a 1% 5% % 1% 5% % OVAb βgal activity A.U. (x1 4 ) 2 1 5 βgal activity A.U. (x1 4 ) 2 1 BSAb 2 hours 4 hours OVAb BSAb OVAb BSAb,1 1 1 1 SIINFEKL (ng/ml) CFSE b Beads Alexa488 (%) 8 6 4 2 ** ** 1:1 5:1 1:1 1:1 CytB c 45 kda 55 kda H2K b αtubulin Beads:cell ratio Supplementary Figure 1 UNCB1 regulates antigen crosspresentation. and BMDCs were stimulated with different concentrations of OVAb or SIINFEKL peptide for 2, 4 or 6 hours before cocultured with CFSElabelled OTI T cells or B3Z hybridoma for 72 hours or 16 hours respectively. T cell activation was monitored by CFSE dilution for OTI T cell proliferation or by measuring βgalactodisade activity for B3Z hybridoma. Graphs show mean ± S.E.M. (n=3) and histograms of CFSE dilution are representative from one experiment out of three. (b) and BMDCs were left to phagocytose streptavidin A488beads (3μm) for minutes at 37 C at different ratios and phagocytic capacity was assessed by flow cytometry. Treatment of cells with cytochalasin D (CytB) was performed as a negative control of phagocytosis. Graph shows mean ± S.E.M. (n=2) via unpaired t test ** P <.1. (c) Immunodetection of MHCI (H2K b ) and tubulin proteins in and BMDCs lysate. Data are representative of two experiments. Supplementary Figure 1 Maschalidi et al.
a * * * * * * * * LAMP1 UNCB1 EEA1 Inset Mean area in late phagosomes (A.U.) 8 6 4 2 LAMP1 Mean area in late phagosomes (A.U.) 8 6 4 2 UNCB1 Mean area in late phagosomes (A.U.) 8 6 4 2 EEA1 b c Phagosomal ROS.5.4.3. *** min.6.4. ** 9 min **** **** DPI DPI Relative Frequency.4.2.1...2.4.6.81. High ROS phag. 2% 35% 1.2 1..6 1.8 2.2.2 2.4 2.62.8 Intracellular ROS (ΔRFU) 3 1 5 2 1 5 1 1 5 OVAb * DPI ** DPI OxyBurst/Alexa568 Ratio d Time (min): kda 2 12 212 phagosomes Rab27a TCL Supplementary Figure 2 Maschalidi et al.
Supplementary Figure 2 Phagosomal ROS production and maturation are altered in DC (a) IF detection of LAMP1 (magenta), UNCB1 (red), EEA1 (green) in BMDC after 2 hours phagocytosis of 3μm beads. Asterisks indicate internalised beads (phagosomes). Quantification of mean area of indicated proteins in late phagosomes (n = cells). Bars = 1 µm. (b) Phagosomal ROS production was monitored by exposing DC to zymosan coupled to OxyBurst and Alexa568 in the absence or presence of DPI (1 µm). (n=4/5/5 (coverslips each) containing 29/3275/3663 () or 2882/397/2957 () phagosomes for min/9 min/dpi; **P <.1; ***P <.1; ****P <.1 via unpaired t test. Relative frequency of high phagosomal ROS in and 3d DC assessed as in left panel. (c) Intracellular ROS production was measured in immature DC loaded with μm DHE and exposed to either OVAcoated beads (OVAb) alone or in the presence of DPI. Graph shows mean ± S.E.M. from 4 independent experiments in triplicate wells via unpaired t test * P <.5; ** P <.1. (d) Phagosomes from or 3d/ 3d DC were magnetically purified after 2 min or 2 hours of cargo internalization. Protein expression of Rab27a was visualized by immunoblot either in total lysate (TCL, 5 μg) or in phagosomes (2 μg). Data are representative of two experiments. Supplementary Figure 2 Maschalidi et al.
a b 8 kda T cells B cells 8 kda BMDC cdc 45 kda 45 kda Supplementary Figure 3 Expression of protein is downregulated in T cells but not in DC. Immunodetection of and (as a loading control) in lysates from (a) T and B cells and (b) BMDC or spleen conventional dendritic cells (cdc) from and mice. Supplementary Figure 3 Maschalidi et al.
a UNCB1FLAG 3d GFP kda 65 kda IP: FLAG b UNCB1 FLAG GFP UNCB1 3d FLAG 35.3 35.5 GFP c Control IgG IgG Control IgG IgG IB: IB: UNCB1 1 nonspecifiic UNCB1 1 UNCB1 IP: IB: IB: UNCB1 IP: 45 d % calcium spots/ cell surface 8 6 4 2 TG Ca 2 TG Ca 2 Supplementary Figure 4 Maschalidi et al.
Supplementary Figure 4 interacts with UNCB1 and its function is impaired in cells. (a) Fibroblasts were transfected with (GFPtagged) and or 3d mutated UNCB1FLAGtagged plasmids and coimmunoprecipitated with antiflag antibody (UNCB1) followed by immunoblotting with antigfp and antiflag antibodies. One representative immunoblot analysis of three independent experiments is shown. (b) Percentage of transfection efficiency of fibroblasts transfected with GFP tagged and or 3d mutated UNCB1FLAG tagged plasmids assessed by flow cytometry. Graph shows representative dot plots from one out of three independent experiments. (c) For Endogenous UNCB1 complexes in DCs were lysed and lysate was immunoprecipitated with protein G beads coated with anti antibody. Immunoprecipitates were thoroughly washed, dissolved by denaturation with 1% SDS and 2% βmercaptoethanol and subjected to SDS PAGE with boiling the samples for immunodetection (left) or without heating the samples for UNCB1 visualization (middle). Immunoprecipitation controls for, UNCB1 and detection in lysate are shown (right). One representative immunoblot analysis of two independent experiments is shown. (d) Representative live imaging of cell surface Ca 2 spots in or DC loaded with Fura2AM and pluronic (see Methods section) treated with thapsigargin (TG) at 5 sec of recording in Ca 2 free Ringer s solution followed by stimulation with Ringer s solution supplemented with 2 mm Ca 2 at sec and imaged for up to 55 sec (left). Bars = 1 µm. Quantification of the percentage of Ca 2 related spots per total cell surface indicate impaired Ca 2 influx in DC (right). Supplementary Figure 4 Maschalidi et al.
a βgal activity (AU) 9 1 4 6 1 4 3 1 4 OVAcoated beads control Stim1silenced control Stim1silenced βgal activity A.U. (x1 4 ) 2 1 Control si b control / H2K b.1.1 1 1 1 SIINFEKL (ng/ml) FcγRIIAGFP Supplementary Figure 5 Silencing in DC inhibits antigen cross presentation (a) and DC silenced or not for were stimulated with OVAb (left panel) or SIINFEKL peptide (right panel) for 2 hours before cocultured with B3Z hybridoma for 16 hours. T cell activation was monitored by measuring βgalactosidase activity. Graphs show mean ± S.E.M. (n=3) (b) Percentage of transfection efficiency of human fibroblasts from control and deficient patient cotransfected with FcγRIIAGFP and H2K b plasmids (right panel) compared to nontransfected fibroblasts (left panel) as assessed by flow cytometry. Graph shows representative dot plots from one out of three independent experiments. Supplementary Figure 5 Maschalidi et al.
a Beads Alexa488 (%) 8 6 4 2 1 beads:cell mcherry mcherry D76A D76A b mcherry mcherry D76A D76A MHCII MHCI CD8 Supplementary Figure 6 active form does not alter DCs phagocytosis or activation. (a) and BMDCs transfected with or D76A were left to phagocytose streptavidin A488beads (3μm) for minutes at 37 C at different ratios and phagocytic capacity was assessed by flow cytometry. Graph shows mean ± S.E.M. (n=2). (b) MHCII, MHCI and CD8 surface expression were assessed in and BMDC transfected with or D76A by flow cytometry. Graphs are representative of two experiments. Supplementary Figure 6 Maschalidi et al.
Figure 2d Time (min): 2 12 2 12 19 1 8 5 VATPase phagosomes Time (min): TCL 2 12 2 12 gp91[phox] phagosomes Time (min): TCL 2 12 2 12 Cystatin C phagosomes 19 19 1 8 1 8 5 5 TCL
Figure 4a UNCB1FLAG GFP 3d 3d 1 75 1 75 5 37 5 37 2 IP: GFP 2 Figure 5b UNCB1FLAG GFP 235 1 1 ΔCt CAD ΔCt CAD 235 1 1 ΔCt CAD ΔCt CAD IP: GFP
Figure 6b Figure 6g Actin Figure 7 UNCB1FLAG D76AGFP 3d 3d 3d 3d 235 1 1 235 1 1 IP: GFP
Supplementary Figure 1 Supplementary Figure 2d Time (min): 2 12 2 12 235 1 1 Rab27a H2K b αtubulin phagosomes TCL Supplementary Figure 3a T cells B cells 1 1 1
Supplementary Figure 3b BMDC cdc 1 1 235 1 1 1 Supplementary Figure 4a Supplementary Figure 4c UNCB1FLAG GFP 3d Control IgG IgG Control IgG IgG 1 75 5 37 2 1 1 IP: 1 1 IB: UNCB1 UNCB1 IB: IP: