JCS031427 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Flow cytometry measurement data for generality of transmembrane protein transfer. The histograms give the raw data summarized in Fig. 4, except those that have been shown in Figs 1, 2, 5. Human cells BE(2)C/CHC(0.2), SKOV3 and A431 or stably transfected CHO cells CHO-CD36⁺, CHO-ICAM1 and CHO-α4/GFP were co-cultured with one another or with other recipient cells PC12 cells, NIH3T3, CHO cells, HUVECs, iHUVECs, LVECs and GMSCs. Protein transfer was assessed by flow cytometry using fluorescent antibodies against the respective transferred protein. Donor cells are represented by dashed pink line, recipient cells by solid black line and the co-culture by thick green line. (H,T) Co-cultures with different ratios of donor cells to recipient cells preserving total cell number in the co-culture. In H, CHO-α4/GFP cells and A431 cells were seeded at the ratio of 1:3 (thick green line) or 3:1 (thick blue line). In T, A431 cells were co-cultured with LVECs at the ratio of 1:3 (thick brown line), 1:1 (thick green line) or 3:1 (thick blue line).

• Supplemental Figure S2 -

  Fig. S2. Cytoplasmic labeling of live cells with a Fluorescein-conjugated dextran. Primary HUVECs were labeled with 10 kDa dextran at a concentration of 10 mg/ml for 2 hours at 37°C. (A) Epifluorescence image of labeled HUVECs; (B) Phase-contrast image of the same cells.

• Supplemental Figure S3 -

  Fig. S3. CHO-ICAM1 cells and CHO cells co-cultured in a 50 cm² flask at the ratio of 1:1 with initial cell number 0.5×10⁶ (thick green line) and 1×10⁶ (thick blue line).

• Supplemental Figure S4 -

  Fig. S4. EFGR transfer from A431 to NIH3T3 cells by altering membrane fluidity. A431 cells were co-cultured with NIH3T3 cells for 2 days without and in presence of 25mM linoleic acid. (Blue line, before co-culture; green line, 2 days after co-culture; red line, 2 days after co-culture in the presence of 25 mM linoleic acid).

• Supplemental Figure S5 -

  Fig.S5. Effect of adhesion molecules on membrane protein transfer. iHUVECs were co-cultured with IBB platelets followed by extensive washing and immunostaining with a PE-conjugated anti-CD36 antibody. TNFα treatment, enhanced surface expression of LFA-1 on iHUVEC cells (data not shown), which results in enhanced transfer of CD36 from IBB platelets to iHUVECs. Anti-human-LFA1 antibody treatment reversed the phenomenon of elevated protein transfer. In the flow cytometry measurements for PE-associated CD36, the black dotted line represents iHUVECs, the green line iHUVECs co-cultured with platelets, the blue dashed line iHUVECs pretreated with TNFα (10 ng/ml for 16 hours) followed by co-culture with platelets, and the pink thick line iHUVECs pretreated with TNFα followed by co-culture with platelets in the presence of anti-human LFA-1 antibody (10 µg/ml).

• Movie 1 -

  Movie 1. Transient membrane adhesion during local cellular deformation in high confluency. iHUVECs were cultured in fresh medium with environmental control under an epifluorescence microscope. The movie was generated with time-lapse microscopy, stitching together 63 images taken under ×40 objective over 62 minutes. White arrows indicate formation of retraction nanotubules. Black arrow indicates transfer of a possible large organelle through a nanotubule.

• Movie 2 -

  Movie 2. Transient membrane adhesion during cellular locomotion in low confluency. SKOV3 cells were cultured in fresh medium with environmental control under epifluorescence microscope. The movie was generated with time-lapse microscopy, stitching together 46 images taken under ×10 objective over 225 minutes. White arrow indicates formation of a nanotubule after cell retraction, after cell forms multiple smaller connections.