Supplementary Figures for TSC1 controls macrophage polarization to prevent inflammatory disorder by Linnan Zhu et al

Supplementary Figures for TSC1 controls macrophage polarization to prevent inflammatory disorder by Linnan Zhu et al

Suppl. Fig. 1 Tissue DN C Proteins kd TSC1-17 TSC 1 loxp bp -48-285 ctin PEMs Neutrophils T cells cells -42 LysMcre PEM DN -7-35 M H 2 O D MDM proteins TSC1 TSC2 kd -17-2 ctin -42 35bp Supplementary Figure 1. Generation and confirmation of myeloid cell-specific TSC1 deficient mice. (), Representative genotyping PCR of and wild-type () mice. (), Representative genotyping PCR of and macrophages. Primary macrophages freshly isolated from the peritoneal cavity (PEMs) of and mice were used. (C), Immunoblotting analysis of TSC1 expression in the sorted macrophages, neutrophils, T cells, and cells from and mice. (D), Immunoblotting analysis of TSC1 and TSC2 expression in the bone marrow cell-derived macrophages (MDMs) of and mice. Two mice from each group were assayed. Results are representative of 3 independent experiments. LysMCre +/- TSC1 loxp/loxp mice were referred to as, whereas mice with LysMCre -/- TSC1 loxp/loxp mice were referred to as control mice. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 2 SS LIN Count mrn expression zhuln 9623 212-9-6 3.LMD zhuln 9621 212-9-6 1.LMD 124 11 768 512 256 Gate 2 83 55 28 1 nti-f4/8-cy5 1 1 1 2 1 3 1 4 1 nti-cd14-fitc 1 1 1 2 1 3 1 4 FL4 LOG FL1 LOG Supplementary Figure 2. CD14 and TLR4 expression of macrophages. (), Representative FCS analysis of CD14 expression in the freshly isolated macrophages of and mice. (), The TLR4 mrn expression in isolated and macrophages was determined by real-time PCR assay. Data are one representative of 3 independent experiments, and presented as mean±sd (n=4 samples/group). 1.4 1.2 1.8.6.4.2 TLR4

TNF-α mrn expression IL-12p4 mrn expression FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG FL2 LOG % of TNF-α+ cells Suppl. Fig. 3 C 1 4.1% 1 3 1 2 1 1 1 3 1 2 1 1 99.69% 18 16 14 12 1 8 6 4 2 LPS (ng ml -1 ).1.1 1 1 1 1 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 4 1 4 FL1 LOG 1.2%.17% 4 FL1 LOG 1.%.% 4 FL1 LOG 1 1 4.%.% 4 FL1 LOG FL1 LOG.1%.%.%.% 1 4 FL1 LOG.1%.% 96.15% 1.2% 4.% 1 3 1 2 1 1.27% 71.69% 1 3 1 2 1 1 3.66% 41.69% nti-tnfα-fitc 1 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 1 4 1 1 1 2 1 3 1 4 FL1 LOG FL1 LOG FL1 LOG FL1 LOG FL1 LOG FL1 LOG 1.% 4.% 1 3 1 2 1 1 28.31% 54.26% 1 3 1 2 1 1 58.31% 38.83% 1.% 4.% 1 3 1 2 1 1 45.74% 49.78% 1 3 1 2 1 1 61.17% 35.58% * * 16 14 12 1 8 6 * 4 * 2 3 6 24 48 3 6 24 48 Time with LPS (h) 1.% 4.% 1 1 5.22% 51.78% 1 1 64.41% 33.14% 9 8 7 6 * 5 4 3 2 1 Conc. of LPS (ng ml -1 ) Supplementary Figure 3. Significantly increased TNF-α and IL-12 production of bone marrow cellderived macrophages (MDMs) after LPS stimulation. Isolated and bone marrow cells were cultured with M-CSF (1 ng ml -1 ) to induce macrophage differentiation for 12 days. MDMs were stimulated with LPS for 6 h to detect inflammatory cytokine production. (), Representative FCS analysis of TNF-α expression in the bone marrow cell-derived macrophages of and mice after different doses of LPS stimulation for 6 h. (), The percentages of TNF-α + cells in the MDMs of and mice were shown after different doses of LPS stimulation for 6 h. (C), TNF-α and IL-12 mrn expression in the and MDMs after LPS (1 ng ml -1 ) stimulation for indicated times were detected by real-time PCR assay. Data are one representative of 3 independent experiments, and presented as mean±sd (n=4 samples per group). P<.1, *P<.1 compared with macrophages. P values were determined using Student s t-tests. 1 3 1 2 1 3 1 2 1.% 4.% 1 3 1 2 1 1 48.22% 54.76% 1 3 1 2 1 1 66.86% 32.36%.% 45.24% 67.63%

Suppl. Fig. 4 LPS: - + - + (3 min) kd TSC1-17 TSC2 p-s6 (235/236) β-ctin -2-32 -42 Supplementary Figure 4. Increased p-s6 activity in macrophages with or without LPS stimulation. Freshly isolated peritoneal macrophages from and mice were stimulated with LPS (1 ng ml -1 ) for 3 min. The levels of TSC1, TSC2, p-s6 and actin in macrophages were detected using western blots. One representative of 3 independent experiments was shown. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 5 mrn expression Percentage(%) Percentage(%) 25 2 15 1 IL-12p4 * * * 7 6 5 4 3 TNF-α * C 5 45 4 35 3 25 2 IL-12p4 * * 5 CMC Rapa CMC Rapa Control LPS 2 1 * CMC Rapa CMC Rapa Control LPS 15 1 5 * CMC Rapa CMC Rapa Control LPS Supplementary Figure 5. Rapa treatment in vivo failed to rescue the enhanced TNF-α and IL-12 production of macrophages after LPS stimulation. and mice were intraperitoneally injected with Rapa (5μg kg -1 body weight) daily for 3 days. Isolated and peritoneal macrophages were stimulated with LPS for 6 h to detect inflammatory cytokine production. (), IL-12p4 mrn expression in and macrophages after LPS (1 ng ml -1 ) stimulation were detected by real-time PCR assay. The percentages of TNF-α + cells () and IL-12p4 + cells (C) in macrophages of and mice were detected by FCS after LPS stimulation for 6 hrs. Data are one representative of 2 independent experiments, and presented as mean±sd (n=4 samples per group). *P<.5, P<.1, *P<.1 compared with macrophages. P values were determined using Student s t-tests.

mrn expression mrn expression (X1 3 ) mrn expression (X1 3 ) uppl. Fig. 6 Loxp LysM 6 5 4 3 2 1 TNF-α IL-12p4 inos mtorko bp -255-169 -7-35 9 8 7 6 mtorko Supplementary Figure 6. Generation and confirmation of myeloid cell-specific mtor deficient mice and significantly increased pro-inflammatory cytokines production of mtorko and MDMs after LPS and/or IFNγ stimulation. (), Representative genotyping PCR of mtorko and wildtype () mice. Primary macrophages freshly isolated from the peritoneal cavity (PEMs) of and mice were used. LysMCre +/- mtor loxp/loxp mice were referred to as mtorko, whereas mice with LysMCre +/- TSC1 loxp/- mice were referred to as mtor +/- mice. (), The mrn expression of TNF-α, IL-12p4 and inos were detected in MDMs of, mtorko and mice by Real-time PCR after LPS (1 ng ml -1 ) and/or IFNγ (5 ng ml -1 ) stimulation for 6 h. Data are one representative of 2 independent experiments, and presented as mean±sd (n=3 samples per group). P<.1 compared with macrophages. P values were determined using Student s t- tests. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27. 3 25 2 mtorko 5 15 4 3 1 2 5 1

Suppl. Fig. 7 nti-tnfα-fitc nti-il-12p4-pe Percentage of TNF-α+ cells (%) Percentage of IL-12 + cells (%) TSC1/mTOR KO 1.7 1.9 2. Control 32.7 44.8 49.2 LPS C Control LPS nti-f4/8-cy5 TSC1/mTOR KO 2.7 5.7 6. 12.7 24.8 34.2 D 6 5 4 3 2 1 45 4 35 3 25 2 15 1 TSC1/mTOR KO Control TSC1/mTOR KO LPS Supplementary Figure 7. TSC1/mTOR KO macrophages produced more TNF-α and IL- 12p4 compared with and mice. Representative FCS analysis () and the percentages () of TNF-α + cells in macrophages of, and TSC1/mTOR KO mice after LPS (1 ng ml -1 ) stimulation for 6 h. Representative FCS analysis (C) and the percentages (D) of IL-12p4 + cells in macrophages of, and TSC1/mTOR KO mice after LPS (1 ng ml -1 ) stimulation for 6 hrs. Data are one representative of 3 independent experiments, and presented as mean±sd (n=3 samples per group). P<.1 compared with the indicated groups. P values were determined using Student s t- tests. 5 nti-f4/8-cy5 Control LPS

Suppl. Fig. 8 Conc. of TNF-α in medium (pg ml -1 ) PD9859 (μm): - - 1 25 5 LPS (1 ng ml -1 ): - + + + + p-erk ctin kd -42/44-42 35 3 25 2 15 1 * * * * KO 5 Supplementary Figure 8. The effect of ERK inhibitor (PD9859) on ERK activity and TNF-α production of and macrophages after LPS stimulation. (). Isolated macrophages were pre-cultured with different doses of PD9859 for 1 h and then cultured with LPS for an additional 3 min. The ERK activity was determined by western blot assays. (). TNF-α level was measured in the culture medium of LPS-treated and macrophages in the presence of PD9859 and Rapa. Isolated and macrophages were pre-cultured with PD9859 (5 μm) and Rapa (1 nm) for 1h and 9 min respectively, and then cultured with LPS for additional 6 h. The medium levels of TNF-α were assayed by ELIS. Data are one representative of 2 independent experiments, and presented as mean±sd (n=3 samples per group). P<.1, *P<.1 compared with group or among the indicated groups. P values were determined using Student s t-tests. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 9 Rapa(h): 2 72 2 72 p-erk ERK ctin LPS: + + + + + + LY2942 (1 μm): + + LY2942 (2 μm): + + LY2942 (5 μm): + + LPS (1 ng ml -1 ): + + + + p-erk kd -42/44-42/44-42 kd -42/44 Supplementary Figure 9. The effect of mtor and PI3K inhibitors on ERK activity of and macrophages during LPS stimulation. () Isolated and macrophages were precultured with Rapa (1 nm) for indicated times and then cultured with LPS (1 ng ml -1 ) for additional 3 min. (), Isolated and macrophages were pre-cultured with different concentration LY2942 for 1 h and then stimulated with LPS (1 ng ml -1 ) for additional 3 min. The ERK activity was determined using western blot assays. Data are one representative of 2 independent experiments. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27. ctin -42

Suppl. Fig. 1 TSC2 ctin TSC2KO kd LPS: - + - - + - IL-4: - - + - - + kd -17 p-erk -42/44-42 ctin -42 Supplementary Figure 1. The expression of TSC2KO MEFs and the ERK activity in and TSC2KO MEFs after IL-4 and LPS stimulation. (). Deficiency of TSC2 expression in TSC2KO MEFs as determined by western blots. (). Increased ERK activity of TSC2KO MEFs after IL-4 (1 U ml -1 ) or LPS (1 ng ml -1 ) treatment. and TSC2KO MEFs were IL-4 or LPS for 3 min. The phosphorylation of ERK was determined using western blot assays. Data are one representative of 3 independent experiments. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 11 mrn expression mrn expression 6 4 2 3 25 2 15 1 5 rg 1 Fizz1 Ym1 12 2 1 15 8 1 2 3 4 5 rg1 Ym1 Fizz1 25 12 * * 3 6 24 48 6 4 5 * 2 * * 2 15 1 5 1 2 3 4 5 Time after IL-4 treatment (h) 3 6 24 48 Time with IL-4 (h) 1 8 6 4 2 3 6 24 48 Supplementary Figure 11. Significantly decreased rg1, Ym1 and Fizz1 expression of isolated primary macrophages or MDMs after IL-4 stimulation. () Primary macrophages freshly isolated from the peritoneal cavity (PEMs) of and mice were treated with IL-4 for the indicated time points. The mrn expression of rg1, Ym1, and Fizz1 were determined by real-time PCR. () Isolated and bone marrow cells were cultured with M-CSF (1 ng ml -1 ) to induce macrophage differentiation for 12 days. The derived macrophages were then stimulated with IL-4 (1 U ml -1 ) for indicated times. The expression of rg1, Ym1, and Fizz1 were determined by real-time PCR. Data are one representative of 3 independent experiments, and presented as mean±sd (n=4 samples per group). P<.1, *P<.1 compared with cells at the same time point. P values were determined using Student s t-tests. * * 1 1 2 3 4 5 * * *

Suppl. Fig. 12 SS LIN SSC Count zhuln 9623 212-9-6 3.LMD 124 zhuln 9621 212-9-6 1.LMD 92 768 69 512 Gate 2 46 256 23 1 nti-cd124-pe 1 nti-f4/8-cy5 1 1 1 2 1 3 1 4 1 1 1 2 1 3 1 4 FL4 LOG FL2 LOG Supplementary Figure 12. CD124 (IL-4Rα) expression on and macrophages. Representative FCS analysis of CD14 expression in the freshly isolated macrophages of and mice. Results presented are one representative of 3 independent experiments.

mrn expression(x1 3 ) mrn expression mrn expression Suppl. Fig. 13 7 6 5 4 3 2 1 rg1 Fizz1 Ym1 25 3 * 2 25 2 * 15 15 1 1 * 5 5 * IL-4: - + - + - + - + (48 h) Rapa: - - + + - - + + (48 h) rg1 β-actin -4kD -42kD C D 35 3 25 2 15 1 5 rg1 Fizz1 Ym1 35 16 3 * 14 * * 25 12 * * * 1 * 2 * * 8 * 15 * 6 1 4 * 5 2 1 1 1 1 1 1 1 1 1 15 1 5 mtorko Control IL-4 Conc. of Rapa (nm) Ym1 Fizz1 rg1 4 35 3 25 2 15 1 5 mtorko Control IL-4 8 7 6 5 4 3 2 1 mtorko Control IL-4 Supplementary Figure 13. The effects of mtor on IL-4-induced M2 polarization. Isolated and peritoneal macrophages were pre-treated with 1 nm of Rapa for 9 min, and then stimulated with IL-4 (1 U ml -1 ) for an additional 48 hrs. (), The mrn expression of rg1, Ym1, and Fizz1 were determined by real-time PCR and (), the expression of rg1 protien was determined by western blot assays. (C), Isolated and peritoneal macrophages were pretreated with different doses of Rapa for 9 min, and then stimulated with IL-4 (1 U ml -1 ) for an additional 48 h. The expression of rg1, Ym1, and Fizz1 were determined by real-time PCR. (D), Isolated, mtorko and peritoneal macrophages stimulated with IL-4 (1 U ml -1 ) 48 h. The expression of rg1, Ym1, and Fizz1 were determined by real-time PCR. Data are one representative of 2 independent experiments, and were presented as mean±sd (n=4 samples per group). *P<.5, P<.1, *P<.1 compared with control macrophages. P values were determined using Student s t- tests. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 14 mtorko IL-4: - + - + kd p-stt6 C/EPβ -11-38 ctin -42 Supplementary Figure 14. The effect of mtor on C/EPβ protein. Isolated and macrophages were stimulated with IL-4 for 3 min. The p-stt6 and C/EPβ were determined using western blot assays. Data are one representative of 3 independent experiments. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 15 C/EPβ ctin GFP C/EPβ kd -38-42 Supplementary Figure 15. Over-expression of C/EPβ in MDMs. one marrow cell-derived and macrophages were infected with GFP-denovirus or CEPβ-denovirus for 24 h. Cells were lysed and the C/EPβ expression level was determined using western blot assays. Data are one representative of 2 independent experiments. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Suppl. Fig. 16 rg1 mrn expression Ym1 mrn expression Fizz mrn expression Control IL-4: - + + + - + + + (3 min) PD9859: - - 2 72 - - 2 72 (h) kd p-stt6-11 p-kt38-6 p-kt473-6 C/EPβ -38 ctin -42 6 5 4 3 2 1 * P>.5 * Control IL-4 IL-4+PD PD 25 2 15 1 5 * P>.5 * Control IL-4 IL-4+PD PD 1 9 8 7 6 5 4 3 2 1 * P>.5 * Control IL-4 IL-4+PD PD Supplementary Figure 16. The effect of ERK on M2 polarization (), Isolated macrophages were pre-cultured with PD9859 (5 μm) for indicated time and then stimulated with IL-4 (1 U ml -1 ) for 3 min. The p-stt6, p-kt and C/EPβ expression level were determined using western blot assays. (), Isolated and macrophages were pre-cultured with PD9859 (5 μm) for 3 min and then stimulated with IL-4 (1 U ml -1 ) for 48 h. The mrn expression of rg1, Ym1, and Fizz1 were determined by real-time PCR. Data are one representative of 3 independent experiments, and presented as mean±sd (n=3 samples per group). *P<.1 compared with macrophages. P values were determined using Student s t-tests. Images have been cropped for presentation; full size blot is shown in Supplementary Figure 27.

Cell no. of mlns (X1 7 ) % in mlns Cell no. of MCs (X1 7 ) % in MCs Cell no. of spl (X1 8 ) % in spleens Cell no. of plns (X1 7 ) % in plns Cell no. of thy (X1 8 ) % in thymus WCs (X1 3 μl -1 ) % in WCs Suppl. Fig. 17 4 2 1 8 6 4 2 C 12 8 4 D 6 4 2 2 15 1 5 E Spleen plns mlns F 3 2 1 G 4 3 2 1 1 8 6 4 2 H 1 5 I 4 3 2 1 1.5 1.5 J 1 5 K 5 4 3 2 1 2 1 L 1 5 M 35 3 25 2 15 1 5 Supplementary Figure 17. The detection of immune cell subpopulations in about 4 weeks old and mice. Four-to-5 week-old and mice were assayed. The percentages and cell numbers of CD4 + T cells, CD8 + T cells, cells, monocytes, DCs, NKs and granulocytes in the thymus (-), peripheral blood (C-D), spleen (F-G), plns (H-I), mlns (J- K), and bone marrow (L-M) were measured by FCM. (,E). Macroscopic examination of the thymus, spleen, plns and mlns in and mice. Data are one representative of 2 independent experiments, and presented as mean±sd (n=4 samples per group).

Suppl. Fig. 18 Supplementary Figure 18. Macroscopic examination of the digestive system in and sick mice. Eight-to-1 weeks old and littermates were assayed.

Suppl. Fig. 19 liver 2X Supplementary Figure 19. Immune cell infiltration in tissues of mice with a myeloid cell-specific TSC1 deficiency. H&E staining of liver (), lung () and intestine (C) tissues from age-matched and mice was presented. Scale bar, 1 μm. More than 3 mice with similar alteration were observed. lung 2X C intestine 1X

Suppl. Fig. 2 Conc. of IL-12p4 (pg ml -1 ) 3 25 2 15 1 Supplementary Figure 2. Macroscopic examination of livers and IL-12p4 levels in the sera of in LPS-treated and mice. LPS (5 mg per kg body weight) was i.p. injected in age-matched and mice. (), Macroscopic examination for the haemorrhaging in liver from LPS-treated and mice was shown. (), The sera were harvested by 6 h after LPS treatment for IL-12p4 detection using ELIS assay. Data are one representative of 2 independent experiments, and presented as mean±sd (n=5 mice per group). P<.1 compared with mice. P values were determined using Student s t-tests. 5 Control LPS

Mouse survival (%) TNF-α (pg ml -1 ) Suppl. Fig. 21 C 1 8 6 4 P<.1 2 2 4 6 8 1 Time after LPS injection (h) D 1 8 6 4 2 Control * LPS Supplementary Figure 21. Mice with a myeloid cell-specific TSC1 deficiency were sensitive to endotoxin shock. (), Kaplan-Meier plots of and mouse survival after LPS injection (2.5 mg per kg body weight) were shown. P values were determined using Log-rank tests. (), Macroscopic examination for the haemorrhaging in liver from LPS-treated and mice was shown. (C), H&E staining of liver and kidney tissues from LPS-treated and mice were presented. Scale bar, 2 μm. (D), TNF-α levels in the sera of and mice treated with or without LPS for 2 h were detected by ELIS assays. Each assay was performed more than two times. Data were shown as Mean+SD (N=6 mice per group). *P<.1 compared with control mice. P values were determined using Student s t- tests. Liver Kidney

Suppl. Fig. 22 RasGTPase Raf1 MEK1/2 TSC1/2 complex X mtor C/EPβ Supplementary Figure 22. proposed and simplified model of the regulation of TSC1/2 complex on macrophage polarization. TSC2 directly inhibits RasGTPase and subsequently attenuates M1 response via Raf1- MEK-ERK pathway. TSC2 directly inhibits RhebGTPase and subsequently mtor activity. mtor inhibits M2 polarization through C/EPβ pathway. The deficiency of TSC1/2 in macrophages will break the M1/M2 balance so that mice suffer from an autoinflammatory disorder. In addition, The identification of RasGTPase as a new target molecule also offered one more interacting node for the crosstalk between Ras-ERK and KT-TSC1/2- mtor pathways. Erk1/2 M1 polarization M2 polarization Inflammation

Suppl. Fig. 23 Supplementary Figure 23. Original western blots for images used in Figure 1. Each antibody produced a clear band at the expected size. locks indicate the specific bands used in the main figure. For detection of total proteins and β-actin, the Odyssey imaging system was used and relevant portions of the blot were scanned.

Suppl. Fig. 24 Supplementary Figure 24. Original western blots for images used in Figure 2. Each antibody produced a clear single band at the expected size. locks indicate the specific bands used in the main figure.

Suppl. Fig. 25 Supplementary Figure 25. Original western blots for images used in Figure 3. Each antibody produced a clear band at the expected size. locks indicate the specific bands used in the main figure. For detection of total proteins and β-actin, the Odyssey imaging system was used and relevant portions of the blot were scanned.

Suppl. Fig. 26 Supplementary Figure 26. Original western blots for images used in Figure 4. Each antibody produced a clear band at the expected size. locks indicate the specific bands used in the main figure. For detection of total proteins and β-actin, the Odyssey imaging system was used and relevant portions of the blot were scanned.

Suppl. Fig. 27 Supplementary Figure 27. Original western blots for images used in Supplementary Figures. Each antibody produced a clear band at the expected size. locks indicate the specific bands used in the main figure.