40 likes | 182 Views
Primary cBMT. p18+/+. Long-term Engraftment. Secondary cBMT. p18-/-. Long-term engraftment. irradiated. p18+/+. irradiated. (re-challenged with equal amount of p18+/+ cells).
E N D
Primary cBMT p18+/+ Long-term Engraftment Secondary cBMT p18-/- Long-term engraftment irradiated p18+/+ irradiated (re-challenged with equal amount of p18+/+ cells) • Semi-quantitative PCR analysis for ratios of the two competitive cell populations in bulk cells or different lineages based on relative intensities of the two genotypic bands on the gels; • Single cell or colony PCR for the absolute representation of each genotype in the stem or progenitor compartment based on the appearance of a distinct band on the gel. p18+/+ (600 bp) p18-/- (400 bp) Fig S1
Fig S2a Sorting strategies for CD34-LKS cells R4 Propidium Iodide R1 Pulse Width Side Scatter Counts R2 R3 Forward Scatter Forward Scatter Forward Scatter Lineage-PE-Cy7 Gated in R1+R2+R3+R4 R5 Sca-1-PE c-Kit-APC Gated in R1+R2+R3+R4+R5 R6 Counts CD34-FITC
Fig S2b 11 Gy p18+/+ 105 Sca-1- cells CD45.1/CD45.2 5-14 weeks CD45.1 Different doses of stem cells PE-CD45.1 p18-/- CD45.2 FITC-CD45.2
Fig S3 Decreased apoptosis of p18 -/- donor cells was not observed 2 days following bone marrow transplantation Gated in CFSE+ cells Whole Lin- Lin+ 104 104 104 103 103 103 102 102 102 p18+/+ 9.93% 5.18% 4.52% 101 101 101 1.74% 0.37% 1.35% 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104 104 104 103 103 103 p18-/- 102 102 102 14.41% 7.82% 6.25% 101 101 101 3.75% 0.87% 2.82% 100 100 100 DAPI 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Annexin V