G(q/11)-coupled P2Y₂ nucleotide receptor inhibits human keratinocyte spreading and migration

Supplemental Data

Files in this Data Supplement:

• Movie 1 - ( 06-7476SuppMovie1.mov; 3.14 MB) ATP induces a transiant lamellipodium collapse and a prolonged inhibition of its dynamics. Hacat cells, cultured in serum-containing medium, were imaged on the temperature-controlled stage of an inverted microscope at 37°C. Phase contrast images were acquired every 2 minutes, 20 minutes before and 90 minutes after ATP addition (100 μM). This movie displays 6 frames per second.
• Figure S1 - ( 06-7476FigS1.tif; 4.0 MB) Comparison of the distribution of α6, α3 and αv integrins in HaCat cells cultured in growth factor-containing medium in the presence (UTP) or the absence (Control) of 10 μM UTP. α6 integrins are localized in hemidesmosome-like structures that are not affected by extracellular nucleotides. α3 integrins are concentrated at the cell-cell junction and at the lamellipodium edge (white arrows). Note that in UTP-treated cells, α3 is only found at the cell-cell contacts. UTP also induce the disorganisation of αv-containing focal contacts.
• Figure S2 - ( 06-7476FigS2.tif; 1.57 MB) A) ATP and UTP dowregulate in a dose dependent manner Erk_1,2 and Akt phosphorylation by serum growth-factors. Serum-starved HaCat cells (ctrl) were stimulated for 10 minutes with 10 % serum (FCS) in the presence of the indicated concentration of either ATP or UTP. The phosphorylation level of Akt and Erk_1,2 was evaluated by immunoblot using antibodies against phospho-Akt (ser-473) and phospho-Erk_1,2 (thr-202/tyr-204). B) ATP and UTP delay growth factor-induced activation of Erk_1,2 in MK cells. Serum-starved MK cells were stimulated for the indicated period of time with either serum alone (FCS) or supplemented with ATP (FCS+ATP) or UTP (FCS+UTP) (100μM). The amount of phospho-Erk_1,2 (thr-202/tyr-204) in the cell lysates was revealed by immunoblot. Western blot with anti-tubulin antibody was performed as loading control.