a b Pml F/F Prrx1-re X Prrx1-re-Pml F/F Relative CFU-F numbers 2. Prrx1-Cre-PML +/+ n.s. d early passages e f 1.4 n.s. Adsorbane Adsorbane.8.6.4.2.8.6.4.2 1 2 3 4 days in ulture late passages 2 4 6 8 1 12 days in ulture Osteoblasts ALP staining Pml +/+ Pml -/- Relative expression Pml mrna BM-MSCs MSCs CD51+Sa1+ Stroma CD51-Sa1- HSCs Prrx1-Cre Pml +/+ Prrx1-Cre Pml F/F Adipoytes Oil-Red-O staining Pml +/+ Pml -/- Prrx1-Cre Pml +/+ Prrx1-Cre Pml F/F Relative adipoyti olonies/ total olonies Prrx1-Cre Pml +/+ Prrx1-Cre Pml F/F 1.2.8 Relative expression Fabp4 mrna 2 15 1 5 Prrx1-Cre-PML +/+ A. Prrx1-Cre-PML +/+ Relative expression Alp mrna 25 2 15 1 5 A. Prrx1-Cre-PML +/+ O. Prrx1-Cre-PML +/+ O. Supplementary Figure 1 Pml regulates the biology of MSCs. (a) Shemati overview of the experimental design used to generate Pml-floxed mie. (b) Expression levels, assayed by RT-qPCR, of Pml mrna in the different populations of mesenhymal ells of Prrx1-Cre-Pml +/+ or Prrx1-Cre-Pml F/F mie; HSCs are used as ontrol. Cells olleted from n=3 mie for eah genotype were pooled together prior to RNA extration. () CFU-F olonies forming the apaity of MSCs derived from Prrx1-Cre- Pml F/F or Prrx1-Cre-Pml +/+ mie (n=7). (d) Proliferation of MSCs derived from Pml +/+ or Pml -/- mie either at early passages in vitro, or at late passages. One representative experiment is shown. (e) Differentiation of MSCs derived from Pml +/+ or Pml -/- mie into osteoblasts or adipoytes. Osteoblasts (on the left) were stained with Alkalyne Phosphatase (ALP), while adipoytes (on the right) were stained Oil-Red-O. The hart on the right shows the quantifiation of Oil-Red-O positive olonies (n=4). (f) Capaity of Prrx1-Cre-Pml +/+ or Prrx1-Cre-Pml F/F MSCs to differentiate to adipoytes (upper hart, A= MSCs triggered to adipogenesis), or into osteoblasts (lower hart. O= MSCs triggered to osteogenesis) one triggered with speifi fators. The hart on the upper right panel shows the relative expression of Fabp4, while the one on the bottom shows the expression of Alp. One representative experiment ± SEM is shown.
a CD48 Gated on CD34- KLS ells Gated on KLS ells Prrx1-Cre-Pml +/+ Prrx1-Cre-Pml F/F Prrx1-Cre-Pml +/+ Prrx1-Cre-Pml F/F C-kit CD15 CD34 Gated on Lin- ells Prrx1-Cre-Pml +/+ Prrx1-Cre-Pml F/F CD34- KLS ells 2. Prrx1-Cre n.s. Prrx1-Cre PmlF/F C-kit Sa1 b BA+ + leukemi ells 1 st Reipients 2 nd Reipients Endosteal BM (E-BM) Pml +/+ Pml -/-.9%.4% Inner BM (I-BM) Pml +/+ Pml -/- 21.7% 8.5% numbers of BA++KLS ells numbers of BA++KLS ells 1 st Reipients 2 nd Reipients % survival % survival 15 1 5 15 1 5 BCR/ABL leukemi ells 2 4 6 8 days BCR/ABL leukemi ells 2 4 6 8 days Control Control Supplementary Figure 2 Pml expressed in MSCs regulates only marginally non-ellautonomously HSCs. (a) Representative plots showing the hematopoieti sub-populations in Prrx1- Cre-Pml +/+ or Prrx1-Cre-Pml F/F mie. The upper left plot shows CD15+CD48-CD34-KLS ells; the one on the right shows CD34-KLS ells, while the one on the bottom shows KLS ells. The hart on the right shows the quantifiation of CD34-KLS ells in Prrx1-Cre-Pml +/+ or Prrx1-Cre-Pml F/F mie (n=5 mie analyzed). (b) Relative perentages (representative plots on the left) and relative total numbers (harts on the right) of BA + + KLS ells present at the endosteal bone marrow (E-BM), or within the inner avity of the bone marrow (I-BM) in seond reipients Pml +/+ or Pml -/- (n=2 mie analyzed). () Survival urves of serially transplanted Prrx1-Cre-Pml F/F or ontrol mie with BA+ + ells. The upper panel shows the survival of primary reipients, while the bottom panel shows the survival of seondary reipient mie.
a ~ 1 passages Day 1% oxygen Day 12 leukemi ells 1, 1, Total leukemi ells and LICs Sorting MSCs Freshly isolated MSCs b wild type SCL-tTA/BCR-ABL gated on Lin- ells KLS ells RV- Fusion gene DNA Pre-leukemi ells + HoxA9 Meis- 1 MLL- AF9 Expansion in wild type reipients + leukemi ells SERIAL o-ultures with MSCs ( or ) BA+KLS ells LV- numbers of BA++ ells d 1 st o-ulture HoxA9-Meis1++ ells 9.8% 6.1% 2.5 2. MLL-AF9-+ ells numbers of BA++KLS ells +kit+ ells 3 2 1 1 st o-ulture e 3 rd o-ulture 12% 4.9% Br/Abl HoxA9+ Meis1 +kit+ ells 2.5 2. 2.5%.6%
Supplementary Figure 3 Pml expressed in MSCs regulates non-ell-autonomously LICs. (a) Shemati overview of MSCs ulturing onditions, and of the o-ultures with MSCs and leukemi ells. (b) Shemati overview of the experimental design to generate leukemi ells that express HoxA9-Meis1,, or +BCR/ABL. () BA++ ells, HoxA9+Meis1 leukemi ells and leukemi ells derived from the first o-ultures with MSCs Pml +/+ or Pml -/- (n 4). (d) Relative numbers and perentages of BA++KLS ells, HoxA9- Meis1 ++ ells, and ++ ells in the first o-ultures with Pml +/+ or Pml -/- MSCs. Quantifiations are represented in the harts on the right, while representative plots are shown on the left (n 3). (e) Representative plots showing perentages of BA++KLS ells, HoxA9-Meis1 ++ ells, and ++ ells in serial o-ultures with Pml +/+ or Pml -/- MSCs.
b From o-ultures with MSCs Pml +/+ a Relative number of olonies HoxA9-Meis1 Relative number of olonies From o-ultures with MSCs Pml -/- % survival 15 PML+/+ PML-/- 1 5 d 1x 4x From o-ultures with MSCs Pml +/+ 1x Untreated AS 2 O 3 6x 1x From o-ultures with MSCs Pml -/- 1x Pml Pml Β-atin shscr shpml Β-atin e Relative number of olonies % survival 15 1 5 1 15 2 5 Methyl ellulose Untreated AS 2 O 3 shscr shpml days 1ells PML+/+ 1ells PML-/- 2ells PML+/+ 2ells PML-/- 1 2 3 4 days
Supplementary Figure 4 Pml expressed in MSCs regulates leukemi ells in a non-ellautonomous manner. (a) Relative number of olonies in methyl-ellulose, generated by leukemi ells derived from o-ultures with MSCs Pml +/+ or Pml -/-. HoxA9-Meis1 leukemi ells are shown on the left, while leukemi ells are shown on the right. One experiment ± SEM is shown. (b) H&E of leukemi blasts in the blood of mie transplanted with HoxA9-Meis1 leukemi ells after o-ulture with or MSCs. The survival urve of these mie is shown on the right. () H&E of leukemi blasts in the blood of mie transplanted with leukemi ells after oulture with or MSCs. The survival urve of these mie is shown on the right. (d) Western blot analysis of Pml expression in MSCs upon knok-down with shrna and upon treatment with AS 2 O 3. (e) Methyl-ellulose assay performed with leukemi ells () whih have been o-ultured with MSCs, while treating with AS 2 O 3, or performed with leukemi ells o-ultured with MSCs transdued with an shrna targeting Pml. One experiment ± SEM is shown.
a 1 st o-ulture 2 nd o-ulture BCR/ABL H+M Start Treatment 1 st o-ulture d Treatment: Anti-CXCR2 Anti-IL6R End 1 st o-ulture: LICs Start 2 nd o-ulture BCR/ABL H+M Day Day 1 Day 4 Day 8 f +Sa1+kit+ ells +kit+ ells Anti-CXCR2 Anti-IL6R Analysis of LICs N.T. HoxA9+Meis1 N.T. b acxcr2 BCR/ABL BCR/ABL ail6r HoxA9+Meis1 15% 9.89% 1.2% 6.9% acxcr2+ail6r 16% 9% 1 st o-ulture 2 nd o-ulture + + Not Treated (N.T.) CXCL1+IL6 reombinant Total numbers of leukemi ells x1 3 4 e 3 2 1 + +kit+ ells MSCs Pml +/+ N.T MSCs Pml +/+ acxcr2+ail6r + N.T. 12% MSCs Pml -/- N.T 15.4% 7.9% 6.2% Total numbers of leukemi ells x1 3 3 2 1 MSCs Pml -/- CXCL1+IL6 + 17.3% acxcr2+ail6r acxcr2+ail6r % of +kit+ ells Total numbers of leukemi ells x1 3 8 6% 6 4 2 Pml +/+ Pml -/- Pml +/+ acxcr2+ail6r Pml -/- CXCL1+IL6 +
Supplementary Figure 5 Pml regulates leukemi ells non-ell-autonomously through IL6/IL6R and CXCL1/CXCR2 pathways. (a) Shemati overview of the experimental design for treatment of the o-ultures with anti-il6r and anti-cxcr2 antibodies. (b) Total number of leukemi ells not treated () or treated with anti-il6r, anti-cxcr2 singularly or in ombination. One representative experiment ± SEM is shown. () Total numbers of BA++KLS ells after the treatment with anti- IL6R or/and anti-cxcr2, singularly or in ombination. Not treated ells () were used as ontrol. The hart on the left shows the quantifiation, while representative plots are shown on the right. One representative experiment ± SEM is shown. (d) Chart on the left shows the numbers of HoxA9- Meis1++kit+ ells after the first o-ulture and treatment with anti-il6r or/and anti-cxcr2 antibodies (singularly or in ombination) One representative experiment ± SEM is shown; representative plots are shown on the right. (e) Chart on the left shows the numbers of MLL-AF9+ +kit+ ells after the first o-ulture and treatment with anti-il6r or/and anti-cxcr2 antibodies (singularly or in ombination) One representative experiment ± SEM is shown; representative plots are shown on the right. (f) Shemati overview of the experimental design is shown on the left. Coultures of MSCs Pml +/+ and HoxA9-Meis1 + + leukemi ells were untreated () as ontrol, or treated with anti-cxcr2 in ombination of anti-il6r. Co-ultures with MSCs Pml -/- and HoxA9- Meis1 + + leukemi ells were untreated as ontrol, or treated with reombinant Il6 and Cxl1 proteins. Leukemi ells were then re-plated onto the new MSCs (Pml +/+ or Pml -/- ); seondary oultures were analyzed. Perentages of +kit+ ells in the different onditions are shown in the plots and the hart on the right. One experiment ± SEM is shown.
Supplementary Figure 6 Original blots of Figure 5