Immunity, Volume 38 Supplemental Information Poly IC Triggers a Cathepsin D- and PS-1-Dependent Pathway to Enhance Cytokine Production and Mediate Dendritic Cell Necroptosis Jian Zou, Taro Kawai, Tetsuo Tsuchida, Tatsuya Kozaki, Hiroki Tanaka, Kyung-Sue Shin, Himanshu Kumar, and Shizuo Akira
14
Embed
Home: Cell Press · Web viewHuman Flag-IPS-1, Myc-Cathepsin D, Myc-TBK1, Myc-FADD, Flag-Caspase-8 full-length (FL) and Flag-Caspase-8 a.a. 1-237 (C8 1-237) were amplified by PCR and
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Immunity, Volume 38
Supplemental Information
Poly IC Triggers a Cathepsin D- and PS-1-DependentPathway to Enhance Cytokine Productionand Mediate Dendritic Cell Necroptosis
(A) GM-DCs derived from wild-type (WT) or IPS-1-deficient (IPS-1 KO) mice were
stimulated with poly IC (pIC) for the indicated periods. Cell lysates were blotted with
an anti-phosphorylated IRF3 antibody.
(B) FL-DCs were stained with anti-CD11b-PE, anti-B220-APC and anti-CD24-FITC
antibodies. B220-CD11b+ cells were separated into CD11bhiCD24lo or
CD11bloCD24hi cDCs by cell sorting.
(C) Total RNA prepared from CD11bhiCD24lo and CD11bloCD24hi cells was
subjected to RT-PCR analyses for the expression levels of Ifih1 (MDA5), Tlr3
(TLR3) or Actb (-actin).
(D) CD11bloCD24hi cells derived from WT, TLR3-deficient (TLR3 KO) or TRIF-
deficient (TRIF KO) mice were stimulated with poly IC, and the concentrations of
IFN- and IL-12p40 in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3).
(E) GM-DCs (left) or CD11bloCD24hicDCs (right) derived from WT or IPS-1 KO mice were cocultured with CD8+ T cells derived from OT-I mice in the presence of
OVA and poly IC or CpG ODN for 4 d. The concentrations of IFN- in the culture
supernatants were measured by ELISA. Data represent means ± SD (n=3).
(F) GM-DCs were stimulated with high molecular weight poly IC (H) or low
molecular weight poly IC (L) with (right) or without (left) transfection. The
concentrations of IFN- in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3). C: control.
Figure S2, Related to Figure 4
(A) Lysosomes were isolated from WT or IPS-1-deficient (KO) GM-DCs with or
without poly IC stimulation. The lysosomes were lysed and blotted with anti-
Cathepsin D or anti-LAMP1 antibody.
(B) CD11bhiCD24lo and CD11bloCD24hi cells derived from WT or KO mice were
stimulated with poly IC or LPS, and immunostained with an anti-Cathepsin D
antibody.
(C) GM-DCs were stimulated with poly IC and immunostained with anti-Cathepsin
D and anti-IPS-1 antibodies.
Figure S3, Related to Figure 5
(A) GM-DCs were treated with the indicated siRNAs. Total RNA was prepared and
analyzed for the expressions of Ctsd (Cathepsin D [Cat D]), Ctsb (Cathepsin B [Cat
B]), Ctsl (Cathepsin L [Cat L]) and Actb (-actin) by RT-PCR.
(B) GM-DCs treated with the indicated siRNAs were stimulated with poly IC. The
concentrations of IFN- (left panel) and IL-6 (right panel) in the culture
supernatants were measured by ELISA. Data represent means ± SD (n=3).
(C) GM-DCs treated with the indicated siRNAs were stimulated with LPS. The
concentrations of IL-6 in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3).
(D) CD11bhiCD24lo or CD11bloCD24hi cells with or without Pep A pretreatment
were cocultured with CD4+ or CD8+ T cells derived from OT-II or OT-I mice in the
presence of OVA and poly IC, respectively. The concentrations of IFN- in the
culture supernatants were measured by ELISA. Data represent means ± SD (n=3).
(E) Schematic representation of Caspase-8. The arrows indicate the cleavage sites of
Caspase-8 by Cathepsin D (upper). The lower panels show sequence alignments of
mutants of Caspase-8.
(F) HEK293 cells were transiently transfected with Flag-Caspase-8, Flag-Caspase-8
L237A, or Flag-Caspase-8 L237A-M383A-L443A, together with Myc-empty or
Myc-Cat D. Cell lysates were immunoprecipitated with an anti-Flag antibody and
immunoblotted with an anti-Flag antibody.
Figure S4, Related to Figure 6
(A) GM-DCs pretreated with z-VAD-fmk were stimulated with poly IC. The cells
were stained with a FITC-anti-Annexin V antibody and PI. The population of dead
cells was analyzed by flow cytometry. Data represent means ± SD (n=3).
(B) GM-DCs were treated with the indicated siRNAs. Total RNA was prepared and
analyzed for the expressions of Ripk3 (Rip-3) and Actb (-actin) by RT-PCR.
(C) GM-DCs pretreated with the indicated inhibitors were stimulated with poly IC.
The cell viability was measured by cell titer-Glo luminescent cell viability kit. Data
represent means ± SD (n=3).
Figure S5, Related to Figure 7
(A) GM-DCs treated with the indicated siRNAs were stimulated with poly IC. The
concentrations of HMGB1 in the culture supernatants were measured by ELISA.
Data represent means ± SD (n=3).
(B) GM-DCs were stimulated with poly IC together with HSP70, oxLDL, acLDL or
acBSA at the indicated concentrations. After 24 h, the concentrations of IFN- in the
culture supernatants were measured by ELISA. Data represent means ± SD (n=3).
(C) GM-DCs derived from WT or MyD88 and TRIF doubly deficient (DKO) mice
were stimulated with poly IC together with recombinant HMGB1 at the indicated
concentrations. After 24 h, the concentrations of IFN- in the culture supernatants
were measured by ELISA. Data represent means ± SD (n=3).
(D) RAW 264.7 cells were stimulated with 50 μg/ml of Etoposide for 24 h. The
supernatants were collected and then incubated with streptavidin beads with or
without biotinylated poly IC for 4 h. The bound proteins were eluted by heat
incubation and analyzed by immunoblotting with an anti-HMGB1 antibody.
(E) Recombinant HMGB1 was preincubated with an anti-HMGB1 neutralizing
antibody, and then IP with streptavidin beads with or without biotinylated poly IC for
4 h. The bound proteins were eluted by heat incubation and analyzed by
immunoblotting with an anti-HMGB1 antibody.
Supplemental Experimental Procedures
Reagents and Plasmids
High and low molecular weight poly IC were purchased from InvivoGen.
LysoTracker Red DND-99 and Alexa Fluor 488-conjugated rabbit anti-goat IgG
antibody were purchased from Invitrogen. LPS from Salmonella minnesota Re-595,