1. CD34+ cells from paroxysmal nocturnal hemoglobinuria (PNH) patients are deficient in surface expression of cellular prion protein (PrP c) 
Antonio M Risitano, Karel Holada, Guibin Chen, Jan Simak, Jaroslav G Vostal, Neal S Young, Jaroslaw P Maciejewski 
Experimental Hematology 
Volume 31, Issue 1, Pages (January 2003)
DOI: /S X(02)

2. Figure 1
Surface and intracellular PrPc expression on leukemic cell lines: PrPc is expressed on surface of WT but not PIG-A-deficient cells; intracellularly, PrPc is present in both WT and PIG-A-deficient cells. A–F and G–J: surface expression on K562 and Jurkat cells, respectively; K–P: intracellular expression in K562 cell line. A–P: flow cytometry, log FITC fluorescence vs log PE fluorescence. All the samples were stained with CD55 and CD59 mAbs PE-conjugated to distinguish between GPI-AP-deficient and phenotypically normal cells. A, D, G, I, K, N: FITC isotypic controls. B, E, H, J, L, O: anti-PrPc mAb C, F: anti-PrPc mAb 6H4; M, P: anti-PrPc mAb competing peptide. Q, R: intracellular PrPc expression by Western blot. Q: anti-PrPc mAb 6H4; R: anti-PrPc + competing peptide.
Experimental Hematology  , 65-72DOI: ( /S X(02) )

3. Figure 2
PrPc is expressed on surface of hematopoietic progenitor cells from normal controls and AA patients. A–E: normal control (NC) #1; F–J: NC #2; K–O: example of aplastic anemia patient (AA#1). A, F, K: CD3 (log ECD fluorescence) vs CD34 (PeCy-5 fluorescence); gate A: CD34+ cells. B, G, L and C, H, M: in gate A, CD55/CD59 (log PE fluorescence) vs isotypic control and vs anti-PrPc mAb 1562 (log FITC fluorescence), respectively. D, I, N: CD55/CD59 (log PE fluorescence) vs CD34 (log PeCy-5 fluorescence); gate B: phenotypically normal (CD55+/CD59+) CD34+ cells. E, J, O: in gate B, log FITC fluorescence histogram, isotypic control (dotted line) vs anti-PrPc mAb 1562 (continuous line); in E, the third histogram (dashed line) represents staining with anti-PrPc mAb competing peptide.
Experimental Hematology  , 65-72DOI: ( /S X(02) )

4. Figure 3
In normal CD34+ cells, PrPc is expressed on surface via the GPI-anchor; PrPc expression is similar in different maturational stages of CD34+ cells, and is not modulated by various cytokines. A–E: CD55/CD59 and PrPc expression prior to and after PIPLC digestion; A and C: CD55/CD59 (log PE florescence) vs CD34 (log PeCy-5 fluorescence), prior to and after PIPLC; B and D: CD55/CD59 (log PE florescence) vs anti-PrPc mAb 1562 (log FITC fluorescence), prior to and after PIPLC; E: anti-PrPc mAb 1562 histogram (log FITC fluorescence), prior to (continuous line) vs after (dotted line) PIPLC-digestion. F–J: PrPc expression on different maturational stages of CD34+ cells. F: CD3 (log ECD fluorescence) vs CD34 (PeCy-5 fluorescence); gate A: CD34+ cells. G and H: in gate A, CD38 (log PE fluorescence) vs isotypic control and vs anti-PrPc mAb 1562 (log FITC fluorescence). I: CD38 (log PE fluorescence) vs CD34 (log PeCy-5 fluorescence); gate B: more mature/committed (CD38+) CD34+ cells; gate C: more immature (CD38−/ low) CD34+ cells. J: anti-PrPc mAb 1562 histogram (log FITC fluorescence) on mature (gate B) CD34+ cells (dotted line) vs immature (gate C) CD34+ cells (continuous line). K–N: PrPc expression on purified CD34+ cells after addition of different cytokines; anti-PrPc mAb 1562 (continuous line) and isotypic control (dotted line) histogram (log FITC fluorescence). K: untreated CD34+; L: Fas-Ligand (CH11 mAb) 0.5 μg/mL; M: IFN-γ 1000 UI/mL; TGF-β 2 ng/mL. O: BFU-E (grey columns) and CFU-GM (black columns) with or without incubation with anti-PrPc mAb 1562; numbers are expressed as colonies for 1000 CD34+ plated cells.
Experimental Hematology  , 65-72DOI: ( /S X(02) )

5. Figure 4
Differential PrPc expression on hematopoietic progenitor cells from PNH patients: PrPc is reduced on type 2 PNH CD34+ cells, completely absent on type 3 PNH CD34+ cells, but present on residual phenotypically normal CD34+ cells. Boxes 1–5: PNH patient #1; boxes 6–10: PNH patient #2; boxes 11–15: PNH patient #3. Boxes 1, 6, 11: CD3 (log ECD fluorescence) vs CD34 (PeCy-5 fluorescence); gate A: CD34+ cells. Boxes 2, 7, 12 and 3, 8, 13: on gate A, CD55/CD59 (log PE fluorescence) vs isotypic control (log FITC fluorescence) and vs anti-PrPc mAb 1562 (log FITC fluorescence), respectively. Boxes 4, 9, 14: CD55/CD59 (log PE fluorescence) vs CD34 (log PeCy-5 fluorescence); gate B: phenotypically normal (CD55+/CD59+) CD34+ cells; gate C: type 2 PNH (CD55low/CD59low) CD34+ cells; gate D: type 3 PNH (CD55−/CD59−) CD34+ cells. Boxes 5, 10, 15: anti-PrPc mAb 1562 histogram (log FITC fluorescence) on phenotypically normal (gate B) CD34+ cells (continuous line) vs type 2 PNH (gate C) CD34+ cells (dashed line) vs type 3 PNH (gate D) CD34+ cells (dotted line).
Experimental Hematology  , 65-72DOI: ( /S X(02) )

6. Figure 5
Differential PrPc expression on hematopoietic progenitor cells from PNH patients: PrPc is reduced on type 2 PNH CD34+ cells, completely absent on type 3 PNH CD34+ cells, but present on residual phenotypically normal CD34+ cells. Boxes 1–5: PNH patient #4; boxes 6–10: PNH patient #5; boxes 11–15: PNH patient #6. Boxes 1, 6, 11: CD3 (log ECD fluorescence) vs CD34 (PeCy-5 fluorescence); gate A: CD34+ cells. Boxes 2, 7, 12 and 3, 8, 13: on gate A, CD55/CD59 (log PE fluorescence) vs isotypic control (log FITC fluorescence) and vs anti-PrPc mAb 1562 (log FITC fluorescence), respectively. Boxes 4, 9, 14: CD55/CD59 (log PE fluorescence) vs CD34 (log PeCy-5 fluorescence); gate B: phenotypically normal (CD55+/CD59+) CD34+ cells; gate C: type 2 PNH (CD55low/CD59low) CD34+ cells; gate D: type 3 PNH (CD55−/CD59−) CD34+ cells. Boxes 5, 10, 15: anti-PrPc mAb 1562 histogram (log FITC fluorescence) on phenotypically normal (gate B) CD34+ cells (continuous line) vs type 2 PNH (gate C) CD34+ cells (dashed line) vs type 3 PNH (gate D) CD34+ cells (dotted line).
Experimental Hematology  , 65-72DOI: ( /S X(02) )