Fig 2: Cytokines regulated by RanBP1 in lung cancer. (A) Identification of secreted factors regulated by RanBP1 using cytokine arrays. A549 cells are used, and the expression of RanBP1 is suppressed using si-RNA. (B) Comparison of the gene expression of cytokines regulated by RanBP1. (C) Analysis of cytokines regulated by RanBP1 using WB. (D) Comparison of the sphere formation ability after each neutralizing antibody treatment. The p value is less than 0.0001 versus the IgG Ab value. (E) Comparison of migration and invasion ability of cells after each neutralizing antibody treatment. Experiments performed in triplicate. (F) CSC marker protein analysis in A549 after treatment with IL-18 neutralizing antibody. (G) Confirmation of the expression levels of EMT marker proteins after treatment with the IL-18 neutralizing antibody. (H) Expression analysis of RanBP1 regulated by IL-18 using neutralizing antibodies. Error bars represent mean ± SD. Triplicate samples. * p < 0.05, ** p < 0.001, versus control. Scale bar = 50 µm.

Fig 3: CASP4/5 preferentially cleave IL-1ß at D27 and IL-18 at D36.(A,B) HEK 293T cells were transiently co-transfected with the indicated constructs. After 24 h, samples were harvested and analyzed by immunoblotting. (C) Schematic of inflammatory substrates and representative cleavage sites by inflammatory caspases. Data are representative of three or more independent experiments. *Represents non-specific bands in immunoblot.

Fig 4: Dimerization of ?CARD DmrB-CASP1/4/5/11 mediates processing of inflammatory substrates.(A,B) HEK 293T Cells stably expressing ?CARD DmrB-CASP1,-4, or -5 and GSDMD-V5 (A) or IL-18-V5 (B) were treated with AP20187 (1 µM) for 1 h or 24 h. Cell death was measured by LDH and samples were analyzed by immunoblotting. (C) HEK 293T cells stably expressing ?CARD DmrB-CASP1,-4, or -5 and GSDMD-V5 were transiently transfected with the indicated IL-1ß constructs for 24 h before the addition of AP20187 (1 µM) for 24 h. Cell death was measured by LDH and samples were analyzed by immunoblotting. (D) HEK 293T cells stably expressing ?CARD Dmrb-CASP11 were transiently transfected with the indicated constructs. After 24 h, samples were treated with AP20187 (1 µM) for 24 h then analyzed for LDH release and immunoblotting. Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. *Represents non-specific bands in immunoblot. Data are representative of two or more independent experiments.

Fig 5: LPS activated non-canonical inflammasomes cleave IL-1ß and IL-18.(A) HEK 293T cells stably expressing IL-18-V5 and either CASP4 or CASP5 were transfected with LPS (25 µg/mL) for 24 h before samples were collected and analyzed by immunoblotting. (B,C) HEK 293T cells stably expressing 2xFLAG-CASP11 were transiently transfected with the indicated constructs. After 24 h, samples were transfected with LPS (25 µg/mL) for 24 h then analyzed for LDH release (B) and immunoblotting (C). Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. Data are representative of two or more independent experiments. *Represents non-specific bands in immunoblot. The small m or h represents mouse or human versions of the proteins respectively.