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. 2019 Apr 11;15:28–38. doi: 10.1016/j.isci.2019.04.013

Figure 3.

Figure 3

Characteristics of GRAPHIC Signal

(A and B) Time-lapse images of GRAPHIC signal in constructing (A) or disrupting (B) intercellular contact between n-GRAPHIC-expressing LLCPK1 cells (red nuclei) and c-GRAPHIC-expressing LLCPK1 cells (blue nuclei). Upper panels are merged images of bright-field (differential interference contrast), RFP and BFP fluorescence. Bottom panels are GFP fluorescent images. In (A), two cell lines first contacted at time 0. In (B), EDTA was administrated at time 0 (final concentration; 5mM).

(C) Quantification of relative membrane retraction velocity during 10–12 min after EDTA ion chelation. H2B-mCherry-expressing (without GRAPHIC) LLCKP1 cells (n = 51) were used as control. Membrane retraction velocity of GRAPHIC was calculated between n- and c-GRAPHIC-expressing cells (n = 33). Student's unpaired t test.

(D-F) GRAPHIC signal still remains in completely dissociated cells. Dissociated (with 5 mM EDTA, without trypsin) LLCPK1 cells from single culture of RFP+ (n-GRAPHIC expressing) or BFP+ (c-GRAPHIC expressing) cell line and co-culture of both cell lines were subjected to flow cytometry (single cultured RFP+; n = 4751, single cultured BFP+; n = 4851, co-cultured RFP+; n = 6062, co-cultured BFP+; n = 5131). Both microscope observation (D and E) and histogram of GFP intensity (F) of sorted cells showed co-culture dependent GRAPHIC signal in dissociated single cell. Scale bars, 20 μm.