4~oVfrNPo3KFT2Txeoxb9hsc0MIIl6KdK2sfLIfeMcCpcAnuhsJodQDmTLVh9wC~8lPXRRlk0v30UjDxlnkDbfFbTnvI5NQ-Z0DryuA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows immunoblot images detecting biotinylated proteins associated with E-cadherin–TurboID and N-cadherin–TurboID constructs under biotin treatment conditions. Panel B outlines the analysis scheme for the E- and N-cadherin BioID data, detailing the filtering criteria for proteins. Panel C presents a Venn diagram showing the overlap and specific proteins for E-cadherin and N-cadherin. Panel D consists of bar graphs depicting cell proliferation data for Can 10 cells transfected with NuMA or RhoA siRNAs, with data points representing means and standard deviations from three independent experiments. Panel E shows immunoblot images of lysates from siRNA-transfected cells, with antibodies against LGN, 4.1R, 4.1G, and α-tubulin, and molecular weights of marker proteins indicated in kilodaltons. Panel F displays representative confocal images of Can 10 cells transfected with various siRNAs, stained with DAPI and antibodies against aPKC and 4.1G. Panel G includes bar graphs quantifying polarity and basal cell structures in single and double knockdown cells, with data points representing means and standard deviations from four independent experiments. Panel H presents rose diagrams and combined scatter and box-and-whisker plots showing spindle angle distributions relative to the basal cell, with values from two independent experiments analyzed using the Wilcoxon rank-sum test with Holm correction.
Identification of E- and N-cadherin interactors, roles of NuMA and its interactors LGN and Band 4.1 in BC elongation, and E-cadherin function in spindle orientation, related to Fig. 4. (A) BioID analysis of E- and N-cadherin interactors. Streptavidin blot of Can 10 cells expressing TurboID-tagged E- or N-cadherin. MWs of marker proteins are indicated in kDa. (B) Analysis scheme for the E- and N-cadherin BioID data. Raw iBAQ values of proteins with ≥2 unique peptides were log2-normalized and filtered using two sequential criteria. Proteins were retained if, compared to both negative controls [biotin(−) and untransfected], they showed (1) significant differential expression [adjusted P < 0.05, fold change (FC) >2] or (2) detection in both replicates with complete absence in controls. (C) Venn diagram of filtered proteins showing overlap between E-cadherin and N-cadherin, as well as those specific to each. Numbers in the circles indicate overlapping proteins (620), E-cadherin–specific proteins (133), and N-cadherin–specific proteins (81). (D) Depletion of NuMA or RhoA does not affect cell proliferation. Can 10 cells transfected with NuMA or RhoA siRNAs were cultured for 36 h or 72 h, stained with DAPI and WGA, and analyzed for cell proliferation. Data represent means ± SD from three independent experiments. (E) Knockdown efficiencies of LGN, 4.1R, and 4.1G in Can 10 cells. Lysates from siRNA-transfected cells were analyzed by immunoblotting using antibodies against LGN, 4.1R, 4.1G, and α-tubulin. The asterisk marks the AGS3 isoform of LGN. MWs of marker proteins are indicated in kDa. (F) LGN, 4.1R, and 4.1G function in the same pathway to promote BC elongation. Shown are representative confocal images of Can 10 cells transfected with LGN siRNA alone or in combination with both 4.1R and 4.1G siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and 4.1G. (G) Quantification of polarity and BC structures in single and double knockdown cells. Data represent means ± SD from four independent experiments (≥742 cells per condition). (H) E-cadherin depletion alters mitotic spindle orientation. Rose diagrams (left) and combined scatter and box-and-whisker plots (right) show spindle angle distributions relative to the BC. Values are from two independent experiments (≥84 cells per condition) and were analyzed using the Wilcoxon rank-sum test with Holm’s correction. Scale bars, 20 µm. P values are indicated at the top of each graph; n.s., not significant. iBAQ, intensity-based absolute quantification; MWs, molecular weights. Source data are available for this figure: SourceData FS3.