|
|
Blood, Vol. 106, Issue 5, 1652-1659, September 1, 2005
Haploinsufficiency, rather than the effect of an excessive production of soluble CD95 (CD95 TM), is the basis for ALPS Ia in a family with duplicated 3' splice site AG in CD95 intron 5 on one allele
Blood Roesler et al.
106: 1652
Supplemental Figures for: Roesler et al, Vol 106, Issue 5, 1652-1659
Files in this Data Supplement:
- Figure S1. Apoptosis of activated patient PBMCs (PDF, 104 KB) -
Patient PBMCs were activated for 5 days (2 days PHA, 3 days IL-2) and incubated in the absence (A,C) and presence (B,D) of agonistic mAb CH-11 for another 24 hours. (A-B) Patient; (C-D) healthy donor; n=5. Abscissa: Binding of FITC-annexin; ordinate: DNA staining with propidium iodine; representative result.
- Figure S2. sFas failed to protect Jurkat cells from soluble super FasL induced apoptosis (PDF, 11 KB) -
(A) Cells were incubated in supernatant from wt EBV-transformed B-cells (dark gray bars), in supernatant from patient EBV-transformed B-cells (filled bars), in serum from a healthy donor (open bars), and in patient serum (light gray bars) before FasL was added. Apoptosis was measured 20 hours later. For sFas concentrations see Figure 5. Data represent more than five independent experiments. (B) 10% PKH26 stained Jurkat cells were mixed with 90% EBV-transformed B-cells and sFas containing supernatant from the patient (gray bars) or cells from a healthy donor (open bars). Super FasL was added carefully to the supernatant after the cells had sedimented to the bottom of the wells; incubation time: 20 hours. Absolute cell numbers and percent stained cells were calculated; the relative loss of stained cells is shown.
- Figure S3. Overexpression of Fas-del6 did not protect Jurkat cells from agonistic mAb Apo1 (or super FasL, not shown) induced apoptosis (PDF, 40 KB) -
(A) Expression of AU1 by Jurkat cells after transfection with 10 ăg of different AU1-tagged plasmids as indicated. (B) Fas expression by Jurkat cells after transfection with the same plasmids as in A. Only the transfection with wt-Fas plasmid led to an enhanced surface expression of Fas (bold line). Nontransfected cells or cells transfected with Fas-del6 or empty plasmid all showed the same endogenous Fas expression (gray line, right side). The gray lines on the left side indicate isotype control. (C) Secretion of sFas into the supernatant by Jurkat cells after 2 days of incubation. Plasmids used for transfection are indicated. (D) Induction of a maximal apoptotic response in transfected Jurkat cells by 500 ng/mL agonistic mAb Apo1 and protein A (filled bars; number of viable cells without prior incubation with mAb Apo1, gray bars). (E) Graded apoptotic response of Jurkat cells to mAb Apo1 without protein A at concentrations indicated (transfections: solid line, Fas-del6; dashed line, empty plasmid; dotted line, wt-Fas). A representative result of 4 experiments is shown.
|
|
