Further characterization of the TAF6d signaling hub may therefore provide novel therapeutic avenues to induce controlled tumor cell death irrespective of their p53 status

June 1, 2017

lines 102 pediatric AML patient samples were 14937-32-7 obtained at the time of diagnosis after informed consent from the Oncogenetic laboratory of the Pediatric Oncology and Hematology department of the University Hospital of Gieen. In order to be able to statistically analyze the different cytogenetic subgroups, patient samples were positively selected for t, t, inv and MLL-rearranged samples. This deviates from the general characteristics of the whole study cohort as referred to elsewhere. Please note that the median white blood cell count of our cohort is higher than of the whole study cohort due to the enrichment of inv and MLL-rearranged samples. Patients of these cytogenetic subgroups also have the highest white blood cell counts compared to the other cytogenetic subtypes of the whole study cohort. The frequency of mutations in NPM1, CEBPA and FLT3, shown to be of prognostic importance especially in the normal cytogenetic subgroup, was not available for our patient cohort. 71 samples were obtained from bone marrow with a median blast content of 74%, while 31 pediatric samples were obtained from blasts out of peripheral blood with a median blast content of 87%. The blast content of bone marrow might be underestimated since only the peripheral blast count was available for some patients. Six adulthood AML samples with adequate RNA quality, three carrying translocation t and three carrying translocation t, were obtained from the Munich Leukemia Laboratory at time of diagnosis following informed consent. Those were used to evaluate possible effects specific to our miRNA detection method when comparing differences in expression of the here reported childhood 25279926 leukemia data to previously published data on adulthood acute myeloid leukemia. Cytogenetic characterization was performed routinely for diagnostic purposes on fresh material. All patient samples were stored at 280uC prior to RNA extraction. CD34+ cells were obtained from bone marrow of two healthy donors after informed consent, separated by using immunomagnetic bead separation and stored after separation in liquid nitrogen. AML cell lines KASUMI-1 and NB4 were obtained from the German collection of microorganisms and cell culture and were grown in RPMI 1640 with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/100 mg/ml streptomycin under standard growth conditions. Photoactivatable-ribonucleoside enhanced crosslinking and total cell extracts Prior to UV-crosslinking 1003006106 cells were incubated with 100 mM 49-thiouridine in growth medium in the dark for 14 hours. Subsequently, cells were washed with ice-cold PBS and irradiated on ice with 365 nm UV light. Afterwards, the cells were washed with PBS, collected by centrifugation at 23964788 5006 g for 5 min, flash frozen in liquid nitrogen and stored at MiRNA Expression and Function in Pediatric AML Characteristic Age, y Median Range Sex, no. Female Male White blood cell count, 6103/ml Median Range Bone marrow: blast content median % no. of patients Peripheral blood: blast content median % no. of patients Cytogenetic abnormalities, no. t t t t t other t t inv t normal karyotype other French-American-British classification, no. M0 M1 M2 M3 M4 M4eo M5 M6 not determined doi:10.1371/journal.pone.0056334.t001 Pediatric AML cohort Adult AML cohort 10.3 0.517.9 44.8 34.581.8 51 51 3 3 42.5 0.9440 74 71 87 31 63 6 – 1 2 16 6 5 3 14 13 24 4 14 3 3 – 1 4 23 12 11 9 25 2 15 3 1 2 280uC. Immediately preceding immunoprecipitation cells were lyz