Xio, et l., Supplementry Figure 1 IL-6 (ng/ml) TNF-α (pg/ml) 16 12 8 4 1,4 1,2 1, 8 6 4 2 med Cl / Pm3CSK4 zymosn curdln Poly (I:C) LPS flgelin MALP-2 imiquimod R848 CpG TNF-α (pg/ml) IL-6 (ng/ml) 2 1.6 1.2.8.4 45 3 3 225 15 Cl / Supplementry Figure 1. CBLB inhiits signling vi the dectin-1. (,) ELISA of TNF-α nd IL-6 production y nd Cl / BMDMs nd BMDCs were stimulted with TLR lignds 1 to 9, zymosn, nd curdln for 48 h. For ll ELISA experiments dt re representtive of three independent experiments (iologicl replictes). Error rs re men ± s.d. P <.5, P <.1; unpired two-tiled Student s t test. n = 3 per group, ech with three repeted wells. Nture Medicine: doi:1.138/nm.4141
Xio, et l., Supplementry Figure 2 TNF-α (pg/ml) 45 3 15 Cl / 3 15 1 3 6 1 3 6 1 3 6 1 3 6 Time fter infection (h) Time fter infection (h) IL-6 (pg/ml) 45 TNF-α (pg/ml) 5 4 3 2 1 IL-6 (pg/ml) 45 3 15 TNF-α (pg/ml) 45 3 3 225 15 Cl / IL-6 (pg/ml) 5 4 3 2 1 1 3 6 1 3 6 1 3 6 1 3 6 Time fter infection (h) 4 3 2 1 IL-6 (pg/ml) 35 28 21 14 Time fter infection (h) 7 Supplementry Figure 2. CBLB inhiits TNF-α nd IL-6 production y dendritic cells ut hs limited role in the production of these cytokines y neutrophils upon infection with C. licns yest or hyphe. () ELISA of TNF-α nd IL-6 production in the superntnts collected from BMDCs of nd Cl / mice infected with C. licns yest nd hyphe forms (MOI = 1:1) for 1, 3, nd 6 h. () ELISA of TNF-α nd IL-6, production in the superntnts collected from BM neutrophils of nd Cl / mice infected with C. licns yest nd hyphe forms (MOI = 1:1) for 1, 3, nd 6 h. The dt re representtive of three independent expendents (iologicl replictes). Error rs re men ± s.d. P <.5, unpired two-tiled Student s t test. n = 3 per group, ech with three repeted wells. Nture Medicine: doi:1.138/nm.4141
C c 1. C tr si R l NA C sir l N A si R 1 N A nm 2 nm Xio, et l., Supplementry Figure 3 CBLB.1 d Ctr sirna: + Cl sirna (n M): Yest (h): + + 1 1 1 2 2 2 3 6 3 6 3 6 1..2.2 1. 1.1 1. 1.1 1..9 Hyphe (h): 3 6 3 6 3 Dectin-1 6 1..1.7.9.7.7.7.8.9 Supplementry Figure 3. Humn mcrophges lcking CBLB fil to down-regulte dectin-1 nd dectin-2 expression nd produce more pro-inflmmtory cytokines upon infection with C. licns yest nd hyphe. (,) ELISA of TNF-α nd IL-6 production y MDMs with or without Cl gene silencing, infected with yest () or hyphe () of C.licns (MOI = 1:1), or left uninfected. For ll ELISA experiments dt re representtive. of two independent experiments (iologicl replictes). Error rs re men ± s.d. P <.5, P <.1; unpired two-tiled Student s t test. n = 3 per group, ech with three repeted wells. (c,d) Immunolot nlysis of CBLB, dectin-1 nd dectin-2, nd ctin expression in MDM lystes. Dt re representtive of two independent experiments. Nture Medicine: doi:1.138/nm.4141 Dectin-2
5 15 3 5 15 3 Anti-Dectin-1 Hyphe (min): IP: (U)nDectin-1 1 Cl / 5 15 3 5 15 3 Anti-Dectin-2 Cl / Yest (min): IP: IP: IgG IP: IgG Xio, et l., Supplementry Figure 4 (U)nDectin-2 1 5 5 (K48U)nDectin-1 1 (K48U)nDectin-2 1 5 5 Dectin-1 5 15 3 5 15 3 Anti-SYK Hyphe (min): IP: 25 Cl C373A 15 3 15 3 Anti-SYK d Cl C373A Yest (min): IP: IP: IgG c Dectin-2 IP: IgG 25 15 (K48U)nSYK 1 (K48U)nSYK 15 1 SYK e Cl / Yest (min): 5 15 3 6 5 15 3 6 1. 3.1 2.8 2.6 2.5 1.6 3.5 3.3 3.3 2.5 1. 2.2 3.8 1. 1.1 1.3 2.1 3. 1.9 2.1 SYK SYK SYK psyk p-p65 Cl / Hyphe (min): 5 15 3 6 5 15 3 6 1. 1.2 1.5 1.5 1.7 1.3 1.8 2.2 2.2 2.1 psyk p-p65 1. 1.1 2.6 1.9 2..9 2.4 2.4 2.4 1.7 Supplementry Figure 4. CBLB deficiency impirs uiquitintion of dectin-1, dectin-2, nd SYK in mcrophges induced y C. licns yest nd hyphl infection. (,) Immunolot nlysis of dectin-1 nd dectin-2 uiquitintion of dectin-1 or dectin-2 immunoprecipittes isolted from BMDMs from nd Cl / mice infected with C. licns yest nd hyphe, respectively, y nti-uiquitin nd nti-k48 uiquitin ntiodies. (c,d) Immunolot nlysis of SYK uiquitintion of SYK immunoprecipittes isolted from BMDMs from nd ClC373A mice infected with C. licns yest nd hyphe, respectively, y nti-k48-specific uiquitin ntiody. (e) Immunolot nlysis of phospho-syk nd NF-κB p65 in BMDMs from nd Cl / mice infected with C. licns yest nd hyphe, respectively, y nti-phospho-syk (Y525,Y526) nd nti-phospho-p65 (S536). Dt re representtive of three independent experiments (iologicl replictes). Nture Medicine: doi:1.138/nm.4141
Xio, et l. Suppementry Figur 5 ROS (RLU/1 3 cells) CFU (1 x 1 6 /ml) d Normlized to mode 8 6 4 2 5 4 3 2 1 Cl / Cl C373A 25 5 Time (min) 1 8 6 4 2 Control Cl / ROS (RLU/1 3 cells) % killing 6 5 4 3 2 1 Cl / 1,5 Ctr Cl / 1, 5 Cl C373A 25 5 Time (min) Normlized to mode 1 8 6 4 2 Control Cl / c ROS (RLU/1 3 cells) % killing 3, 2, 1, 1 8 6 4 2 Cl / Cl C373A 25 5 Time (min) / Cl Cl C373A 1 2 1 3 1 4 1 5 Alex Fluor 488 1 2 1 3 1 4 1 5 Alex Fluor 488 Supplementry Figure 5. Loss of CBLB or CBLB C373A muttion enhnces ROS production nd fungl killing ut does not ffect phgocytosis. () ROS production y, Cl / or Cl C373A BMDMs co-cultured with C. licns t MOI = 5:1 nd 2:1, nd monitored in rel time over the indicted time period using the luminol ssy. Vlues re expressed s reltive light units (RLU) per 1, cells nd represent men vlues. () Killing cpcity of or Cl / BMDMs s ssessed y 24 h co-culture with C. licns. Assys were performed in triplicte. Dt re shown s mens ± s.d. (c) ROS production y, Cl /, or Cl C373A BM neutrophils co-cultured with C. licns t 2:1, nd monitored in rel time over the indicted time period using the luminol ssy. Vlues re expressed s reltive light units (RLU) per 1, cells nd represent men vlues (upper pnel). Killing cpcity of, Cl / or Cl C373A BM neutrophils s ssessed y 24 h co-culture with C. licns (lower pnel). (d) Phgocytosis of C. licns. or Cl / BMDMs co-cultured with Alex Fluor 488 leled C. licns. After 45 min of co-culture, or Cl / BMDMs were nlyzed for C. licns uptke y flow cytometry. P <.5, P <.1, Student s t test. Nture Medicine: doi:1.138/nm.4141
Xio, et l. Supplementry Figure 6 Supplementry Figure 6. CBLB C373A muttion significntly reduces fungl urden in kidneys, livers, lungs, nd spleens, nd leds to less myeloid cell recruitment to the kidneys. () Fungl urden in depicted orgns t 48 h fter systemic C. licns infection, shown s colony forming units (CFU)/grm orgn weight. Ech dot represents n individul mouse. () Percentges nd solute numers of hemtopoietic cells in the kidneys from nd Cl C373A mice t 48 h fter infection with C. licns (1 x 1 6 CFU). n = 5 for or Cl C373A mice. Dt re shown s mens ± s.d. nd nlyzed using the Student s t test. P <.5, P <.1. Nture Medicine: doi:1.138/nm.4141
Xio et l, Supplementry Figure 7 c d e Supplementry Figure 7. CBLB C373A muttion fcilittes fungl killing y enhncing ROS ctivity in the lood nd within spleens nd kidneys y monocytes/mcrophges. () Fungl urden of nd Cl C373A in the lood t 2 nd 6 h fter systemic C. licns infection, nd killing cpcity of or Cl C373A PBMCs nd neutrophils s ssessed y 24 h co-culture with C. licns. () Killing cpcity of splenic monocytes nd neutrophils of or Cl C373A mice s ssessed y 24 h co-culture with C. licns. (c) ROS stining in splenic monocytes, mcrophges nd neutrophils infected with 1 x 1 6 CFU of C. licns performed t dy 2 fter infection. (d) ROS stining in monocytes, mcrophges nd neutrophils in the kidneys of nd Cl C373A infected with 1 x 1 6 CFU of C. licns performed t dy 2 fter infection. Killing cpcity of CD45-enriched cells isolted from nd Cl C373A kindneys infected with C. licns. (e) ELISA of TNF-α, IL-6, nd IL-1β levels of kidney homogentes of or Cl C373A mice infected with 1 x 1 6 CFU of C. licns t 48 h fter infection. n = 5 mice per group nd re representive dt from three independent experiments. Error rs, men ± s.e.m. Dt were nlyzed using the Student s t test. P <.5, P <.1. Nture Medicine: doi:1.138/nm.4141
Xio, et l., Supplementry Figure 8 3, 1,6 h 24 h 48 h h 24 h 48 h TNF-α (pg/ml) 2, 1, IL-6 (pg/ml) 1,2 8 4 Fungl urden (CFU/g) 4,5, 3,5, 2,5, 1,5, 5, Cl / Clec7 / Clec7 / Cl / Clec7 / Cl / Cl / Clec7 / Cl / Clec7 / Clec4n / Cl / Clec7 / Cl / Clec7 / Clec7 / Cl / Clec4n / Cl / Clec7 / Clec7 / Cl / Cl / Clec7 / Cl / Clec7 / Supplementry Figure 8. Introducing dectin-1 or dectin-2 deficiency, or oth into Cl / mice significntly reduces serum TNF-α nd IL-6 which correltes with incresed fungl urden in the kidneys. () ELISA of serum TNF-α nd IL-6 t 24 nd 48 h from, Cl /, Clec7 /, Clec4n /, Cl / Clec7 /, Cl / Clec4n /, Clec7 / Clec4n / nd Cl / Clec7 / Clec4n / mice infected with C. licns (3.5 x 1 5 CFU) y i.v. injection. () Fungl urden in the ove mice mesured t 48 h fter infection. Dt re representtive of two independent experiments. Nture Medicine: doi:1.138/nm.4141
Xio, et l., Supplementry Figure 9 c d Supplementry Figure 9. Autontiody, IgG, nd IgE titers in the ser nd IL-17/IFN-γ in the ser nd kidneys of, Cl /, nd Cl C373A mice efore nd fter C. licns infection. (-c) Serum titers of nti-dsdna nd nti-ssdna ntiodies, IgG, IgE, IL-17, nd IFN-γ in, Cl /, nd Cl C373A mice efore or 48 h fter C. licns infection (1 x 1 6 CFU). (d) IL-17 nd IFN-γ in the kidneys of, Cl /, nd Cl C373A mice efore or 48 h fter C. licns infection. n = 5 mice per group nd re representive dt from three independent experiments. Error rs, men ± s.e.m. Dt were nlyzed using the Student s t test. Nture Medicine: doi:1.138/nm.4141
Xio, et l., Supplementry Figure 1 C. licns Cl / Hyphe (min): 5 15 3 6 5 15 3 6 Hyphe (h): 1 3 6 Cl / 1 3 6 Dectin-3 MR Mincle DC-SIGN Supplementry Figure 1. Loss of CBLB stilizes dectin-3, ut not MR, Mincle nd DC-SIGN. Immunolot nlysis of lystes of nd Cl / BMDMs infected with C. licns hyphe for the times indicted with ntiodies ginst dectin-3, MR, Mincle, nd DC-SIGN. Results re representive dt from three independent experiments (n = 3). Nture Medicine: doi:1.138/nm.4141