JCS028183 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Serum factors are not required for scrape-wound-induced polarity. Confluent cells were cultured in DMEM without serum for 24 hours, subject to an experimental scrape wound and fixed 4 hours later. Samples were processed as in Fig. 1B,C. Dashed line indicates random orientation. Means ± s.d. are from two independent experiments (ns, P=0.18; Student’s t-test).

• Supplemental Figure S2 -

  Fig. S2. Serum factors are not required for cell-cell-contact-induced polarity. Cells were cultured in the paired configuration in DMEM with or without serum and fixed 8 hours later. Samples were processed as in Fig. 1I-K. Dashed line indicates random orientation. Means ± s.d. are from two independent experiments (ns, P=0.052; Student’s t-test).

• Supplemental Figure S3 -

  Fig. S3. Mitosis is not responsible for orientation. (A) Number of cells after 24 hours as a percentage of initial number. Cells were treated for 2 hours with PBS or 10 µg/ml mitomycin C. Means ± s.d. are from two independent experiments (**P=0.0054; Student’s t-test). (B) Orientation 24 hours after plating, assessed as in Fig. 1J. Dashed line indicates random orientation. Means ± s.e.m. are from three independent experiments (ns, P>0.05, Student’s t-test).

• Supplemental Figure S4 -

  Fig. S4. Microtubules are responsible for centrosomal positioning. G0-synchronized cells treated with 1% DMSO or 10 µM nocodozole 3 hours, and fixed 8 hours, after plating; processed as in Fig. 1I-M. Bar, 25 µm.

• Supplemental Figure S5 -

  Fig. S5. E-cadherin involvement in cell-cell-contact-induced polarity is serum-independent. Cells infected with Ad-GFP or Ad-ED were G0 synchronized, plated into microwells, and fixed 8 hours after plating; processed as in Fig. 1I-K. Dashed line indicates random orientation. Means ± s.e.m. are from three independent experiments (*P=0.0112; Student’s t-test).

• Supplemental Figure S6 -

  Fig. S6. E-cadherin is required for the maintenance of orientation. Cells plated into microwells and allowed to polarize for 6 hours, then infected with either Ad-GFP or Ad-EΔ. Orientation was measured 24 hours after plating, assessed as in Fig. 1J. Dashed line indicates random orientation. Means ± s.e.m. are from three independent experiments (**P=0.0054; paired Student’s t-test).

• Supplemental Figure S7 -

  Fig. S7. Unpatterned Ad-GFP- but not Ad-EΔ-infected cells display E-cadherin localization to cell-cell contacts. Ad-GFP- or Ad-EΔ-infected cells fixed 24 hours after plating. Bar, 25 µm.

• Supplemental Figure S8 -

  Fig. S8. Actin-cytoskeleton involvement in cell-cell-contact-induced polarity is serum-independent. G0-synchronized cells cultured in the presence of DMSO or 1 µM cytochalasin D and fixed 8 hours after plating; processed as in Fig. 1I-K. Dashed line indicates random orientation. Means ± s.e.m. are from three independent experiments (**P=0.0014; Student’s t-test).

• Supplemental Figure S9 -

  Fig. S9. Myosin-II activity is not required for cell-cell polarity. (A) G0-synchronized cells cultured in the presence of PBS or 50 µM Y27632 and fixed 8 hours after plating; processed as in Fig. 1I-K. (B) G0-synchronized cells treated with 1% DMSO or blebbistatin 3 hours, and fixed 8 hours, after plating; processed as in Fig. 1I-K (ns, P>0.05; ANOVA). Dashed lines indicate random orientation. Means ± s.e.m. are from three independent experiments.

• Supplemental Figure S10 -

  Fig. S10. Cdc42 involvement in cell-cell-contact-induced polarity is serum-independent. siRNA-transfected cells fixed 8 hours after plating; processed as in Fig. 1I-K. Dashed line indicates random orientation. Means ± s.e.m. are from three independent experiments (*P<0.05; ns, P >0.05; ANOVA and Tukey’s HSD).

• Movie 1 -

  Movie 1. Cells display polarized ruffling relative to cell-cell contact. Phase contrast, time-lapse microscopy of cells in the paired configuration. The playback rate is 100×. Bar, 25 µm.

• Movie 2 -

  Movie 2. Cells migrate in a directed fashion following release from pairs. Cells cultured in the paired configuration for 20 hours and released from the pattern via electroactive switching. Dashed lines indicate initial pattern, and text highlights pair shown in Fig. 2. The playback rate is 3000×. Bar, 25 µm.

• Movie 3 -

  Movie 3. Ad-GFP-infected cells display polarized ruffling. Phase contrast, time-lapse microscopy of Ad-GFP-infected cells cultured in the paired configuration. The playback rate is 100×. Bar, 25 µm.

• Movie 4 -

  Movie 4. Ad-EΔ-infected cells do not display polarized ruffling. Phase contrast, time-lapse microscopy of Ad-EΔ-infected cells cultured in the paired configuration. The playback rate is 100×. Bar, 25 µm.