Figure 7. CD73-generated adenosine induces cortical F-actin.
(A and D) Representative immunoblots of total actin isolated from confluent HEC-1-A cells. (A) HEC-1-A cells treated with 50 μM 5′-AMP (untreated) or 50 μM 5′-AMP plus human recombinant CD73 for 50 minutes following 36 hours of hypoxia (1% O2, 5% CO2). DCF (10 μM) was included with treatments and controls (untreated). (D) HEC-1-A cells treated with 50 μM 5′-AMP plus DMSO (untreated) or 50 μM 5′-AMP plus DCF. Graphs in A and D show the densitometric data from 7 independent experiments each. (B and E) G-actin/F-actin ratios. (C and F) Alexa Fluor-conjugated phalloidin staining (F-actin) in HEC-1-A cells treated with human recombinant CD73 (C) or DCF (F). Scale bars: 20 μm. Graphs show F-actin intensity as assessed in 15 or more images (original magnification, ×20) using cellSens Dimension software, Adaptive Threshold. Data are expressed as the mean ± SEM. *P < 0.05 for F-actin and #P < 0.05 for G-actin compared with untreated cells, by 1-way ANOVA with Tukey’s post test (A and D) and by Holm-Sidak post test (B and E). **P < 0.001 for F-actin intensity, by 1-way ANOVA with Tukey’s post test (C and F).