Page 54 - Haematologica June

M.W. Wlodarski et al.
RP gene is haploinsufficient.36 We performed these assays
in CD34+ cells isolated from bone marrow mononuclear
cells (BM-MNC) of 2 patients with truncating RPL15
mutations p.Tyr81*. Compared to healthy control, upregulation on both day 7 and day 9 in the Tyr81*
patients BM-MNC showed a higher rate of cell death and apoptosis (Figures 5B and Online Supplementary Figure S5). FACS analysis revealed CD36 downregulation and CD34
AB
C
D
EFG
HI
956
Figure 5. Erythroid cell culture assays of primary RPL15 c.242dupA erythroid progenitor and precursor cells reveal severe erythroid proliferation defects, differen- tiation delays, and TP53-related apoptosis. (A) Proliferation curve of erythroid cells isolated from CD34+ cells from peripheral blood of 2 individuals with Diamond- Blackfan anemia (DBA) and an RPL15 mutation or a healthy control over nine days in liquid culture medium. (B) FACS analysis results of the percent of dead cells or apoptotic cells staining positive for Annexin V on days 7, 10, and 13 after plating in red cell culture medium. (C) FACS analysis results on days 7 and 9 of the per- cent of cells staining positive for CD36 and CD34. (D) FACS analysis results on days 7 and 9 of cells staining positive for IL3R and GPA. (E) FACS analysis results on days 7 and 9 of cells staining positive for Alpha-4 and Band-3. (F) Real-time PCR results of the ratio between RPL15 mRNA and actin mRNA in cells. (G) Real-time PCR results of the ratio between p21 mRNA and actin mRNA in cells. (H) Western blot analysis of cells from a healthy control or an individual with DBA and a mutation in RPL15 using antibodies against phosphorylated TP53, p21, eL15, or actin on day 7. (I) Western blot analysis of cells from a healthy control or an RPL15 with DBA and a mutation in RPL15 using antibodies against TP53 or actin on days 7 and 9.
haematologica | 2018; 103(6)