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The Supplementary file contains three figures with figure legends. - PowerPoint PPT Presentation


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The Supplementary file contains three figures with figure legends. The files submitted as a 1997-2003 Powerpoint file. a. NS. 10 8. 10 7. # DP cells. 10 6. 10 5. Wt >Wt. Wt>Dll3 -/-. b. 10 8. NS. 10 7. #CD4+ cells. 10 6. 10 5. Wt >Wt. Wt>Dll3 -/-. c. 10 8.

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slide1

The Supplementary file contains three figures with figure legends.

The files submitted as a 1997-2003 Powerpoint file.

slide2

a

NS

108

107

# DP cells

106

105

Wt >Wt

Wt>Dll3-/-

b

108

NS

107

#CD4+ cells

106

105

Wt >Wt

Wt>Dll3-/-

c

108

NS

107

#CD8+ cells

106

105

Wt >Wt

Wt>Dll3-/-

Supplemental Figure 1. Wild type T cells develop normally in a Dll3-/- host. Irradiation chimeras were set up using bone marrow cells from wild type CD45.1+ donor animals and these were transferred to irradiated Dll3+/+(CD45.2) or Dll3-/- (CD45.2) recipient mice. Twelve weeks after the transfer the thymus and spleen were removed and cells analysed by flow cytometry. The data shows the number of CD45.1+ (a) DP cells, (b) CD4+ and (c) CD8+ T cells in the thymus. Unpaired t test was used to test for statistically significant differences between groups. NS = not significant.

Supplemental Figure 1 Hoyne et al

slide3

Supplemental Figure 2

Hoyne et al

Dll3-/- : CD45.1

> insHel

Dll3+/+: CD45.1

> insHel

a

CD4

CD8

Gated on

CD4+ cells

CD4

1G12

b

35

65

55

45

Gated on

DP cells

45

55

30

70

c

CD45.1

CD45.1 cells

100

100

CD45.2 cells

10

10

#CD4 1G12+ (x 106 )

#CD4 1G12+ (x 106 )

1

1

0.1

0.1

0.01

0.01

1

10

100

1

10

100

#DP cells (x106)

#DP cells (x106)

Supplemental Figure 2. Negative selection of autoreactive CD4+ T cells is not affected by Dll3 deficiency. a. Representative FACS dot plots showing CD4 v CD8 expression of total thymocytes from a Dll3+/+ TCR:TCR+CD45.1+ > insHel mice or Dll3-/- TCR+:TCR+CD45.1+ > insHel mice (top panel). Lower panel shows CD4 v 1G12 staining on the CD4 gated thymocyte population. b. Representative histograms showing the proportion of CD45.1+ and CD45.1-ve cells in the DP or CD4+ 1G12+ gates of representative chimaeras. c. Total cell numbers of DP and CD4+ 1G12+ cells in the thymus of individual chimaeric mice. Cells from the Dll3+/+:CD45.1 chimeras (filled squares), and Dll3-/-:CD45.1 chimaeras (open triangles) are shown.

slide4

Dll3+/+

100

100

80

80

60

60

40

40

% T cells

% B cells

20

20

0

0

0

0.1

0.3

1

L-685,458concentration (mM)

Dll3-/-

100

100

80

80

60

60

% B cells

40

40

% T cells

20

20

0

0

0

0.1

0.3

1

L-685,458concentration (mM)

Supplemental Figure 3. T cell production by Dll3-/- precursors is dependent on Notch cleavage by g-secretase. Lineage negative DN cells isolated from the thymus of either Dll3+/+ or Dll3-/- mice were cultured on OP9-Dll1 cells in the presence or absence of the g-secretase inhibitor L-685,458 (Calbiochem, gsi-X) at the indicated concentrations. FACS analysis of Thy1 versus B220 expression was made 8 days after commencement of the culture. The graphs show the frequency of B cells (B220+) (black squares) and T cells (Thy1+) (white squares) arising in the same cultures. Top graph shows the T vs B cell production in Dll3+/+ derived cells and lower graph shows T vs B cells derived from Dll3-/- progenitors. Each point represents the average of 6 replicate wells and is representative of one of three assays performed.

Supplemental Figure 3 Hoyne et al

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