Supplementary Figure 1. Deletion of Smad3 prevents B16F10 melanoma invasion and metastasis in a mouse s.c. tumor model.
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- Ginger King
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1 A B16F1 s.c. Lung LN Distant lymph nodes Colon B B16F1 s.c. Supplementary Figure 1. Deletion of Smad3 prevents B16F1 melanoma invasion and metastasis in a mouse s.c. tumor model. Highly invasive growth patterns () and metastasis to lymph nodes (LN), lung (arrows), and colon (arrow) developed in B16F1 tumor-bearing mice (A) are prevented in mice (B). Representative macroscopic images are shown for groups of 8 mice.
2 A 1 st challenge Rechallenge 1 st challenge Rechallenge Rechallenge Day 15 Day 2 B Tumor volume (mm 3 ) Rechallenged mice Day C NK1.1 NKp46 Tumor tissue of rechallenged mice D Spleen of rechallenged mice Blood of rechallenged mice NKp46-PE NK1.1-APC Supplementary Figure 2. microenvironment reduces cancer progression in the tumor rechallenged mice. (A and B) The growth of B16F1 tumor was significantly suppressed in the mice, qualified by imaging on Days 15 # and 2 (A) and quantified by the tumor volume on day 2 (B). (C) Tumor-infiltrating NK cells are largely increased in the rechallenged mice compared to the mice, which are showed by NKp46 and NK1.1 coimmunofluorescence imaging. (D) Mature NK cells (NKp46 + NK1.1 + ) were increased in spleen and blood of the rechallenged mice compared to the rechallenged mice, which are quantified by 2-colour flow analysis of Day 15 samples. Data represent mean ± SEM for groups of 3 mice. p<.5, compared to B16F1 tumor-rechallenged group analyzed by ANOVA. Scale bar, 1 µm. # 1 st challenged mice were scarified on Day 15 due to the restriction of maximum tumor volume (2mm 3 ) under Animal Ethics Experimental Committee.
3 A Spleen Lung Bone marrow Blood Normal mice NK1.1.39% 1.77%.4% 2.88%.67% 2.72% 2.37% 3.49% B CD49b Spleen Lung Blood B16F1-bearing mice NK1.1 C Spleen NKp46 CD49b.97% 2.56%.8% 9.76%.51% 6.2% Supplementary Figure 3. Smad3-dependent microenvironment promotes cancer by suppressing the NK cell differentiation and maturation in normal and tumorbearing mice. (A and B) Two-color flow cytometry shows that normal mice null for Smad3 develop higher levels of the NK1.1 + CD49b + cell population in the spleen, lung, bone marrow, and peripheral blood (A), which is further increased in tumor-bearing mice (up to a 1-fold increase in lung and peripheral blood), but not in mice. (C) Increased splenic NKp46 + cells are detected in B16F1 tumorbearing mice but not in mice by immunofluorescence imaging. Data represent for groups of 8 mice. Scale bar, 1 µm. NKp46 + cells %
4 A 25.19% 46.75% pg/ml IFN-γ by ELISA 4 2 PE- IFN-γ B C D Cytotoxicity(%) Cytotoxicity(%) Yac-1 LLC B16F E:T E:T E:T Cytotoxicity(%) Supplementary Figure 4: Deletion of Smad3 enhances NK cell anti-cancer cytotoxicity ex-vivo. (A) Flow cytometry and ELISA show that IFN-γ-producing ability by splenic NKp46 + from tumor-bearing mice is largely enhanced. (B- D) The cytotoxicity assay detects that deletion of Smad3 largely enhances the cellkilling activity by splenic NK cells against the NK-sensitive target cells YAC-1 (B), syngeneic LLC (C), and B16F1 (D) cancer cells. Data represent for mean ± SEM from 4 independent experiments. p<.1 compared between groups analyzed by ANOVA.
5 A CD31 + CD31 + area ( % ) ## C B Normal B16F1 (D7) VEGF CD31 Ratio(CD31/) Ratio(VEGF/) CD4 + Foxp3 + Merge Normal VEGF CD31 ### ## B16F1 CD4 + Foxp3 + cells (%) ## D Normal B16F1 (D7) E Normal B16F1 (D7) Pro-MMP2 Act-MMP2 MMP9 CXCR4 MMP13 Supplementary Figure 5. Smad3 facilitates cancer progression by enhancing angiogenesis and Treg immune response in mice. (A) Immunofluorescence and (B) Western blot analysis detect a marked intratumoral angiogenesis identified by numerous CD31+ vessels and higher levels of VEGF and CD31 protein expression in B16F1 melanoma-bearing mice, which is blunted in B16F1 melanomabearing mice on day 7. (C) Two-color immunofluorescence shows that there are numerous CD4+ Foxp3+ cells infiltrating the tumor tissues of B16F1 melanoma-bearing mice, which is absent in B16F1-bearing mice. (D) Western blot analysis detect that there are high levels of intratumoral pro-and active MMP-2, MMP-9, and MMP-13 expression in B16F1 melanoma-bearing mice, which is abolished in mice. (E) Western blot analysis shows that mice null for Smad3 are prevented from a marked upregulation of intratumoral CXCR4 in the B16F1 melanoma tissues. Data represent mean ± SEM for groups of 8 mice. p<.5, p<.1 compared to normal; ##p<.1, ###p<.1 compared to tumor-bearing mice analyzed by ANOVA. Scale bar, 5 and 1 µm.
6 A B C D Ratio(MMP-2/) Ratio(MMP-9/) Ratio(MMP-13/) Ratio(CXCR4/) Normal MMP-2 MMP-9 MMP-13 CXCR4 ### B16F1 ### ### ### Supplementary Figure 6. Deletion of Smad3 suppresses MMPs-mediated matrix degradation and CXCR4 expression in B16F1 tumor bearing mice. (A) Quantification results of the Western blot analysis shows that higher levels of intratumoral proand active MMP-2, MMP-9, and MMP-13 expression in B16F1 melanoma-bearing mice are protected in mice. (B) Quantification results of the Western blot analysis shows that a marked upregulation of intratumoral CXCR4 in B16F1 melanoma-bearing mice is prevented in mice. Data represent mean ± SEM for groups of 8 mice. p<.1, p<.1 compared to normal; ###p<.1 compared to tumorbearing mice analyzed by ANOVA.
7 control Ab + anti-nk1.1 Ab + control Ab + anti-nk1.1 Ab Tumor volume (mm 3 ) Days after B16F1 inoculation Supplementary Figure 7. NK cell depletion restores cancer progression in B16F1 tumor-bearing mice but not in mice. Tumor growth rate is largely inhibited in the mice (blue), which is restored to the level of mice (red) by depleting NK cells with the anti-nk1.1 antibody (Ab). In contrast, treatment with the anti-nk1.1 Ab produces no significant effect on the tumor growth rate on mice. Data represent mean ± SEM for groups of 8 mice. p<.5, p<.1 compared to B16F1 tumor-bearing mice analyzed by ANOVA.
8 Cytotoxicity (%) Cytotoxicity (%) A B Bone marrow-derived NK cells + SIS3 B16F E:T Splenic NK cells + SIS3 B16F E:T C Control + TGF-β1 NKp46-FITC + SIS3 IFNγ-APC Supplementary Figure 8. Deletion and inhibition of Smad3 enhance the cancer killing activity of NK cells. (A) The cytotoxicity assay detects that deletion ( ) or inhibition ( +SIS3, 1µM) of Smad3 largely enhances the cancer killing activity of bone marrow derived NK cells. (B) splenic NK cells treated with SIS3 (1µM for 72h) promote tumor-killing activity of B16F1 melanoma cells. Data represent for mean ± SEM from 3 independent experiments. p<.1, p<.1 compared to Smad3+/+ NK cells with SIS3 treatment analyzed by ANOVA. (C) Flow cytometry shows that deletion ( ) or inhibition (SIS3) of Smad3 largely increases the production of IFNγ + NKp46 + bone marrow derived NK cells with or without TGF-β1 (.5ng/ml), compared to control group ( ) on Day 8 NK differentiation ex vivo. Results shown are representative data of 3 independent experiments.
9 mrna level of E4BP4 on Day 6.3 Ratio(E4BP4/GAPDH).2.1 ### + SIS3 & ### ###. Control sie4bp4 Supplementary Figure 9. Effect of sie4bp4 on the E4BP4 mrna expression in bone marrow cells on Day 6. Bone marrow cells were transfected with nonsense (control) or sirna against mouse E4BP4 (si- E4BP4), or treated with SIS3 (SIS3) on day and day 4 and cells were cultured with the NK cell differentiation medium for 6 days. Expression of E4BP4 mrna was measured by real-time PCR. Data represent mean ± SEM for 3 independent experiments. p<.5 compared to cells; ### p<.1 compared to the individual control group before sie4bp4 treatment; & p<.5 compared to cells analyzed by ANOVA.
10 A Predicted E4BP4 binding site on promoter of IFNγ Mouse Human B.4 Ratio(IFN-γ/GAPDH) ###. sie4bp4 Supplementary Figure 1. E4BP4 knockdown reduces the enhancement of IFNγ transcription on NK cells. (A) An E4BP4 binding site is predicted at the promoter of the evolutionarily conserved region of IFNγ (Ifng) nearby T-bet (TBX5_Q5) binding site in human and mouse genomes (left panel), the sequences of E4BP4 binding site is indicated as blue (right panel). (B) The increased mrna levels of IFNγ in the ex-vivo differentiated NK cells on Day 7 are largely suppressed by E4BP4 knockdown (sie4bp4). Results shown are representative data of 3 independent experiments, p<.1 compared to the cells and ### p<.1 compared to cells analyzed by ANOVA.
11 A B Dosages of SIS3 treatment Control Day 2.5 µg/g 5 µg/g 1 µg/g 12 Control 2.5 µg/g 5. µg/g 1 µg/g Tumor volume (mm3) Days after SIS3 treatment C D SIS3 (µg/g ) Control Survival rate Tumor weight (g) 8 Control µg/g 5 µg/g 4 1 µg/g 2 Control SIS3 (µg/g ) Days after SIS3 treatment Supplementary Figure 11. SIS3 treatment inhibits the progression of invasive lung cancer LLC in Smad3+/+ mice. LLC-luc tumor-bearing Smad3+/+ mice treated with SIS3 are protected from the cancer growth and death in a dose-dependent manner as demonstrated by: the bioluminescent imaging (A), the tumor volumes (B), the tumor weights on day 15 after treatment (C), and the survival rates (D). Data represent mean ± SEM for groups of 8 mice. p<.5, p<.1 compared to control-treated LLC-luc tumor-bearing Smad3+/+ mice analyzed by ANOVA.
12 A C Control Control p-smad3 Pro-MMP2 Act-MMP2 Smad3 MMP9 MMP13 Ratio (P-Smad3/) B Control Ratio Ratio (MMP-2/) (MMP-9/).6 MMP MMP VEGF CD31 Control Ratio (MMP-13/) MMP-13 Control Ratio (VEGF/) VEGF D CXCR4 Control Ratio (CD31/) CD31 Control Ratio (CXCR4/) 1.2 CXCR4.8.4 Control Supplementary Figure 12. SIS3 treatment inhibits angiogenesis and tumor invasive factors in LLC tumor bearing mice. Western blot analysis shows that treatment with SIS3 dose-dependently inhibits intratumoral phosphorylation of Smad3 (A), angiogenesis including expression of VEGF and CD31 proteins (B), pro-and active MMP-2, MMP-9, and MMP-13 expression (C), and CXCR4 expression (D). Data represent mean ± SEM for groups of 8 mice. p<.5, p<.1, p<.1 compared to the control-treated group analyzed by ANOVA.
13 A SIS3 (5µg/g) SIS3 (5µg/g) Control IgG anti-nk1.1 Control IgG anti-nk1.1 Tumor weight (g) # Day 15 Control IgG anti-nk1.1 SIS3 (5µg/g) B SIS3 (5µg/g) Control IgG anti-nk1.1 NKp46-PE NK1.1-APC Supplementary Figure 13. Depletion of NK cells increases cancer progression in SIS3-treated mice. (A) The growth of B16F1 tumor was significantly suppressed in the SIS3-treated IgG group (5µg/g/day, i.p.) compared to the untreated control group, which was partially reversed in the SIS3-treated anti-nk1.1 group (2µg/mouse, weekly i.v.) as qualified by imaging and tumor weight on day 15. (B) Mature NK cells (NKp46 + NK1.1 + ) were increased in the blood of SIS3-treated IgG group compared to the untreated control group, but was largely reduced in the SIS3-treated anti-nk1.1 group as demonstrated by two-colour flow analysis of Day 15 samples. Data represent mean ± SEM for groups of 3 mice. p<.5, p<.1, compared to control group; ## p<.1, compared to the SIS3-treated IgG group analyzed by ANOVA.
14 Ce ll Viability (% of Control) Concentration of SIS3 (µm) Supplementary Figure 14. Effect of SIS3 on proliferation of B16F1 cells. MTT assay shows that addition of SIS3 is able to inhibit B16F1 cell proliferation in a dose-dependent manner. Data represent mean ± SEM for 3 independent experiments. p<.5, p<.1 compared to medium without SIS3 () analyzed by ANOVA.
15 Figure 2c Normal B16F1 82kda 64kda 49kda NKp46(47kDa) 82kda 64kda 49kda 37kda β- actin(43kda) Figure 4a BM NKC Splenic NKC WT KO WT KO 7kDa 55kDa E4BP4 (6kDa) 35kDa 7kDa 55kDa (43kDa) 35kDa Supplementary Figure 15. Full-size scans of Western blots shown in Figures 2 and 4.
16 Figure 5d NC NK cells sie4bp4 sit-bet 7kDa 55kDa 35kDa NKp46 (47kDa) 7kDa 55kDa 35kDa GAPDH () Supplementary Figure 16. Full-size scans of Western blots shown in Figure 5.
17 Figure 7a Figure 7c Control p-smad3 (54kDa) Control Pro-MMP2(72kDa) Act-MMP2(63kDa) 64kDa (43kDa) 18kDa VEGF (42kDa) 115kDa MMP-9 (92kDa) 82kDa MMP-13 (48kDa) 26kDa Smad3 (54kDa) (43kd) Figure 7b Control Figure 7d CD31 (13kDa) 18kDa Control kDa 82kDa (43kDa) CXCR4 (47kDa) (43kd) Supplementary Figure 17. Full-size scans of Western blots shown in Figure 7.
18 Supplementary Figure 5B Supplementary Figure 5D Normal B16F1 (D7) Normal B16F1 (D7) 18kDa 115kDa CD31 (13kDa) 64kDa Pro-MMP2(72kDa) Act-MMP2(63kDa) 82kDa VEGF (42kDa) 82kDa MMP-9 (92kDa) (43kDa) MMP13 (48kDa) Supplementary Figure 5E Normal B16F1 (D7) (43kDa) 64kDa CXCR4 (47kDa) 64kDa (43kDa) Supplementary Figure 18. Full-size scans of Western blots in Supplementary Figure 5.
19 Supplementary Figure 12A Supplementary Figure 12B p-smad3 (54kDa) Control VEGF (42kDa) Control Smad3 (54kDa) CD31 (13kDa) 18kDa 115kDa 82kDa (43kDa) (43kDa) Supplementary Figure 19. Full-size scans of Western blots in Supplementary Figure 12.
20 Supplementary Figure 12C Control Pro-MMP2 (72kDa) Act-MMP2 (63kDa) 64kDa MMP-9 (92kDa) 18kDa 115kDa 82kDa MMP13 (48kDa) (43kDa) Supplementary Figure 12D Control CXCR4 (47kDa) (43kDa) Supplementary Figure 2. Full-size scans of Western blots in Supplementary Figure 12.
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