Live cell imaging of trafficking of the chaperone complex vaccine to the ER. BMDCs were incubated with ER-Tracker Red (1 M) in staining solution for
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1 Live cell imaging of trafficking of the chaperone complex vaccine to the ER. BMDCs were incubated with ER-Tracker Red (1 M) in staining solution for 15 min at 37 C and replaced with fresh complete medium. Cells were pulsed with FITC-labeled complexes for 15 min and washed extensively with PBS. Live cell images were collected using a Zeiss Cell Observer SD spinning disc confocal microscope.
2 Wang H et al., Supplementary Figures
3 Supplementary Figure S1 IP: anti-grp170 IB: Sec61 IB: Grp170 1/20 Input Sec61 Grp170 IB: Grp170 IB: Sec61 IP: anti-sec61 1/20 Sec61 Input Grp170 Supplementary Figure S1. Endogenous Grp170 interacts with Sec61. DC1.2 cell lysates were immunoprecipitated with antibodies against Grp170 (B, left) or Sec61 (B, right), followed by immunoblotting analyses to examine the association of intracellular Grp170 with Sec61. Total cell lysates as the input were also examined by immunoblotting for the indicated molecules.
4 Supplementary Figure S2 A Anti-His Anti-KDEL His-Grp ng His-Grp170 ng His-Grp170 ng His-Grp 30 ng B Calnexin Lamp1 Giantin C His-Grp170 His-Gp Bip -actin ER fractions WCL Control Positive controls Grp170 Gp D Sec61 KDEL EEA1 0 min 60 min Supplementary Figure S2. Association of chaperone complex vaccine with the ER following internalization by DCs. A. Characterization of KNDEL-truncated His-Grp170 protein. Recombinant His-Grp170 protein expressed and prepared using insect cells was examined by immunoblotting with anti-his or anti-kdel antibodies. Similarly prepared His-Gp protein was also included for analysis. B. BMDCs were incubated with FITC-labeled Gp complexed with Grp170 for 30 min. Cells were washed and stained with antibodies against calnexin (ER), Lamp1 (lysosome) or Giantin (Golgi), followed by analysis using a fluorescence microscopy. Scale bar, 5 M. C. BMDCs were treated with or without the complex. ER fractions were isolated using Endoplasmic Reticulum Isolation Kit (Sigma-Aldrich) according to manufacturer's instructions, and analyzed for the presence of His-Grp170 or His-Gp by immunoblotting using anti-his antibodies. Bip and -actin in the samples were also examined. Recombinant His-Grp170 and His-Gp serve as positive controls. D. The ER access of the chaperone vaccine following receptor-mediated endocytosis. BMDCs were incubated with FITC-labeled complexes on ice for 30 min. Cells were washed extensively (set as 0) and incubated at 37 C for additional 60 min. Cells were stained with antibodies for Sec61 (left), KDEL (middle) or EEA1 (right).
5 Supplementary Figure 3 A Sec61 B 15 min KDEL Eeyarestatin I treatment 30 min 60 min C EEA1 IP: anti-luc Input Grp170-Luc cytochalasin D IB: UB min IB: Luc -actin IB: Luc Supplementary Figure S3. Disruption of the ERAD pathway causes the accumulation of internalized complex in the ER. A. BMDCs were pretreated with Eeyarestatin I (8 M) for 2 h and pulsed with FITC-labeled Gp in complexes with Grp170 for 15 min at 37 C. Cells were washed with PBS and chased for the indicated times, followed by immunostaining with antibodies for Sec61. Scale bar, 5 M. B. The ER entry of the vaccine complex is independent of phagocytosis. BMDCs were pretreated with cytochalasin D (10 M) for 2 h and incubated with chaperone-protein complexes. Cells were stained with antibodies against KDEL or EEA1. Scale bar, 5 M. C. Inhibition of phagocytosis does not influence the ubiquitination of antigen-chaperoned by Grp170. BMDCs were incubated with the Grp170-luciferase (Luc) complex in the presence of cytochalasin D. Cells were washed and cultured for 8 h in the presence of lactacystin (20 M). Cell lysates were immunoprecipitated with anti-luc antibodies and the ubiquitination of Luc was examined by immunoblotting with anti-ubiquitin (UB) antibodies.
6 Supplementary Figure S4 A NS B NS C IL-2 (pg/ml) IL-2 (pg/ml) IL-2 (pg/ml) Control MG132 primaquine peptatin A NH 4 Cl D Control Exo A MG132 E Scrambled shrna Sec61 shrna IFN- IFN CD90.1 CD90.1 Supplementary Figure S4. Functional ERAD machinery is required for DC-mediated cross-presentation promoted by the Grp170. A-B. The effects of Exo A and MG132 on Grp170-enhanced cross-presentation are not due to their cytotoxicity. BMDCs were pretreated with Exo A (1 g/ml, A) or MG132 (20 M, B) for 2 h and washed extensively with PBS. Cells were then pulsed with Gp for 4 h, and washed, followed by incubation with CD8 + pmel cells at a ratio of 1:10. T-cell activation was examined using ELISA assays for the levels of IL-2 in the culture supernatant. NS, not significant. C. Inhibition of lysosome activity has little effect on Grp170-enhanced T cell priming. BMDCs were pretreated with MG132 (proteasome inhibitor, 20 M), primaquine (recycling endosome inhibitor, M), peptatin A (lysosome inhibitor, 1 mm), or NH 4 Cl (lysosome inhibitor, 20 M). After pulsing with the complex, cells were washed and cocultured with naïve CD8 + pmel cells. D-E. Disruption of the ERAD pathway or proteasome function abolishes Grp170-enhanced T-cell priming in vivo. BMDCs were pretreated with Exo A or MG132 for 2 h (D) or infected with lentiviruses encoding shrna for Sec61 (E), followed by pulsing with -complex vaccine. Cells were washed and used to immunize mice s.c. (day 0) that had received pmel cells (day -1). Draining lymph node cells were isolated on day 5 and stimulated with Gp peptide. The frequency of IFN- -producing CD CD8 + T-cells was analyzed by intracellular cytokine staining assays.
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