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Blood, Vol. 108, Issue 5, 1661-1667, September 1, 2006
Loss of inhibitory semaphorin 3A (SEMA3A) autocrine loops in bone marrow endothelial cells of patients with multiple myeloma
Blood Vacca et al.
108: 1661
Supplemental materials for: Vacca et al, Vol 108, Issue 5, 1661-1667
Files in this Data Supplement:
- Table S1. Gene expression by RT-PCR in patients’ bone marrow endothelial -
- Figure S1. RT-PCR analysis of VEGF165, SEMA3A, Nrp-1, and plexin-A1 in the ECs of representative patients and HUVECs (JPG, 54.6 KB)
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Histograms show the band intensity evaluated as interior area (or pixel).
- Figure S2. Unleashing basal and VEGF165-driven MMEC chemotaxis by RNA interference for endogenous SEMA3A (JPG, 16 KB)
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By means of Oligofectamine Reagent (Invitrogen, Carlsbad, CA) MMECs were transfected twice (at 0 and 24 hours) with 200 pmol of siRNA duplexes for human SEMA3A (Dharmacon, Lafayette, CO; code no. M-020091-00) and of control nontargeting siRNA (Ctl; code no. D-001206-13-05). Transfection was efficiently performed, as shown in Figure S3. Twenty-four hours after the last transfection, 30,000 MMECs were seeded on the upper side of 8-µm pore filters of Transwell chambers (Corning Costar, Cambridge, MA) coated on the lower side with fibronectin (1 µg/mL; R&D Systems, Minneapolis, MN), and allowed to migrate towards serum-free medium with or without 10 or 20 ng/mL VEGF-A165 (R&D Systems) for 3 hours. Cells on the upper side of the filters were then mechanically removed. MMECs of the filter lower side were then fixed in 8% glutaraldehyde for 30 minutes, stained with 0.1% crystal violet, and counted in 5 oil × 400 immersion fields/membrane. Counts are given as mean ± 1 SD. As shown in the figure, knockdown of endogenous SEMA3A by siRNA unleashes both basal and VEGF165-stimulated MMEC migratory activity, which in average was 3 times higher than that of control cells treated with nontargeting siRNA. The migratory activity peaked at 10 ng/mL VEGF165, whereas it declined at 20 ng/mL according to a bimodal behavior.
- Figure S3. Monitoring the efficiency of SEMA3A mRNA silencing (JPG, 16 KB)
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Data: The SEMA3A transcript in MMECs was dramatically reduced at 24 hours after the last transfection (when the cell chemotactic activity was assessed), and persisted at these very low levels until 72 hours: | | SEMA3A siRNA | | 0h | 1 | | 24h | 0,125 | >| 48h | 0,066 | | 72h | 0,051 |
At 0, 24, 48, and 72 hours after the last transfection with siRNA duplexes for human SEMA3A, MMEC total RNA was extracted with TriReagent (Sigma Chemical, St Louis, MO) according to the manufacturer’s instructions and quantified by means of the RNA 6000 Nano Assay kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany). Equal amounts (1 µg) of total RNA were reverse-transcribed by means of the High Capacity cDNA Archive Kit (Applied Biosystems). SEMA3A and 18S mRNAs were quantitatively analyzed by means of TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) run on an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). The relative quantification method employed was based on the following arithmetic formula:
2—ΔΔCT
where  CT is the normalized signal level in a sample relative to the normalized signal level in the corresponding calibrator sample (0-hour sample). The 18S gene was the endogenous control detector employed for normalization.
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