Supplementalinformation to:
The role of UNC93B1 in surface localization of TLR3 and in cell priming to nucleic acid agonists
Jelka Pohar1, Nina Pirher1, Mojca Benčina1,2, Mateja Manček-Keber1 and Roman Jerala1, 2, 3,4
1 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
2EN-FIST Centre of Excellence, 1000 Ljubljana, Slovenia
3Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia
*Running title:TLR3 regulates UNC93B1 and other endosomal TLRs
4To whom correspondence should be addressed: Prof. Dr. Roman Jerala, Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, P.O. Box 660, 1000 Ljubljana, Slovenia, Tel.:+386 1 47 60 335; Fax: +386 1 47 60 300; E-mail:
SFig.1.Activation of HUVEC cells with poly(I:C) leads to the increased transcription of TLR3 and TLR9.(A, B) HUVEC cells were incubated with poly(I:C) (25 µg/ml), ODN2216 (5 µM) or LPS (25ng/ml) for the indicated time. TLR3, TLR9(A) and IFN-α (B)mRNA transcriptswere determined by real-time PCR. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown. (C) HUVEC cells were either left untreated or stimulated with ODN2216 (5 µM) for 24 h. Histograms of unstained cells (filled areas) or cells stained for cell surface associated TLR3 (solid lines) are shown. The representative data from two experiments are shown.
SFig.2.Overexpression of UNC93B1 does not translocate TLR7 and TLR8 to the plasma membrane. (A) HEK293T cells were transfected with TLR7-YFP (yellow) alone or cotransfected with UNC93B1. Plasma membrane was dyed with CTB- Alexa 555 (magenta). (B) HEK293T cells were transfected with TLR8-HA (cyan) alone or cotransfected with UNC93B1. Plasma membrane was dyed with CTB- Alexa 555 (magenta). Localization of TLR8 was determined by immunohistochemical staining with rabbit anti-HA antibodies and secondary goat anti-rabbit antibodies conjugated with DyeMer 488/615 (D31601, Molecular Probes, Invitrogen). (A, B right) TLR membrane localization was evaluated from plots of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 µm line profiles (n=9). Three representative lines are marked on merged images. Images are selected from two independent experiments. Scale bars, 10.
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SFig.3. Overexpression of TLR3 without UNC93B1 does not markedly elevate the amount of TLR3 at the surface of the plasma membrane. HEK293T cells were transfected with increasing amounts of TLR3 (2µg, 3µg or 4µg). Cell surface and intracellular expression levels of the TLR3 were determined by flow cytometry. Histograms of mock-transfected cells (pcDNA plasmid - filled areas) or cells transfected with TLR3 (solid lines) are shown. Increase of cell population expressing plasma membrane localised TLR3 normalised by the number of cells expressing intracellular TLR3 is shown on right.The representative data from three experiments are shown.
SFig. 4.Overexpression of UNC93B1 increases response to TLR7 and TLR8 agonists.(A) HEK293T cells were transfected with TLR7, TLR8 and TLR9 alone or cotransfected with UNC93B1. Cell lysates were separated by 5% SDS-PAGE gel, blotted and probedusing anti-HA antibodies. The representative data from two experiments are shown. (B, C) HEK293 cells were transfected with TLR7 (B) or TLR8 (C) with increasing amounts of UNC93B1. Cells were cotransfected with NFκBresponsive reporter plasmid and Renilla normalization reporter plasmid. 18h after stimulation with R848 (10µg/ml), luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from the triplicate wells. The representative data from three experiments are shown.
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