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. 2014 May 13;9(5):e97350. doi: 10.1371/journal.pone.0097350

Figure 8. Knockdown of STAT3 reduces the inhibitory effect of IL-22 on H. pylori-induced CCL20.

Figure 8

A, Knockdown of STAT3 with shRNAs. AGS cells were infected with lentivirus carrying STAT3-specific shRNA clone 7 or clone 8 for 24 h. After puromycin selection for another 48 h, total cell lysates were subjected to immunoblotting with anti-STAT3. The membrane was stripped and re-probed with anti-β-actin (upper panel). The cells with STAT3 knockdown were infected with H. pylori in the presence or absence of IL-22, and the culture supernatants were collected for the determination of CCL20 concentration by ELISA. The lower panel shows the inhibitory efficiency (% inhibition), which was calculated as follows: [(CCL20 concentration in H. pylori-infected cells without IL-22 − CCL20 concentration in H. pylori-infected cells with IL-22) / CCL20 concentration in H. pylori-infected cells without IL-22]×100. Data represent the mean ± SEM and reflect one representative of four independent experiments. *, p<0.02 versus sh-GFP. B, Knockdown of STAT3 with siRNAs. Similar to (A) except that siRNA#a and siRNA#b were used to knockdown STAT3. The lower panel shows the inhibitory efficiency, which was calculated as described in (A). Data represent the mean ± SEM and reflect one representative of two independent experiments. *, p<0.002 versus si-control; **, p<0.0001 versus si-control. C, AGS cells were transfected with siRNA#b together with either the expression vector encoding wild-type STAT3 (WT-STAT3) or the expression vector encoding dominant-negative STAT3 (DN-STAT3). Total cell lysates were subjected to immunoblotting with anti-phospho-STAT3 and the membrane was stripped and re-probed with anti-STAT3 (upper panel). At 36-h post-transfection, the transfectants were infected with H. pylori in the presence or absence of IL-22 followed by the determination of CCL20 in cell culture supernatants 6-h post-infection (lower panel). The inhibitory efficiency was calculated as described in (A). Data represent the mean ± SEM from three independent experiments. *, p<0.01 versus vector.