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Blood, Vol. 110, Issue 6, 1970-1981, September 15, 2007
A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA
Blood Ivanov et al.
110: 1970
Supplemental materials for Ivanov et al, Vol. 110, Issue 6, 1970-1981
Files in this Data Supplement:
- Figure S1. Comprehensive description of a novel approach to assess the levels of CpG-ODN associated with the TLR9 complex (JPG, 77.5 KB)
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(A) Graphical representation of the CpG-ODN amplification process. (B) Various amounts of CpG-ODN were used to amplify gradually increasing levels of the dUTP rich template (ten-fold serial dilutions, starting at 1/1015). Control that lacked CpG-ODN was used to determine background parameters for the technique. Optimization and proper controls are critical for success. We have determined that working template dilutions between 1/1018 and 1/1021 from 100 µM initial stock solution, and 22 to 27 cycles at the second phase PCR work well. (C) Increasing levels (100 pg/mL to 100 ng/mL; ten-fold serial dilutions) of CpG-ODN were amplified in the presence or absence of UNG to demonstrate specificity.
- Figure S2. Three-dimensional confocal depiction and analysis of HMGB1’s pre-association with TLR9 in vesicles(JPG, 124 KB)
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(A) Wt and Tlr9−/−BMDMs were stained with anti-TLR9 mAb antibody. Confocal images were acquired by indirect immunofluorescence as described in “Materials and methods, Confocal microscopy.” Nuclear DNA was stained by DAPI. (B) BMDMs were fixed and stained with antimouse-Rhodamine and antirabbit-fluorescein isothiocyanate (FITC). Nuclear DNA was stained by DAPI. (C) Partial z-stack series of confocal images used to reconstruct depth profile of a BMDM stained for HMGB1 (green) and TLR9 (red) shown in figure 3b. (D) Shown are the fluorescent pixel intensity mapped along lines spanning two vesicles (V1 and V2) and a random line as well as the depth pixel intensity profiles within the same regions. The intensity profiles reveal extensive co-localization of TLR9 and HMGB1 within the vesicles but not in the randomly selected region.
- Figure S3. Depletion of HMGB1 by LMB postpones the translocation of TLR9 to early endosomes in response to CpG-ODN (JPG, 159 KB)
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Wt BMDMs were pre-treated with LMB (20 ng/mL) or left untreated for 60 minutes followed by stimulation with CpG-ODN (1018, 5 µg/mL) for the indicated time points. Cells were fixed, permeabilized, and stained with anti-TLR9/Alexa568, anti-HMGB1/Alexa 488 and anti-EEA1/Alexa 647. Confocal images were acquired by indirect immunofluorescence as described in “Materials and methods, Confocal microscopy.”
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