Supplementary Figure 3: Silencing of POP3 specifically affects the AIM2 inflammasome.

(a, c, d) hMΦ were transfected with either control or a, POP3#2 or c, d, POP3 siRNAs and infected with MVA or transfected with poly(dA:dT) as indicated for 16 h and analyzed for a, mature IL-1β and c, IL-6 by ELISA (n = 3 ± s.e.m.) and d, TCL from Figure 3f, were analyzed in parallel for expression of AIM2 and IFI16 by immunoblot. (b, f) THP-1 cells were transfected with siRNAs as above, and infected with MVA, transfected with poly(dA:dT) or treated with MSU or SiO_2, as indicated for 16 h and analyzed for b, IL-1β secretion and f, IFN-b by ELISA (n = 3 ± s.e.m.). (e, g) THP-1 (GFP) and THP-1 (GFP-POP3) cells were analyzed for secretion of e, TNFα and g, IFN-β in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU as indicated by ELISA (n = 3 ± s.e.m.). (h) The POP3 antibody does not cross-react with other POP family members. HEK293 cells were transfected with Myc-tagged POP1, POP2 and POP3 and immunoprobed with our custom POP1, POP2 and POP3-specific antibodies. (i) The POP3 antibody does not cross-react with the related PYDs of AIM2 and IFI16. HEK293 cells were transfected with GFP or RFP-tagged POP3, AIM2-PYD and IFI16-PYD and immunoprobed with our POP3 antibody and with GFP and RFP antibodies as control. ^* denotes a cross-reactive protein. Data are representative of 3 experiments (a), 2 experiments (b-d, f) and 1 experiment (e, g-i). (a) ^*P<0.0001, ^**P=0.0049; (b) ^*P=0.0199, ^**P=0.0495; (f) ^*P<0.0001; (g) ^*P=0.0031, ^**P=0.0161, ^***P=0.0013;.