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Blood, Vol. 109, Issue 5, 1825-1833, March 1, 2007
Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells
Blood Forde et al.
109: 1825
Supplemental materials for: Forde et al, Vol 109, Issue 5, 1825-1833
Files in this Data Supplement:
- Figure S1. Treatment of CD133+ cells with the CD164 103B2 mAb does not affect spontaneous migration (PDF, 25.4 KB) -
In the transwell migration assay using CD133+ cells, cells that migrated to CXCL12 on fibronectin were normalized to 100%, and migration in the absence of CXCL12 was measured. This was significantly lower than when CXCL12 was present (*P < .05). In the absence of CXCL12, no significant difference was observed in the migration of untreated cells or cells treated with the 103B2 CD164 antibody (22.9% ± 0.3% and 20.9% ± 1.4%, respectively).
- Figure S2. Transwell migration of CD133+ cells and Jurkat cells in the presence and absence of fibronectin (PDF, 131 KB) -
(A) CD133+ cells, untreated or treated with the indicated antibodies, were induced to migrate to CXCL12 with the use of inserts that were uncoated or coated with 20 µg/mL fibronectin. In the absence of fibronectin, only 27.2% ± 3.4% of cells migrated to CXCL12 compared with the normalized control (P < .05; n = 3). No significant difference was observed when cells were treated with the relevant antibodies (P > .05), indicating that fibronectin is required for a CD164-induced reduction in migration to CXCL12. (B) In Jurkat cells, migration was induced as in panel A, and similar results were observed with 25.1% ± 2.9% cell migration compared with the normalized control (P < .05). Incubation with antibodies to CD164 did not significantly reduce this migration to CXCL12 in the absence of fibronectin (P > .05; n = 3).
- Figure S3. Jurkat cell migration to alternative chemokines (PDF, 74 KB) -
(A) Transwell migration assay in which cells were induced to migrate to the indicated chemokines in 3 independent experiments. Significant migration was observed to CXCL12 and CCL5 compared with migration in the absence of a chemokine (P = .001 and .028, respectively). No significant migration was observed to any other chemokine (P > .05), although CCL17 and CCL22 did induce 9.6% ± 0.6% (P = .065) and 10.9% ± 1.1% (P = 0.12) migration, respectively, compared with 3.6% ± 3.2% migration in the absence of a chemokine. (B) Migration to CXCL12, CCL5, CCL17, and CCL22 in the presence of the CD164 mAb 103B2. Migration to CXCL12 was significantly reduced when the cells were incubated with the mAb, but this inhibition of migration by 103B2 was not observed when the cells were induced to migrate to CCL5, CCL17, and CCL22 (P > .05).
- Figure S4. CD164-1 and CD164-2 siRNA duplex design and knockdown of CD164 protein levels in the HS-5 cell line (PDF, 154 KB) -
(A) Schematic diagram of the CD164 protein and the location of CD164-1 and CD164-2 siRNA duplex design in exon 1 and in the 3′ UTR region, respectively. (B) Western blot of CD164 levels in HS-5 cells treated with control-1, control-2, CD164-1, or CD164-2 siRNA duplexes or that were mock transfected or untransfected.  -Tubulin was included as a loading control.
- Figure S5. Transwell migration of CD133+ cells and Jurkat cells in the presence and absence of fibronectin, during which cells were treated with control and CD164 siRNA (PDF, 45.8 KB) -
(A) CD133+ cells were nucleofected only or were nucleofected with control-2 or CD164-2 siRNA and induced to migrate to CXCL12 on coated or uncoated inserts. Only 33.8% ± 3.9% of cells migrated in the absence of fibronectin compared with the normalized control (P < .05; n = 3). Migration without fibronectin was not significantly altered in cells treated with control or CD164 siRNA (2.2% ± 4.9% and 4.9% ± 1.2% difference, respectively, compared with cells that were nucleofected only; P > .05, n = 3). (B) This trend was also observed in Jurkat cells, in which only 37.6% ± 1.8% of cells migrated in the absence of fibronectin compared with the normalized control (P < .05; n = 3). Migration without fibronectin, in cells treated with control or CD164 siRNA was not significantly changed (2.1% ± 0.9% and 4.6% ± 3.5% difference, respectively, compared with cells that were nucleofected only; P > .05, n = 3).
- Figure S6. Binding of the 103B2 CD164 antibody or nucleofection with siRNA duplexes does not induce apoptosis of CD133+ cells or alter CXCR4 receptor expression (PDF, 57.8 KB) -
(Ai) Percentages of apoptotic cells (black bars) and live cells (gray bars) are shown in CD133+ cells after 4-hour incubation in the presence of 50 µg/mL CD164 or control antibodies, as indicated. Apoptotic cells were detected as costaining for Annexin V and PI. CD43 was used as a positive control. Data represent mean ± SEM of 3 independent experiments. (ii) Incubation with the CD43 antibody, MEM-59, induced apoptosis, as was expected in primitive cells, and this resulted in a significant reduction in CD133+ migration to CXCL12 in the transwell migration assay. (Bi) CD133+ cells were untreated or treated with CD164 antibodies (N6B6 and 103B2) or control antibodies (mIgG2a and mIgG3 respectively) for 4 hours at 37°C and were stained with anti-CXCR4 antibody (12G5, mIgG2a or 6H8, mIgG1). MFI of untreated cells was normalized to 100%, and MFI of antibody-treated cells was compared with this value (n = 3). (ii) Knockdown of CD164 by RNAi did not change CXCR4 protein levels. CD133+ cells were nucleofected only or were nucelofected with control or CD164 siRNAs and, after 48-hour incubation, were immunoblotted for CXCR4 and CD164 protein expression. CXCR4 expression was not altered despite knockdown of CD164 levels. -Tubulin was used as a loading control. (iii) CXCR4 surface expression was not altered after nucleofection with control (dark gray line) or CD164 (light gray line) siRNA duplexes. Black line represents the isotype control.
- Figure S7. Coimmunoprecipitation of CXCR4 with CD164, VLA-4, and VLA-5 in Jurkat cells in response to CXCL12 stimulation in the presence and absence of fibronectin (PDF, 230 KB) -
(A) Immunoprecipitation in the presence of fibronectin, during which cells were stimulated with CXCL12 on fibronectin for 0, 1, 10, and 30 minutes. Immunoprecipitated CXCR4 was immunoblotted with mAbs to (i) CD164, (ii) VLA-4, (iii) VLA-5, and (iv) CXCR4. L = 50 µg total cell lysate. (B) Immunoprecipitation in the absence of fibronectin, during which cells were stimulated with CXCL12 for 0, 1, 10, and 30 minutes and were immunoblotted as described in panel A. Densitometry analysis of CD164, VLA-4, and VLA-5 protein levels were calculated as percentages of the respective total immunoprecipitated CXCR4 protein for panels A and B and showed that fibronectin enhanced coprecipitation with CXCR4. For example, a 4.4-fold increase in CD164 immunoprecipitated with CXCR4 was observed in the presence of fibronectin at the 10-minute time point compared with its absence.
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