Skip to main content
. 2022 Jan 20;298(3):101611. doi: 10.1016/j.jbc.2022.101611

Figure 5.

Figure 5

TRIM34 regulates IAV induced NLRP3 inflammasome and programmed cell death pathways via ZBP1 in vitro.A, scramble-KO THP-1 macrophages (ZBP1+/+) and ZBP1-deficient THP-1 macrophages (ZBP1−/−) were transfected with siRNA-control (si-ctrl) or siRNA-TRIM34 for 24 h. Then, cells were infected with IAV (MOI = 1) for 24 h or left uninfected. IL-1β and IL-18 protein levels in cell culture supernatants were determined by ELISA. B, ZBP1+/+ and ZBP1−/− THP-1 macrophages were transfected with siRNA-control (si-ctrl) or siRNA-TRIM34 for 24 h. Then, cells were infected with IAV (MOI = 1) for 24 h or left uninfected. Mature IL-1β and p20 in supernatants or pro-IL-1β and pro-Casp-1 in lysates were determined by Western blot. C and D, experiments were performed as described in (A) and (B), except that cells were transfected with vector control or Flag-TRIM34. E, ZBP1+/+ and ZBP1−/− THP-1 macrophages were transfected with siRNA-control (si-ctrl) or siRNA-TRIM34 for 24 h. Then, cells were infected with IAV (MOI = 1) for 24 h or left uninfected. IL-6 and TNF-α protein levels in cell culture supernatants were determined by ELISA. F, experiments were performed as described in (E), except that cytosolic and nuclear extracts were prepared and subjected to Western blot analyses. Lamin A and β-tubulin were used as markers for nuclear and cytosolic fractions, respectively. G and H, experiments were performed as described in (E) and (F), except that cells were transfected with vector control or Flag-TRIM34. I, ZBP1+/+ and ZBP1−/− THP-1 macrophages were transfected with siRNA-control (si-ctrl) or siRNA-TRIM34 (right panel) for 24 h. Then, cells were infected with IAV (MOI = 1) for 24 h or left uninfected. Cell death was quantified by LDH release. J, experiments were performed as described in (I), except that the pro and cleaved forms of caspase-8, caspase-3, and caspase-7 were determined by Western blot. K and L, experiments were performed as described in (I) and (J), except that the cells were transfected with vector control or Flag-TRIM34. M and N, experiments were performed as described in (J) and (L), except that MLKL and GSDMD were determined by Western blot. All experiments were repeated at least three times. Bar graphs present means ± SD, n = 3 (∗∗p < 0.01, n.s., not significant). GSDMD, gasdermin D; IAV, influenza A virus; KO, knockout; MLKL, mixed-lineage kinase domain-like protein; TRIM, tripartite motif; ZBP1, Z-DNA-binding protein 1.