Fig 2: IAV infection activated JNK via the DAI/SPAG9/JNK pathway. (A) HEK293T cells were transfected with increasing concentrations of DAI or treated with the JNK inhibitor SP600125 (50 μM) as a negative control. Phosphorylated and total JNK1/2 were analyzed by immunoblotting. (B) Wild-type (WT) or SPAG9−/− HEK293T cells were transfected with the indicated plasmids, and phosphorylated and total JNK1/2 were analyzed by immunoblotting. (C) WT, DAI−/−, and SPAG9−/− RAW264.7 cells were infected with IAV (PR8; MOI = 2), and phosphorylated and total JNK1/2 were analyzed by immunoblotting. (D) Domain architecture of DAI and schematic of DAI constructs used in this study. (E) HEK293T cells were transfected with the indicated DAI constructs, and phosphorylated and total JNK1/2 were analyzed by immunoblotting.

Fig 3: SPAG9 promoted IAV-induced cell death. (A) Cell death observed in bright-field microscopy (magnification, ×20). Arrowheads indicate dead cells. RAW264.7 cells were infected with IAV (PR8) at an MOI of 2 for 16 h. (B) Cell death was assessed by PI staining (magnification, ×10). RAW264.7 cells were infected with IAV (PR8) at an MOI of 2 for 12 h, and the cells were then stained with PI. (C) Quantification of cell death. WT or SPAG9−/− RAW264.7 cells were infected with IAV (PR8) at an MOI of 2, and cell death was assessed at indicated times postinfection. (D) Effects of SPAG9 knockout on IAV replication. WT or SPAG9−/− RAW264.7 cells were infected with IAV (PR8) at an MOI of 2, and supernatants were collected at the indicated time points postinfection for evaluation of virus titers using plaque assays. P values were determined by Student's t test. NS, not significant; *, P < 0.05; ***, P < 0.001.

Fig 4: The SPAG9-JNK1/2 pathway is critical for enhancing interactions among RIPK1, RIPK3, and DAI. (A) Wild-type (WT) HEK293T cells were cotransfected with SPAG9-Flag and HA-RIPK1, RIPK3, DAI, HA–caspase-6, or HA–caspase-8 for 36 h. Anti-Flag immunoprecipitation was performed to detect RIPK1, RIPK3, DAI, HA–caspase-6, or HA–caspase-8. (B) SPAG9−/− HEK293T cells were cotransfected with RIPK1, RIPK3, DAI, and increasing concentrations of SPAG9-Flag for 36 h, and anti-RIPK3 immunoprecipitation was performed to detect RIPK1, RIPK3, or DAI. (C) WT, SPAG9−/−, and RIPK3−/− RAW264.7 cells were infected with IAV (PR8; MOI = 2) for 12 h, and anti-RIPK3 immunoprecipitation was performed to detect RIPK1, RIPK3, or DAI. (D) WT HEK293T cells were transfected with siRNA against JNK1/2 (25 nM), and at 24 h posttransfection, the cells were cotransfected with RIPK1, RIPK3, and DAI transfection siRNA for 36 h. Anti-RIPK3 immunoprecipitation was performed to detect RIPK1, RIPK3, and DAI. (E) WT and RIPK3−/− RAW264.7 cells were transfected with siRNA against JNK1/2 (25 nM), and after 24 h posttransfection, the cells were infected with IAV (PR8; MOI = 2). After 12 h, anti-RIPK3 immunoprecipitation was performed to detect RIPK1, RIPK3, or DAI. IB, immunoblotting.

Fig 5: SPAG9 is a direct target gene of miR-9-5p. (A) Schematic diagram of the predicted binding sites between miR-9-5p and SPAG9, and the mutation in seeded region of SPAG9-mut. (B) Relative luciferase activities were detected in SW1736 and 8505C cells cotransfected with SPAG9-wt or SPAG-mut and miR-NC mimics or miR-9-5p mimics. (C) SW1736 and 8505C cells were transfected with miR-NC mimics or miR-9-5p mimics, and their lysates were incubated with anti-Ago2 or anti-IgG, followed by the measurement of SPAG9 mRNA enrichment by reverse transcription-quantitative PCR assay. (D) Western blot analysis of SPAG9 expression in SW1736 and 8505C cells transfected with miR-NC mimics, miR-9-5p mimics, anti-miR-NC or anti-miR-9-5p. *P<0.05 vs. NC, or as indicated. 3′-UTR, 3′-untranslated region; Ago2, Argonaute2; miR-9-5p, microRNA-9-5-p; mut, mutant; NC, negative control; SPAG9, sperm-associated antigen 9; wt, wild type.