Figure 2.
Identification of IL-27Rα and JAM2 as RELA-activation responsive genes. RELA was downregulated in KMS18 cells using retroviral-mediated shRNA knockdown. Cells expressing shRELA and control shRen could be identified as YFP+dsRed+ (shRNA) in comparison with YFP+dsRed− counterpart cells (WT) that do not express shRNA (supplemental Figure 3A). On day 0, day 7, and day 12 after induction of shRNA, cells were stained for IL-27Rα, JAM2, or isotype control and assessed via flow cytometry. (A-B) Representative histograms are shown for (A) IL-27Rα and (B) JAM2 on day 12. Graphs show triplicate mean fluorescence intensity values ± standard deviation for IL-27Rα or JAM2 from shRNA (YFP+dsRed+) normalized to WT (YFP+dsRed−) from corresponding shRen, shRELA#1 and shRELA#2 expressing cells. Similar findings were also observed in U266 and JJN3 cell lines (supplemental Figure 3B). (C-D) On day 5 after induction of shRNA expression, cells were stimulated with 50 ng/mL rIL-27 for 30 minutes before fixing and intracellular staining for phosphorylated STAT1 (p-STAT1) and p-STAT3. shRNA– (YFP+dsRed-) and shRNA+ (YFP+dsRed+) cells from shRen and shRELA-transduced cells were compared for the extent of p-STAT1 and p-STAT3 expression. Representative histograms and triplicate values ± standard deviation are shown. ∗P < .05; ∗∗∗P < .001using one-way ANOVA and Tukey multiple comparison test. ANOVA, analysis of variance; ns, nonsignificant; WT, wild type.

Identification of IL-27Rα and JAM2 as RELA-activation responsive genes. RELA was downregulated in KMS18 cells using retroviral-mediated shRNA knockdown. Cells expressing shRELA and control shRen could be identified as YFP+dsRed+ (shRNA) in comparison with YFP+dsRed− counterpart cells (WT) that do not express shRNA (supplemental Figure 3A). On day 0, day 7, and day 12 after induction of shRNA, cells were stained for IL-27Rα, JAM2, or isotype control and assessed via flow cytometry. (A-B) Representative histograms are shown for (A) IL-27Rα and (B) JAM2 on day 12. Graphs show triplicate mean fluorescence intensity values ± standard deviation for IL-27Rα or JAM2 from shRNA (YFP+dsRed+) normalized to WT (YFP+dsRed−) from corresponding shRen, shRELA#1 and shRELA#2 expressing cells. Similar findings were also observed in U266 and JJN3 cell lines (supplemental Figure 3B). (C-D) On day 5 after induction of shRNA expression, cells were stimulated with 50 ng/mL rIL-27 for 30 minutes before fixing and intracellular staining for phosphorylated STAT1 (p-STAT1) and p-STAT3. shRNA– (YFP+dsRed-) and shRNA+ (YFP+dsRed+) cells from shRen and shRELA-transduced cells were compared for the extent of p-STAT1 and p-STAT3 expression. Representative histograms and triplicate values ± standard deviation are shown. ∗P < .05; ∗∗∗P < .001using one-way ANOVA and Tukey multiple comparison test. ANOVA, analysis of variance; ns, nonsignificant; WT, wild type.

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