Struct were washed and harvested with PBS, then subjected to FACS analysis using FACSCanto II (BD Bioscience).Immunoblotting and AntibodiesThe polyclonal antibody used to detect human IRF-3 in native PAGE and anti-human IRF-3 polyclonal antibodies for immunostaining were described previously [35]. Other antibodies were obtained from the following Title Loaded From File sources: Anti-human NF-kB antibody (sc-109), anti-human TRAF6 (sc-8409), and anti-human MFN1 (sc-50330) from Santa Cruz Biotechnology, anti-HA-Tag (6E2) from Cell Signaling, and anti-human Actin (A-1978) from Sigma.Supporting InformationFigure S1 Microarray analysis of mRNAs induced by oligomerized IPS-1 CARD or IPS-1. HeLa cells stably expressing FK-IPS or FK-IPS CARD were stimulated with AP20187 for the indicated time. Total RNA extracted from these cells was subjected to analysis using a DNA microarray (Genopal, Mitsubishi Rayon) of interferon-stimulated genes and interferon genes. Relative mRNA levels using a control expression as 1.0 are shown. (PDF) Figure S2 FK-IPS DCARDDTM forms speckle like aggregates in the cytoplasm. HeLa cells stably expressing FK-IPS DCARDDTM were mock treated or treated withImmunofluorescence MicroscopyFor immunofluorescence analysis, cells were fixed with 4 paraformaldehyde for 10 min, permeabilized with acetone: methanol (1:1), and blocked with 5 mg/ml of BSA in PBST (0.04 Teen20 in PBS) for 1hour. Cells 26001275 were incubated with relevant primary antibodies overnight at 4uC, then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen). To label mitochondria, cells were incubated for 30 min at 37uC with MitoTracker Red CMXRos according to the manufacturer’s instructions (Molecular Probes). Fluorescence images were obtained by Leica Microsystems AF6500 (Leica).Delimitation of Critical Domain in IPS-AP20187 for 3 h and stained with mitoTracker (mitochondria) and anti-HA antibody. Fluorescent microscopic images of FKIPSDCARDDTM and mitochondria are shown. (PDF)Figure S3 MFN1 is dispensable for signaling induced by forced oligomerization of IPS-1. MEFs of MFN12/2 or +/ + were transiently transfected with p-125Luc (reporter for IFN-b promoter activity) together with the indicated FK-IPS fusion constructs. Cells were treated with or without AP20187 for 6 h. Relative luciferase activities were determined as described in Materials and Title Loaded From File Methods. A representative result of at least two independent experiments is shown. Error bars indicate standard error of triplicate samples. (PDF) Figure S4 FK-IPS 400?08 can activate IRF-responsiveisolation of soluble and insoluble fractions by differential centrifugation. B and C. Immunoblot analysis of soluble/ insoluble fractions separated by differential centrifugation. FKIPS DCARD stable cells were cultured for 3 h in the absence or presence of AP. Cell lysates were separated by differential centrifugation. FK-IPS DCARD and endogenous MFN1, TRAF6, and actin were detected by immunoblotting. (PDF)Figure S7 Involvement of CARD9 in NF-kB dependent pathway. A. HeLa FK-IPS#48 cells were transfected with N.C. siRNA or CARD9 targeted siRNA for 48 h, and the knockdown of CARD9 was analyzed by RT-PCR. B, C and D. HeLa FKIPS#48 cells were transfected with N.C. siRNA or CARD9 targeted siRNA for 48 h, then mock treated or treated with AP20187 for 3 h. Cellular RNA were extracted and analyzed for IFN-b (B), Il-6 (C) or Il-1b (D) mRNA by qPCR. Representative data of at least two independent experiments are shown. Error bars: standard err.Struct were washed and harvested with PBS, then subjected to FACS analysis using FACSCanto II (BD Bioscience).Immunoblotting and AntibodiesThe polyclonal antibody used to detect human IRF-3 in native PAGE and anti-human IRF-3 polyclonal antibodies for immunostaining were described previously [35]. Other antibodies were obtained from the following sources: Anti-human NF-kB antibody (sc-109), anti-human TRAF6 (sc-8409), and anti-human MFN1 (sc-50330) from Santa Cruz Biotechnology, anti-HA-Tag (6E2) from Cell Signaling, and anti-human Actin (A-1978) from Sigma.Supporting InformationFigure S1 Microarray analysis of mRNAs induced by oligomerized IPS-1 CARD or IPS-1. HeLa cells stably expressing FK-IPS or FK-IPS CARD were stimulated with AP20187 for the indicated time. Total RNA extracted from these cells was subjected to analysis using a DNA microarray (Genopal, Mitsubishi Rayon) of interferon-stimulated genes and interferon genes. Relative mRNA levels using a control expression as 1.0 are shown. (PDF) Figure S2 FK-IPS DCARDDTM forms speckle like aggregates in the cytoplasm. HeLa cells stably expressing FK-IPS DCARDDTM were mock treated or treated withImmunofluorescence MicroscopyFor immunofluorescence analysis, cells were fixed with 4 paraformaldehyde for 10 min, permeabilized with acetone: methanol (1:1), and blocked with 5 mg/ml of BSA in PBST (0.04 Teen20 in PBS) for 1hour. Cells 26001275 were incubated with relevant primary antibodies overnight at 4uC, then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen). To label mitochondria, cells were incubated for 30 min at 37uC with MitoTracker Red CMXRos according to the manufacturer’s instructions (Molecular Probes). Fluorescence images were obtained by Leica Microsystems AF6500 (Leica).Delimitation of Critical Domain in IPS-AP20187 for 3 h and stained with mitoTracker (mitochondria) and anti-HA antibody. Fluorescent microscopic images of FKIPSDCARDDTM and mitochondria are shown. (PDF)Figure S3 MFN1 is dispensable for signaling induced by forced oligomerization of IPS-1. MEFs of MFN12/2 or +/ + were transiently transfected with p-125Luc (reporter for IFN-b promoter activity) together with the indicated FK-IPS fusion constructs. Cells were treated with or without AP20187 for 6 h. Relative luciferase activities were determined as described in Materials and Methods. A representative result of at least two independent experiments is shown. Error bars indicate standard error of triplicate samples. (PDF) Figure S4 FK-IPS 400?08 can activate IRF-responsiveisolation of soluble and insoluble fractions by differential centrifugation. B and C. Immunoblot analysis of soluble/ insoluble fractions separated by differential centrifugation. FKIPS DCARD stable cells were cultured for 3 h in the absence or presence of AP. Cell lysates were separated by differential centrifugation. FK-IPS DCARD and endogenous MFN1, TRAF6, and actin were detected by immunoblotting. (PDF)Figure S7 Involvement of CARD9 in NF-kB dependent pathway. A. HeLa FK-IPS#48 cells were transfected with N.C. siRNA or CARD9 targeted siRNA for 48 h, and the knockdown of CARD9 was analyzed by RT-PCR. B, C and D. HeLa FKIPS#48 cells were transfected with N.C. siRNA or CARD9 targeted siRNA for 48 h, then mock treated or treated with AP20187 for 3 h. Cellular RNA were extracted and analyzed for IFN-b (B), Il-6 (C) or Il-1b (D) mRNA by qPCR. Representative data of at least two independent experiments are shown. Error bars: standard err.
