Figure 6. Cx31R42P mediates the lucifer yellow uptake.

(A) Dye uptake with high Ca²⁺ _o (High Ca²⁺) and 15 min after high Ca²⁺ _o is washed (Wash-out). Images of Hoffman modulation contrast (A1, 3, 5, 7, 9, 11, 13, 15) and fluorescence (A2, 4, 6, 8, 10, 12, 14, 16) are shown. Cells tested include control Hela, Cx31WT (WT) and Cx31R42P (R42P) cells. Note that round dead cells were stained by both lucifer yellow (LY) and Dextran-FITC (DX). Hela and Cx31WT cells do not take up LY no matter high Ca²⁺ _o treatment is washed out or not (1–8). Cx31R42P cells take up LY (11, 12) but not DX (15, 16) after high Ca²⁺ _o is washed out. No LY uptake is observed in Cx31R42P stable cell line continuously to be incubated under high Ca²⁺ _o condition (9, 10). (B) Pharmacological analysis of Cx31R42P hemichannels. Cells expressing Cx31R42P were pretreated with either control solvent DMSO, 200 µM connexin hemichannel blocker FFA, 10 µM P2X7 ATP receptor blocker KN-62, or 100 µM ROS scavenger BHA for 30 min. Note that FFA (3, 4) and BHA (7, 8) completely block LY uptake, but KN-62 shows little effect on LY uptake (5, 6). Scale bars = 20 µm. (C) Quantification of cells uptaking LY or DX. (D) Quantification of Cx31R42P cells uptaking LY after treatment with various pharmacological inhibitors. Error bars represent SEM. Two stars: P<0.01, three stars: P<0.001.