Fig 2: Relocation of Lyn to the plasma membrane by cell-cell interactions. (a,c) Cells cultured as described below were stained with rabbit anti-Lyn and anti-E-cadherin antibodies. Scale bars, 10 µm. (a) MDCK/TR/Lyn cells cultured as confluent monolayers (polarized cells) were treated with 1 µg/ml Dox for 8 h, detached by trypsinization and subsequently cultured in a spinner flask with calcium- and serum-free IMDM (single cell suspension culture). Single suspended cells were transferred into polyHEMA-coated culture dishes and cultured in IMDM-5% bovine serum (calcium replenishment) for the indicated time (static suspension culture). (b) Single suspended cells cultured as described in (a) (single cell suspension culture) were transferred into polyHEMA-coated culture dishes and cultured in IMDM-5% bovine serum (calcium replenishment and static suspension culture) for 20 h. Cells were stained with anti-Lyn antibody and propidium iodide. The number of cells exhibiting predominant accumulation of Lyn to endomembranes was counted. The data represent the mean ratio ± S.D. from three independent experiments in which at least 50 cells for each category were scored. The significant differences are calculated by one-tailed Student’s t test. (c) Cells cultured as described in (a) (single cell suspension culture) were transferred into polyHEMA-coated culture dishes and cultured for 16 h in IMDM containing 5% bovine serum (static suspension culture, Control), IMDM containing 5% bovine serum in the presence of 4 mM or 10 mM EGTA (static suspension culture, Calcium deprivation), or serum-free IMDM (static suspension culture, Serum-free). Scale bars, 10 µm.

Fig 3: Endomembrane localization of Lyn through Rab11-positive endosomes. (a,b) Double immunofluorescence staining was performed using mouse monoclonal or rabbit polyclonal anti-Lyn antibody (green) together with the indicated antibody (red). The Pearson’s R value determined from the green and red signals in the region of interest (ROI; blue square) is shown in each image. Scale bars, 10 µm. (a) MDCK/TR/Lyn cells sparsely cultured (non-polarized cells) were treated with 1 µg/ml Dox for 10 h. (b) MDCK/TR/Lyn cells cultured to confluence (polarized cells) were treated with 1 µg/ml Dox for 8 h, detached with trypsin and subsequently cultured in a spinner flask with calcium- and serum-free IMDM containing 1 µg/ml Dox for 2 h. (c) MDCK/TR/Lyn cells transiently transfected with the indicated plasmid were cultured to confluence and then treated with 1 µg/ml Dox for 8 h, detached with trypsin and subsequently cultured in a spinner flask with calcium- and serum-free IMDM for 2 h. (i) HA-tagged Rab11-wt (wild-type Rab11) and HA-tagged Rab11(S25N) (dominant-negative Rab11) were stained with rabbit anti-HA antibody (red). The images of GFP-conjugated Rab5a(S34N) (dominant-negative Rab5) and GFP-conjugated Rab7(N125I) (dominant-negative Rab7) were pseudocoloured in red. Lyn was stained with mouse monoclonal anti-Lyn antibody (green). The Pearson’s R value determined from the green and red signals in the ROI (blue square) is shown in each representative image of Rab11- or Rab11(S25N)-expressing cell. Scale bars, 10 µm. (ii) The plots represent the Pearson’s R values determined from Lyn and Rab11-wt or Lyn and Rab11(S25N) in the ROI in each cell. n, cell number.

Fig 4: Translocation of Lyn to endomembranes by calcium deprivation. (a) MDCK/TR/Lyn cells cultured to confluence (polarized cells) were treated with 1 µg/ml Dox for 8 h, then treated with 4 mM EDTA or 4 mM EGTA for 15 min and subsequently cultured in calcium- and serum-free IMDM for 2 h (calcium deprivation). Cells were fixed and stained with rabbit anti-Lyn antibody (green) and anti-E cadherin antibody (red). Scale bars, 10 µm. (b) MDCK/TR/Lyn cells cultured to confluence (polarized cells) were treated with 1 µg/ml Dox for 8 h, then treated with 4 mM EDTA for 15 min and further incubated in calcium- and serum-free IMDM for 2 h (calcium deprivation), and subsequently incubated with IMDM containing 5% bovine serum for 10 min and 120 min (calcium replenishment). Cells were stained with anti-Lyn and anti-E-cadherin antibodies. Scale bars, 10 µm. (c) MDCK/TR/Lyn cells cultured to confluence (polarized cells) were treated with 1 µg/ml Dox for 8 h, then treated with 4 mM EDTA for 15 min and further incubated in calcium- and serum-free IMDM for 2 h (calcium deprivation), and subsequently incubated with IMDM containing 5% bovine serum for 20 h (calcium replenishment). Cells were stained with anti-Lyn antibody (green) and propidium iodide. The number of cells in which Lyn was predominantly accumulated to endomembranes was counted. The data represent the mean ratio ± S.D. from three independent experiments in which at least 50 cells for each category were scored. The significant differences are calculated by one-tailed Student’s t test.

Fig 5: Establishment of an MDCK cell line expressing inducible Lyn. MDCK/TR/Lyn cells were grown in sparse and confluent cultures, which generates non-polarized cells and polarized cells, respectively. The cells were subsequently treated with 1 µg/ml doxycycline (Dox) for the indicated times. Whole cell lysates were subjected to Western blot analysis using the indicated antibodies. TR, tetracycline repressor. Molecular size markers are shown in kDa. The amount of p53/p56 Lyn in Dox-treated cells is expressed as the value relative to that in cells without Dox treatment after normalization with actin levels. For better clarity and conciseness of the presentation, cropped blots are shown. The full-length blots are presented in Supplementary Fig. S6.