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STEM_1416_sm_SuppFigure1.pdf1470KSupplementary Figure 1. Abi3bp knockdown regulates MSC cell biology, differentiation. (A) MSC or MSC-GFP-Akt1 cells were transfected with the indicated constructs. After 3 days RNA extracted to measure Abi3bp mRNA levels by qPCR. Protein extracts were immunoblotted for Akt1. N=3. Significance shown for comparisons with control cells, *** p≤0.001. (B) Wild-type and Abi3bp knockout MSCs showed senescence as evidenced by β- galactosidase staining n=6. Scale bar 100 microns. (C) Top: vinculin staining in MSC-GFP-Akt1 cells. Bottom: quantification for wild-type and knockout Abi3bp MSC isolation 1. Scale bar 100 microns. (D) The indicated cell-types were stained for phospho-paxillin (Y118), paxillin, vinculin, phalloidin or DAPI one day post-seeding. Foci intensity, number, and area as well as phalloidin intensity and cell area were determined. N=9-12. Foci number is expressed as a function of cell area. p-paxillin/paxillin ratio is shown. Mean intensity of foci. P-paxillin/paxillin ratio is shown. Heat map of p-paxillin, paxillin, and vinculin staining as well as the p-paxillin/vinculin ratio and cell area. N=9-12. Significance between control and Abi3bp knockdown/knockout *p≤0.05, *** p≤0.001. (E) MSCs were differentiated down the chrondrogenic pathway (N=3). Scale bar 1mm. (F) Scratch assay for MSC-GFP-Akt1 cells. Scale bar 200 microns.
STEM_1416_sm_SuppFigure2.tif2994KSupplementary Figure 2. Cell surface marker analysis of MSCs used in this study. MSCs used in this study were analyzed by flow cytometry for MSC (CD44, Sca-1, CD29, CD51), hematopoietic (CD45, CD11b, c-Kit, CD31) and endothelial (CD34) markers.
STEM_1416_sm_SuppFigure3.tif729KSupplementary Figure 3. Abi3bp knockdown affects proliferation in both MSC-GFP-Akt1 and MSC cells. (A) Growth curves MSC, MSC-GFP, and MSC-GFP-Akt1 cells as determined by MTS assay, n=5-6. (B) MTS assay for MSC-GFP-Akt1 cells expressing control or shRNA-91. (C) MSC cells expressing either control scrambled or Abi3bp shRNA-sh83/sh91 were incubated with BrdU for 6 hours one day post-seeding. After flow cytometry the percentage of cells in S, G0/G1- and G2/M-phases were calculated. N=3. Significance shown for the comparison between Abi3bp knockdown and control cells *p≤0.05, ** p≤0.01, *** p≤0.001. (D) BrdU ELISA for MSC-GFP-Akt1 cells expressing either scrambled or Abi3bp shRNA n=3. * p≤0.05. (E) MSC and MSC-GFP-Akt1 cells were treated with concentrated media prepared from HEK293 cells expressing either the control or myc-tagged Abi3bp vector at the indicated concentrations for two days. Cell number was determined by MTS assay. The data is presented as the percentage increase in cell number when compared to untreated cells. Significance shown for comparisons between control- and Abi3bp- conditioned media *p≤0.05, ** p≤0.01.
STEM_1416_sm_SuppFigure4.tif615KSupplementary Figure 4. ERK regulation of MSC-GFP-Akt1 cell proliferation. (A) Quantification of day 2 immunoblots for wild-type and Abi3bp knockout MSCs. (B) Cell number for MSC-GFP-Akt1 cells stably expressing either a control or myc-DDK tagged cyclin-d1 vector, was determined at day 0 and day 2 post seeding using a MTS assay (left panel, n=5). One day post-seeded cells were incubated with BrdU for 4 hours. Flow cytometry was performed to determine the number of cells in S-phase (middle panel, n=4). Significance shown for comparison between groups *** p≤0.001. A representative immunoblot showing over-expression of cyclin-d1 is shown far right. (C) One day post-seeded MSC-GFP-Akt1 cells, expressing scrambled or ERK2 shRNA were incubated with BrdU for 6 hours. After flow cytometry the percentage of cells in G0/G1- and G2/M-phases were calculated. N=4. Significance shown for comparisons between knockdown and control cells ** p≤0.01, *** p≤0.001.
STEM_1416_sm_SuppFigure5.tif1239KSupplementary Figure 5. MEK dependence for MSC proliferation. (A) MSC-GFP-Akt1 cells, one day post-seeding, were incubated with 10μM U0126, 10μM PD98059, or equivalent volume of vehicle for the indicated times. Extracts (7.5μg) were probed for p-ERK1/2, cyclin-d1, and actin. Intensities were normalized to the actin loading control and the normalized intensity at time zero was taken to be 1. N=4. Significance shown for comparisons between vehicle or inhibitor groups and untreated cells (0hr) *p≤0.05, ** p≤0.01, *** p≤0.001. (B) MSC-GFP-Akt1 or MSC cells, one day post-seeding, were incubated with 10μM U0126, 10μM PD98059, or equivalent volume of vehicle. After BrdU incubation cells were fixed. Flow cytometry was performed to determine the number of cells in S-, G0/G1- and G2/M-phases. N=3 for U0126 (Akt1 cells), n=4 for PD98059 (Akt1 cells), n=4 for U0126 (MSCs). Significance shown for comparisons between vehicle or inhibitor groups and untreated cells *** p≤0.001. (C) Quantification of p-paxillin immunoblot density for day 2 post-seeded wild-type and Abi3bp knockout MSCs.
STEM_1416_sm_SuppFigure6.tif570KSupplementary Figure 6. Abi3bp requires paxillin for negative regulation of proliferation and is important for Akt mediated cell transformation (A) Quantification of paxillin and vinculin siRNA knockdown. (B) MSC-GFP-Akt1 cells, one day post-seeding, were incubated with either 10μM PP2, or an equivalent volume of vehicle for the indicated times. Extracts were probed for phospho-ERK1/2, cyclin-d1, and actin. N=4
STEM_1416_sm_SuppFigure7.tif1980KSupplementary Figure 7. Abi3bp is expressed in most tissues and is important for MSC transformation. (A) RNA expression determined by qPCR. Relative expression to brain shown. N=4 (N=2 for knockout lung). (B) Colony formation of MSC-scrambled shRNA, MSC-GFP-Akt1- scrambled shRNA, and MSC-GFP-Akt1-sh83 shRNA cells in soft agar. After 21 days colonies were photographed. Left, colonies were counted at x10 under a phase contrast microscope. N=5. Significance between groups is shown on the figure *** p≤0.001. Right, representative photographs. Scale bar 400 microns.
STEM_1416_sm_SuppFigure8.pdf1300KSupplementary Figure 8. Bone marrow CD45negCD44posCD105pos cells express MSC markers. (A) Bone marrow CD45negCD44posCD105pos cells from both wild-type and Abi3bp knockout mice were analyzed for various markers including murine MSC markers CD29 and Sca-1. These cells lacked hematopoietic and endothelial cell markers (CD45, CD31, CD34) as well as the pericyte marker PDGFRβ. (B) Bone marrow aspirates from wild-type and Abi3bp knockout mice were sub-divided after isolation. One half was analyzed immediately by flow cytometry, the other half cultured for 12 hours after which the cells were removed by nonenzymatic means and analyzed for CD45 and CD44.
STEM_1416_sm_SuppFigure9.pdf1070KSupplementary Figure 9. Isolated lung and liver CD45negCD73posCD90posCD105pos cells differentiate into cartilage, fat, bone, and smooth muscle. (A) Lung and liver CD45negCD73posCD90pos CD105pos cells were isolated and cultured. Under specific culture conditions these cells differentiated into chondrocytes, adipocytes, osteoblasts, and smooth muscle as expected for MSCs. Scale bar either 100 microns (chondrocytes, adipocytes, and smooth muscle) or 200 microns (bone). (B) Flow cytometry analysis of the kidney CD45negCD73posCD90posCD105pos population (top panels) and proliferation in the lung (bottom panel).
STEM_1416_sm_SuppFigure10.tif1473KSupplementary Figure 10. Abi3bp is important for the check and balance of MSC proliferation, adhesion/adhesion dynamics, and cellular transformation.
STEM_1416_sm_SuppTable1.pdf29KSupporting Information
STEM_1416_sm_SuppInfo.pdf114KSupporting Information

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