Supplementary Information
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The TSC-mTOR pathway regulates macrophage polarization Vanessa Byles1,3, Anthony J. Covarrubias1,3, Issam Ben-Sahra1, Dudley W. Lamming2, David M. Sabatini2, Brendan D. Manning1, and Tiffany Horng1
1
Department of Genetics & Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 2
Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Biology, MIT, Cambridge, MA 02139; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139; Broad Institute of Harvard and MIT, Seven Cambridge Center, Cambridge, MA 02142; The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139 3
These authors contributed equally to this work
Corresponding author: Tiffany Horng Harvard School of Public Health 655 Huntington Ave, II-115 Boston, MA 02115 Ph: (617) 432-7526 F: (617) 432-5236
[email protected]
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Supplementary Figures S1-S9 Supplementary Table S1 Su Su
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Supplementary Figure S1. Increased Cell Size and Granularity in Tsc1Δ/Δ BMDMs Flow cytometry analysis of Tsc1fl/fl and Tsc1Δ/Δ BMDMs stained with macrophage markers CD11b and F4/80. Histogram plots are shown as % of maximum. Red= Tsc1fl/fl and Blue= Tsc1Δ/Δ
IL-10
0.5 24 hr
IL-10
*
3 2 1
0.5 0.0
24 hr
2.5
Tsc1fl/fl Tsc1!/!
2.0 1.5 1.0 0.5 0.0 S e Ig RB ate C d G -S + R L PS B C + LP S un LP S S tre Ig RB ate C d G -S + R B LPS C + LP S LP S
1.0
IL-10
Il-12 p40 Relative Expression
1.5
*
12 hr
un tre at ed
IL-10
2.0
NS
0
0
2.5
NS
*
4h r
1
4
tr
0
NS
S tre Ig RB ate C d G -S + R B LPS C + LP S L un P S SR trea te Ig B C d G -S + R B LPS C + LP S LP S
1
12 hr
0.0
Il-12 p35 Relative Expression
2
**
NS
un
3
S tre Ig RB ate C d G -S + R B LPS C + LP S un LP S SR trea t Ig B C ed G -S + R L PS B C + LP S LP S
Relative Expression
NS
1.0
Relative Expression
2
Il-10 4
***
un tre at ed
24 hr
12 hr
8h r
4h r
un tre at ed
IL-10
***
3
NS
un
NS
0
un
1.5
NS
Socs3
24 hr
NS
4
4h r
NS
Relative Expression
Relative Expression
**
b
NS
Bcl3 **
1
2.0
8h r
un tre at ed
IL-10
Tsc1fl/fl Tsc1"/"
**
8h r
0.0
Stat3
2
*
NS
0.5
24 hr
12 hr
8h r
4h r
un tre at ed
NS
1.0
0.0
3
Relative Expression
0.5
NS
2.5
un tre at ed
1.0
1.5
24 hr
1.5
2.0
12 hr
NS
12 hr
NS
2.5
8h r
2.0
*
4h r
***
NS
Relative Expression
Relative Expression
2.5
Tgf-!
NS (p= 0.056)
4h r
Mrc1
Arg1
8h r
a
Supplementary Figure S2. Impaired M2c but not M2b Polarization in Tsc1Δ/ Δ BMDMs a. M2c gene expression in Tsc1fl/fl and Tsc1Δ/Δ BMDMs stimulated with 10ng/ml IL-10 for the indicated times (n=2 representative experiments). b. M2b gene expression of Tsc1fl/fl and Tsc1Δ/Δ BMDMs stimulated simultaneously with LPS and non-opsonized (SRBC) or opsonized SRBC (IgG- SRBC) for 6h (Data representative of 3 independent experiments). Graphs are shown as mean ± SEM, *p
