Fig. 6: α-Catenins interact with LC3 via two LIR regions.

a Representative images of endogenous CTNNA3–LC3 colocalization in MCF10A cells treated with BafA1. The control cells were treated with DMSO. Scale bars are 10 µm. The graphs on the right show the image intensities for all channels (CTNNA3, LC3 and DAPI). Scale bars are 10 µm. The experiment was repeated twice with similar results. b Quantification of colocalization for the experiment in (a). The Pearson’s and Mander’s (fraction of CTNNA3 overlapping LC3) coefficients were quantified for n = 45 cells (DMSO) and n = 38 cells (BafA1). Bars represent the mean ± s.e.m. (****P < 0.0001; two-tailed t-test). c Representative confocal images of endogenous CTNNA3–LC3 colocalisation in MCF10A cells exposed to either Tat-scrambled or Tat-beclin1 peptide (20 µM, 48 h). Scale bars are 10 µm. The graph on the right shows the fluorescent intensity for each channel (red-CTNNA3, green-LC3 and blue-DAPI) along the indicated distances. d Co-immunoprecipitation of endogenous LC3 with CTNNA1 in BafA1-treated MCF10A cells. The experiment was repeated three times with similar results. e Co-immunoprecipitation of endogenous LC3 with wild-type and Flag-CTNNA1 mutants in BafA1-treated MCF10A cells. MCF10A cells were initially transfected with empty Flag (control vector), wild-type Flag-CTNNA1 or the indicated Flag-CTNNA1 mutants. The Flag-tagged proteins were pulled down using the Flag-Trap technology. The experiment was repeated three times with similar results. f Co-immunoprecipitation of endogenous LC3 with wild-type and mEm-CTNNA1 mutants. MCF10A cells were transfected with empty mEm (control vector), wild-type mEm-CTNNA1 or the indicated mEm-CTNNA1 mutants. The mEm-tagged proteins were pulled down using the GFP-Trap technology. The experiment was repeated three times with similar results. g 3D structure of the LIR region (417 KEYAQV 422) and its conservation among various species of α-catenins. h Representative images of mEm-CTNNA1—endogenous LC3 colocalization in MCF10A cells transiently transfected with either wild-type or Y419A-V422A mEm-CTNNA1. Scale bars are 10 µm. i The Pearson’s (PC), Overlap (OC) and Mander’s (M1—fraction of LC3 overlapping mEm-CTNNA1, and M2—fraction of mEm-CTNNA1 overlapping LC3) coefficients for MCF10A cells treated as in (h). Thirty-one wild-type cells and 21 Y419A-V422A mutant cells were quantified over three independent experiments. Bars represent the mean ± s.e.m. (****P < 0.0001, ***P < 0.001; two-tailed t-test). Exact P values for asterisks: i 0.0009.