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. 2017 Dec 26;9(5):6282–6297. doi: 10.18632/oncotarget.23676

Figure 2. LIMD1 is upregulated by NFκB and IRF4 in virus-transformed cells.

Figure 2

(A) LIMD1 expression is correlated with NFκB activity in B and T cell lines. (B) LMP1 induces LIMD1 expression. BJAB and Akata stable cell lines expressing LMP1 or control were generated by transfecting with pLXCN/Flag-LMP1 expression and control plasmids and selected with 2 mg/ml G418 for two weeks, and then subjected to immunoblotting analysis for LIMD1 protein. LMP1 in DG75 stable cells was induced by 1 µg/ml doxycycline for 48 h before collection. All cells were treated with 5 µM MG132 for 6 h before collection. (C) Knockdown of IRF4 in IB4 cells decreases LIMD1 protein level in EBV-transformed cells. IB4 stable cell lines expressing pTRIPz/shIRF4 or control were induced with 1 µg/ml doxycycline, and IRF4 and LIMD1 protein levels were evaluated. (D) Inhibition of NFκB activity decreases LIMD1 protein levels in virus-transformed cells. NFκB activity was inhibited with the NFκB-specific inhibitor Bay11-7085 at the concentration of 2.5 µM for 48 h. (E) IRF4 depletion by IRF-specific shRNA downregulates LIMD1 mRNA expression in JiJoye cells. JiJoye cells stably expressing control and IRF4 shRNAs were treated with 1 µg/ml doxycycline to induce shRNA expression for 96 h. Total RNAs were extracted for qPCR to quantitate IRF4 and LIMD1 mRNA expression. The average mRNA levels of the duplicates in shControl-expressing cells were set to 100%.