Mark J. Kiel, Melih Acar, Glenn L. Radice, and Sean J. Morrison

Transcription

1 Cell Stem Cell, Volume 4 Supplemental Data Hematopoietic Stem Cells Do Not Depend on N-Cadherin to Regulate Their Maintenance Mark J. Kiel, Melih Acar, Glenn L. Radice, and Sean J. Morrison SUPPLEMENTAL EXPERIMENTAL PROCEDURES Supplemental N-cadherin excision analysis Alternate primer sequences for amplification of N-cadherin alleles were: N-cadherin NC1f 5 -ACC AGC TCG CTC TCA TTG G; N-cadherin NC1r 5 -GCT ATC CGG CAC ATG GAG; N-cadherin NC16f 5 -CCC TCA ACT CCT CCA GTA GC and N-cadherin NC16f 5 - AGC TGA GCT CGG TGT TTT CC. PCR primers NC1f and NC1r were designed to amplify a product containing the ATG start site of exon 1. PCR primers NC16f and NC16r were designed to amplify exon 16 of N-cadherin. Products were separated using 2% agarose gels to confirm the presence or absence of these amplicons. Products were sequenced and confirmed to correspond to N-cadherin. 5-fluorouracil administration A single intraperitoneal dose of 5-fluorouracil (Adrucil, Pharmacia and Upjohn Co., Kalamazoo, MI; 150mg/kg) was administered to Mx-1-Cre + N-cadherin fl/- and littermate control mice one day following 2 weeks of pipc injections. Isolation of HSCs using collagenase treatment and calcium-containing medium Bone marrow cells were flushed from the long bones (tibias and femurs) with Hank s buffered salt solution containing calcium and magnesium, supplemented with 2% heatinactivated calf serum (GIBCO). Flushed bones were crushed with scissors and incubated for 90 minutes in 0.125% collagenase II (GIBCO) at 37 o C. Bones were thoroughly triturated and filtered through nylon screen (45 um, Sefar America, Kansas City, MO) to obtain a single cell suspension before being washed and centrifuged. Staining for MNCD2 on HSCs was performed as described (see Methods section) with the exception that calcium and magnesium containing medium was used. Western blot analysis Cells were lysed in RIPA lysis buffer containing protease inhibitors (Roche), loaded onto NuPAGE 7% Tris-acetate gels (Invitrogen, Carlsbad CA) and electrotransferred onto Immobilon Transfer PVDF membranes (Millipore, Temecula CA) for 2-3 hours. Membranes were blocked in 5% non-fat milk protein in TBS (20mM Tris ph8.0, 150mM NaCl) buffer with 0.05% Tween- 20 (TBS-T) for 1 hour at room temperature. Membranes were incubated in primary anti-ncadherin MNCD2 antibody overnight at 4 o C at a dilution of 1:2,500. The membrane was washed 5 times for 1 hour in TBS-T and incubated in peroxidase-conjugated goat anti-rat IgG (catalogue 1

2 number: ; Jackson Immunoresearch, West Grove PA) at a dilution of 1:10,000 for 2 hours at room temperature. The membrane was then washed 5 times for 1 hour in TBS-T and 2 times for 30 minutes in TBS. Bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford IL). For the ß-actin loading control blot, anti-β-actin primary antibody (clone C4, Santa Cruz, Santa Cruz CA) was used at a dilution of 1:1,000 at 4 o C overnight. A fraction of the cell extracts used for the MNCD2 N-cadherin blot were loaded for the ß-actin blot. Postnatal day 0 (P0) forebrain cells were isolated, counted and serially diluted into RIPA buffer as a positive control for N-cadherin expression. Sorted CD45+ and surface IgM+ bone marrow cells from Mx-1-Cre + N-cadherin fl/- mice or littermate controls were diluted in RIPA buffer. Lineage - Sca-1 + c-kit + (LSK) cells from Mx-1-Cre + N-cadherin fl/- mice or littermate controls were sorted by flow-cytometry into HBSS +. Cells were then centrifuged at 500g for 5 minutes, the supernatant was aspirated and the pellet resuspended in <30ul of RIPA lysis buffer. Figure S1: Excision of N-cadherin from HSCs was confirmed by PCR using independent sets of primers. CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + bone marrow HSCs from pipctreated Mx-1-Cre + N-cadherin fl/- mice or littermate controls were cultured in methylcellulose for 14 days. Genomic DNA was then extracted from individual colonies and examined for N- cadherin deletion using primers that amplify ~200 bp of N-cadherin exon 1 and 5 -UTR (A) or ~300 bp of N-cadherin exon 16 (B). None of the DNA from colonies of pipc-treated Mx-1- Cre + N-cadherin fl/- mice retained exon 1, the region of N-cadherin deleted by Cre-mediated recombination (Kostetskii et al., 2005). In contrast, exon 1 was readily amplified from DNA from control littermate colonies (A). As expected, N-cadherin exon 16 was readily amplified from all colonies as this sequence is unaffected by Cre-mediated recombination (B). Sequencing of each band confirmed the specific amplification of N-cadherin. 2

3 Figure S2: Recovery of hematopoiesis following 5-fluorouracil-induced myelosuppression was not affected by N-cadherin deficiency. Peripheral blood, bone marrow, and spleen were examined in Mx-1-Cre + N-cadherin fl/- mice (black bars) or littermate controls (white bars) 10 days after a single dose of 5-fluorouracil (5-FU) that was administered following 2 weeks of pipc treatment. No statistically significant differences were observed in the concentration of white blood cells (WBC), erythrocytes (RBC) or platelets (Plts) from the blood of Mx-1-Cre + N- cadherin fl/- mice as compared to littermate controls (A). We also did not observe any differences in the composition or cellularity of bone marrow (B) or spleen (C) between Mx-1-Cre + N- cadherin fl/- mice and littermate controls. Although HSC surface markers transiently change their expression levels in the days immediately following 5-FU treatment (Randall and Weissman, 1998; Venezia et al., 2004) and it is uncertain whether these markers return to normal 10 days after 5-FU treatment, we assessed the content of CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + cells by flow-cytometry (D). We observed no difference in the frequency (E, G) or absolute number (F, H) of CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + cells in the bone marrow (E, F) or spleen (G, H) of Mx-1-Cre + N-cadherin fl/- mice or littermate controls. Data are from 2 to 5 mice per genotype. All error bars represent SD. N-cadherin deletion was confirmed by PCR of Mac-1 + myeloid cells from Mx-1-Cre + N-cadherin fl/- mice (I). 3

4 Figure S3: MNCD2 antibody did not detectably stain HSCs and the staining that was observed in whole bone marrow was unaffected by N-cadherin deletion. MNCD2 staining was compared to streptavidin-only negative control (first column) in whole bone marrow cells (A), CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + HSCs (B), and Flk2 - lineage - Sca-1 + c-kit + HSCs (C). Staining above background was only observed in whole bone marrow (A). In all cases, staining was unaffected by N-cadherin deficiency (third column shows Mx-1-Cre + N-cadherin fl/- mice after pipc treatment). N-cadherin deficiency was confirmed by PCR in pipc-treated Mx-1- Cre + N-cadherin fl/- mice (D). 4

5 Figure S4: MNCD2 antibody did not stain HSCs isolated from 2 month old, 1 year old or 2 year old mice. MNCD2 staining (red lines) was compared to streptavidin-only negative control (black lines) in CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + HSCs (A) and Flk2 - lineage - Sca-1 + c-kit + HSCs (B). An independent experiment, in which 2000 HSCs from 2 year old mice were analyzed with an independent aliquot of MNCD2 antibody yielded similar results (C). No staining above background was observed in either population at any age. Data are representative of 4 independent experiments. 5

6 Figure S5: MNCD2 antibody did not stain HSCs isolated by collagenase-treatment of bone and subsequently stained in calcium-containing medium. MNCD2 staining (red lines) in calcium-containing medium was compared to streptavidin-only negative control (black lines) in CD150 + CD48 - CD41 - lineage - Sca-1 + c-kit + HSCs (A and C) and Flk2 - lineage - Sca-1 + c-kit + HSCs (B) following collagenase-treatment of bones. The experiment in (C) was performed with a different aliquot of MNCD2 antibody as compared to that used in panels A or B. Similar results were obtained using medium without calcium and using traditional bone marrow flushing techniques. Either way, no staining above background was observed in either population. Results are from two independent experiments. 6

7 Figure S6: MNCD2 antibody strongly stained surface IgM + B-cells but that staining was unaffected by N-cadherin deletion. B220 hi surfaceigm + B-cells (A) were strongly stained by biotinylated MNCD2 antibody plus streptavidin-pharred (MNCD2, B, red lines) as compared to streptavidin-pharred control (2 only, B, black lines). B220 hi surfaceigm + B-cells from pipc treated Mx-1-Cre + N-cadherin fl/- mice showed a similar level of staining by MNCD2 antibody (B, right plot) as compared to cells isolated from littermate control mice (B, left plot). PCR analysis of genomic DNA from MNCD2 + bone marrow cells isolated from Mx-1-Cre + N-cadherin fl/- mice 5 months following initial pipc treatment showed complete excision of N-cadherin confirming N-cadherin deficiency in these cells (C). These results demonstrate that when used for flowcytometry, MNCD2 binds to something other than N-cadherin in the bone marrow. Results are from two independent experiments. 7

8 Figure S7: Western blot using MNCD2 antibody revealed N-cadherin expression in as few as 2,000 neonatal forebrain cells but no N-cadherin expression in 500,000 CD45 + bone marrow cells or in 10,000 Lineage - c-kit + Sca-1 + (LSK) cells. The only technique by which MNCD2 has been shown to specifically bind N-cadherin is Western blot (Radice et al., 1997). To independently test whether the MNCD2 staining observed on bone marrow cells by flowcytometry reflects N-cadherin expression, we performed a Western blot using MNCD2 antibody. A) N-cadherin was readily detected in as few as 2,000 neonatal forebrain cell equivalents (lane 1: 2x10 5 cell equivalents; lane 2: 2x10 4 cell equivalents; lane 3: 2x10 3 cell equivalents). In contrast, whether we used 5x10 5 CD45 + cells (lane 4), 10 5 sigm + B cells (lane 5), or 10 4 LSK cells (lane 6) from wild-type bone marrow or pipc-treated Mx-1-Cre + N-cadherin fl/- mice, this band could not be detected. ß-actin staining was performed as a loading control and could be detected in all samples except 2,000 neonatal forebrain cells (B; see long exposure to detect the ß-actin band in LSK cells). 8

9 Figure S8: N-cadherin expression cannot be detected within Lineage - c-kit + Sca-1 + (LSK) cells by Western blot. In case N-cadherin was expressed at very low levels within LSK cells, we examined the expression of N-cadherin by Western blot using up to 100,000 LSK cells. A) Although N-cadherin expression was readily detected in 30,000 P0 forebrain cells using MNCD2 antibody (lanes 1-3), no N-cadherin expression was detected in either 125,000 whole bone marrow (WBM) cells or in 30,000 to 100,000 LSK cells from control (wt; Mx-1-Cre - N- cadherin fl/- ) or Mx-1-Cre + N-cadherin fl/- cells (ko; lanes 4-8). Aliquots of the same samples were analyzed in a second Western blot for ß-actin levels, as a loading control. B) N-cadherin deletion was confirmed by PCR using genomic DNA from the WBM and LSK cells used in (A). The upper band represents the unrecombined floxed allele while the lower band represents the deleted allele. 9

10 Figure S9: Bone marrow cells that stain MNCD2 + by flow-cytometry do not express N- cadherin when analyzed by Western blot. To determine whether MNCD2 + bone marrow cells identified by flow-cytometry express N-cadherin protein, cells from wild-type C57BL mice that stained with MNCD2 antibody were sorted by flow-cytometry and analyzed for N-cadherin expression by Western blot using MNCD2 antibody. A) N-cadherin expression was readily detected in P0 forebrain cells but was not detected in whole bone marrow (WBM) cells or in cells that stained MNCD2 + by flow-cytometry. Both RIPA buffer soluble and insoluble protein fractions were analyzed. Aliquots of the same samples were analyzed in a second Western blot for ß-actin levels, as a loading control. These data indicate that MNCD2 antibody staining by flow-cytometry does not reflect N-cadherin expression. 10

11 SUPPLEMENTAL REFERENCES Kostetskii, I., Li, J., Xiong, Y., Zhou, R., Ferrari, V.A., Patel, V.V., Molkentin, J.D., and Radice, G.L. (2005). Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure. Circ Res 96, Radice, G.L., Rayburn, H., Matsunami, H., Knudsen, K.A., Takeichi, M., and Hynes, R.O. (1997). Developmental defects in mouse embryos lacking N-cadherin. Developmental biology 181, Randall, T.D., and Weissman, I.L. (1998). Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: resting stem cells or mystery population? Stem cells (Dayton, Ohio) 16, Venezia, T.A., Merchant, A.A., Ramos, C.A., Whitehouse, N.L., Young, A.S., Shaw, C.A., and Goodell, M.A. (2004). Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS biology 2, e