Page 164 - 2019_06-Haematologica-web

A.C. Glembotsky et al.
AB
C
D
E
G
F
Figure 1. Transcriptome of RUNX1-deficient megakaryocytes (MK) reveals downregulation of TREML1 and ITGA2. (A and B) Venn diagram representation of the overlap between genes deregulated in patient MK (BII-2, BIII-1, AII-1 and AII-2), between genes deregulated in shRUNX1-transduced MK (shRUNX1_1_A, shRUNX1_1_B and shRUNX1_2), and between genes deregulated in both patient and shRUNX1-transduced MK. (A) A total of 598 genes were up-regulated in patient MK (n=4) and 289 genes were up-regulated in shRUNX1-transduced MK (n=3). A total of 43 genes were found to be up-regulated in both patient and shRUNX1- transduced MK. (B) A total of 492 genes were down-regulated in patient MK (n=4) and 499 genes (527 probes) were down-regulated in shRUNX1-transduced MK (n=3). A total of 61 genes were found to be down-regulated in both patient and shRUNX1-transduced MK. shRUNX1_1 was used in two experiments (shRUNX1_1_A and shRUNX1_1_B) and shRUNX_2 in one. (C) Differentially down-regulated genes and functions associated with different biological processes. Gene ontology (GO) terms and differentially down-regulated genes are represented as nodes based on their kappa score >0.4. The node size represents the GO terms enrichment sig- nificance. (D) Microarray data for ITGA2 and TREML1 in MK from patients (AII-1 and AII-2, carrying the R174Q mutation, and BII-2 and BIII-1, with the R139X muta- tion) relative to healthy subjects (n=4) (horizontal dashed line). (E) Real-time polymerase chain reaction (RT-PCR) analysis of RUNX1, ITGA2 and TREML1 transcript levels normalized to PPIA during normal MK in vitro differentiation. Cells were analyzed on day (D)6, D9, and D13 of culture. Data represent Mean±Standard Deviation (SD) of three independent experiments. (F) RT-PCR analysis of RUNX1, TREML1 and ITGA2 transcript levels normalized to PPIA in MK transduced with lentiviruses encoding shRUNX1 (shRUNX1_1 and shRUNX_2) relative to control shRNA (shSCR). Data represent Mean±SD of three independent experiments. (G) RT- PCR analysis of RUNX1, TREML1 and ITGA2 transcript levels normalized to HPRT and relative to control (CTRL) MK. CTRL MK: MK transduced with control lentivirus expressing Cherry and lentivirus expressing shSCR-GFP; shRUNX1_2: MK transduced with control lenvtivirus expressing Cherry and lentivirus expressing shRUNX1_2 and GFP; RUNX1mut/shRUNX1_2: MK transduced with lentivirus expressing RUNX1mut and Cherry and with lentivirus expressing shRUNX1_2 and GFP. RUNX1mut: WT RUNX1 cDNA was cloned into the lentivirus pRRL_EF1a_MCS/PGK-Cherry and mutation in four nucleotides, keeping the same aminoacids, was introduced to avoid recognition of the cDNA by shRUNX1_2. Data represent Mean±SD (n=2).
1246
haematologica | 2019; 104(6)