FIG 2.

Knockdown (KD) of CD73 expression in T84 intestinal epithelial cells impairs intracellular replication of and translocation by Salmonella. (A) Lentiviral shRNA-mediated KD of CD73 (shCD73) in T84 intestinal epithelial cells results in diminished CD73 mRNA relative to the short hairpin nontargeting control (shNTC). **, P < 0.005 as determined by Student's t test. (B) CD73 KD results in diminished phosphohydrolysis of 1,N⁶-etheno-5′-AMP to 1,N⁶-ethenoadenosine, similar to the effect of the specific CD73 inhibitor adenosine 5′-(α,β-methylene)diphosphate (APCP). ***, P < 0.0005 for conversion by APCP-treated monolayers as well as CD73 KD monolayers relative to the control, as determined by Student's t test. (C) Initial invasions of control and CD73 KD monolayers by Salmonella are equivalent, as determined by a gentamicin protection assay. Monolayers were harvested at 1 h postinfection, as described in Materials and Methods. (D) Intracellular replication of Salmonella is decreased after CD73 KD in T84 cells at 8 h postinfection compared to that in control cells in an in vitro gentamicin protection assay. Recovered CFU were normalized to the CFU recovered at 1 h postinfection. **, P < 0.005 as determined by two-way ANOVA. (E) Translocation of Salmonella bacteria across polarized T84 monolayers on semipermeable supports is reduced 100-fold after CD73 KD. Data represent the means and standard deviations of results from at least three independent experiments. ***, P < 0.0005 as determined by Student's t test.