Supplementary Materials for

Guanabenz inhibits TLR9 signaling through a pathway that is

independent of eIF2α dephosphorylation by the GADD34/PP1c complex

Jessica Perego, Andreia Mendes, Clarisse Bourbon, Voahirana Camosseto,

Alexis Combes, Hong Liu, Thien-Phong Vu Manh, Alexandre Dalet, Lionel Chasson,

Lionel Spinelli, Nathalie Bardin, Laurent Chiche, Manuel A. S. Santos, Evelina Gatti,*

Philippe Pierre*

*Corresponding author. Email: gatti@ciml.univ-mrs.fr (E.G.); pierre@ciml.univ-mrs.fr (P.P.)

Published 23 January 2018, Sci. Signal. 11, eaam8104 (2018)

DOI: 10.1126/scisignal.aam8104

The PDF file includes:

Fig. S1. GBZ inhibits TLR9 signaling in B cells.

Fig. S2. TMPD and CpG synergize to promote inflammation.

Fig. S3. GBZ does not inhibit endosomal acidification or protein synthesis.

Fig. S4. GBZ and 25-HC do not reduce TLR9 abundance in pDCs.

Table S1. List of genes in DCs with increased expression in response to poly(I:C)

or P. mirabilis that is inhibited by GBZ.

Table S2. List of the primers used for gene expression analysis by qPCR.

Table S3. List of the antibodies used for sorting DC1, DC2, and pDCs from

Flt3L-DC culture.

Table S4. List of the antibodies used for flow cytometry.

www.sciencesignaling.org/cgi/content/full/11/514/eaam8104/DC1

Fig. S1. GBZ inhibits TLR9 signaling in B cells.

(A) Mouse splenic B cells were purified and activated with ODN 1826 (100 nM) in the presence of

GBZ, chloroquine or clonidine (50 µM). 24h after activation, cells were harvested and screened for

the up-regulation of the activation markers CD86 and CD69 by flow cytometry. (B) Human primary

B cells were purified from blood and activated either with 5 µM ODN 2006 or 2% serum from

lupus patients. GBZ was added at 50 µM. TNF secretion was then measured at different time points

or at 16h for the cells activated with serum. (C) Mean Fluorescence Intensity (MFI) for phospho-S6

detected by intracellular phospho-flow cytometry in CpG-A activated human primary B cells

treated or not with GBZ. Data of n=3 experiments were normalized to non-treated cells. Statistical

significance was assigned using ANOVA (A) or two tailed Student’s t test (B and C) on at least n=3

independent experiments (* p<0.05; ** p<0.005; ****p<0.0001).

Fig. S2. TMPD and CpG synergize to promote inflammation.

(A) Mice were injected at day 0 with 500 µL of TMPD (or PBS for control mice), then injected at

day 16 with ODN 1585 (50 µg/20 g). Mice were sacrificed 24h after CpG injection. The different

immune subsets present in the peritoneal cavity were analyzed by flow cytometry. (B) Mice were

injected at day 0 with 500 µL of TMPD (or PBS for control mice) and at day 16 with ODN 1585

(50 µg/20 g). Mice were sacrificed 24h, 48h or 5 days after CpG injection. The expression of ISGs

and cytokine-encoding genes such as IFNa4, TNF, C25H, ISG15, OASL1 in PECs was determined

by qPCR. n values in (B) indicate the number of mice and also apply to (A, one representative

experiment presented).

Fig. S3. GBZ does not inhibit endosomal acidification or protein synthesis. (A) HEK 293 cells

expressing hTLR9 were activated with ODN 2006 (2.5 µM), GBZ (50 µM) or bafilomycin

(200nM). Dextran 40 MW coupled to a pH sensitive FITC fluorochrome was internalized by

incubating the cells at 37 °C for different time points. The experimental protocol was adapted from

(39). FITC MFI was detected by flow cytometry. This is one representative experiment out of n=3

independent experiments. (B) A20 cells were treated with ODN 2006 (2.5 µM), GBZ (50 µM) or

chloroquine (50 µM) for 24h. Cells were immunoblotted for the invariant chain using IN-1

supernatant and actin as a loading control. This is one representative result out of n=3 independent

experiments. (C) Western Blot of eIF2α-P and total eIF2α in GM-CSF DCs treated with 1μM ODN

1585 for 6h, in the presence of 25 or 50 μM of GBZ. This is one representative result out of n=3

independent experiments. (D) Protein translation intensity was monitored in mouse Flt3L-derived

pDCs, differentiated from both FVB wild type and GADD34ΔC/ΔC mice, using puromycin

incorporation and detection with anti-puromycin antibody by flow cytometry (n=3 independent

experiments).

Fig. S4. GBZ and 25-HC do not reduce TLR9 abundance in pDCs.

CAL-1 cells were stimulated with 3 µM ODN 2216 in the presence of GBZ (50 µM) or 25-HC (100

nM). (A) qPCR monitoring of TLR9 mRNA induction (n=3 independent experiments). (B) Mean

Fluorescence Intensity (MFI) of TLR9 expression measured by flow cytometry (n=3 independent

experiments). (C) TNF mRNA expression in CAL-1 cells stimulated with 3 µM ODN 2216 for 3h,

with the addition of GBZ (50 µM) and 25-HC (100 nM) (n=3 independent experiments).

Table S1. List of genes in DCs with increased expression in response to poly(I:C) or P.

mirabilis that is inhibited by GBZ.

Table S2. List of the primers used for gene expression analysis by qPCR.

Table S3. List of the antibodies used for sorting DC1, DC2, and pDCs from Flt3L-DC culture.

Table S4. List of the antibodies used for flow cytometry.