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Blood, Vol. 113, Issue 20, 5010-5018, May 14, 2009
Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells
Blood Nightingale et al.
113: 5010
Supplemental materials for: Nightingale et al
Files in this Data Supplement:
- Figure S1. Confluency/cell number of plated HUVECs affects the size of the releasable pool of VWF (JPG, 49.7 KB)
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Untransfected HUVECs were plated out in serial dilutions (1/2) and allowed to grow for 48h. Samples of freshly-changed media were acquired after 30min with and without 100ng/ml PMA and then the cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. The most confluent cells are to the right of the x-axis whereas the least confluent are to the left. Less confluent HUVEC have a smaller fraction of total VWF as a releasable pool.
- Figure S2. Consecutive rounds of nucleofection result in some cell death which can affect the confluency of cells but no toxic affect of oligonucleotide is apparent with respect to VWF secretion or cell morphology (JPG, 91.3 KB)
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(A) HUVECs were either treated in 2 consecutive rounds with a control oligonucleotide 1 (200pmol) with buffer alone or trypsinized and re-plated. Samples of media were acquired after 30min with and without 100ng/ml PMA, the cells were then lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. (B) Confocal images were acquired of immunofluorescence staining of treated HUVECs with VWF (blue), MyRIP (red) and phalloidin conjugated to Alexa 488nm (green). Bars correspond to 25µm. There is a small reduction in the regulated release of VWF from nucleofected cells compared to untransfected cells, likely due to the difference in confluency (some cell death occurs at transfection). No RNA specific differences were noted in transfected cells and no morphological difference is apparent.
- Figure S3. Rab27a depletion increases stimulated release of VWF following treatment with histamine or forskolin (JPG, 48.5 KB)
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(A, B) HUVECs were treated in 2 consecutive rounds with a siRNA oligonucleotide 1 (200pmol) directed against Rab27a or the same concentration of a control oligonucleotide. Samples of media were acquired after 30min with and without 0.1µM histamine (A), 100ng/ml PMA or 10µM forskolin (B) and then the cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF.
- Figure S4. Rab27a-GFP overexpression increases stimulated release of VWF following treatment with PMA (JPG, 68.2 KB)
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(A) HUVECs were transfected with 2µg WTRab27a-GFP, the constitutively active mutant Rab27aQ78L-GFP,1 the dominant negative construct Rab27aN133I-GFP,2 empty vector pEGFP or mock transfected (with water). After 24h samples of media were acquired after 30min with and without 100ng/ml PMA and then the cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. The results are mean determinations from 4 separate experiments and standard error of the mean is shown (B) Representative confocal images were acquired of immunofluorescence staining of cells transfected with GFP construct, VWF (red) and DAPI. Bars correspond to 50µm.
- Figure S5. Rab27a effector expression in HUVEC, HEK and MNT-1 cells (JPG, 110 KB)
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(A) RNA and cDNA was prepared from HUVEC, HEK and MNT-1 cells. Specific primers were used to amplify (40cycles) 100–300bp products of Rab27a effectors and actin from an equal concentration of cDNA. The products were analyzed on 2% agarose gels and imaged using ethidium bromide and UV transillumination. (B) Table to show the cycle time (CT) at which logarithmic production of product occurred. The lower the CT the higher the level of product present. Values given are mean values of triplicate determinations with standard deviations.
- Figure S6. The increase in VWF release following Rab27a depletion is dependent on actin as it is inhibited by Cytochalasin E (JPG, 86.7 KB)
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(A) HUVECs were treated in 2 consecutive rounds with a siRNA oligonucleotide 1 (200pmol) directed against Rab27a or the same concentration of a control oligonucleotide. Cells were then pre-incubated with media containing 1µm Cytochalasin E (CCE) or media alone for 15min. Samples of media were acquired after 30min with and without 100ng/ml PMA again containing, where appropriate, CCE and then the cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. (B) Confocal images were acquired of immunofluorescence staining of cells treated with or without CCE for 15min with VWF (red), and phalloidin conjugated to Alexa 488nm (green). Bars correspond to 10µm.
- Figure S7. The increase in VWF release following Rab27a depletion is dependent on microtubules as it is inhibited by nocodazole (JPG, 71.7 KB)
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(A) HUVECs were treated in 2 consecutive rounds with a siRNA oligonucleotide 1 (200pmol) directed against Rab27a or the same concentration of a control oligonucleotide. Cells were then pre-incubated with media containing 10µg/ml nocodazole or media alone for 15min. Samples of media were acquired after 30min with and without 100ng/ml PMA again containing, where appropriate, nocodazole and then the cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. (B) Confocal images were acquired of immunofluorescence staining of cells treated with or without nocodazole for 15min with VWF (red), and tubulin (green). Bars correspond to 10µm.
REFERENCES
1. Johnson JL, Ellis BA, Noack D, Seabra MC, Catz SD. The Rab27a-binding protein, JFC1, regulates androgen-dependent secretion of prostate-specific antigen and prostatic-specific acid phosphatase. Biochem J. 2005;391:699–710.
2. Ramalho JS, Anders R, Jaissle GB, Seeliger MW, Huxley C, Seabra MC. Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models. BMC Cell Biol. 2002;3:26.
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