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Fig. 6 | Clinical Epigenetics

Fig. 6

From: Epigenetic down-regulation of the HIST1 locus predicts better prognosis in acute myeloid leukemia with NPM1 mutation

Fig. 6

H1-3 KD promotes granulocytic differentiation in ATRA-treated AML cell lines. a qPCR expression analysis of the indicated histone H1 genes in shCtrl and shH1-3 (KD#1) without (Dox-) or with 6-days-induction of doxycycline (Dox+). Data represent three independent Dox inductions. Results are normalized on HPRT and expressed in fold change (FC) between Dox+ and Dox− conditions. b Immunoblot of H1-3, H1, and H3 in chromatin-bound extracts from shCtrl and KD#1 upon (+) or not (−) doxycycline (Dox). c, d Cell surface analysis of granulocytic markers consecutive to H1-3 KD in OCI-AML3 clones. c Percentage of CD11b positive cells in shCtrl and KD#1 upon doxycycline induction (Dox+) or not (Dox−) without ATRA (wo ATRA) or with 72 h of ATRA-treatment (0.5 μM or 1 μM). d Percentage of CD11b-CD11c cells upon 72 h of ATRA-treatment (0.5 μM). Data represent three independent experiments. Statistical significance was estimated using Mann Whitney test *p < 0.05; **p < 0.005. NS non-significant. e, f Cytological analysis of shCtrl and KD#1 clones upon doxycycline and after 96 h of ATRA treatment (0.5 μM). e May–Grünwald Giemsa coloration. Black arrows are pointing cytoplasmic azurophilic granules. f Quantification of cells with cytoplasmic granules. Results are presented as a percentage of positive cells (more than two granules). Statistical significance was estimated using Mann–Whitney test *p < 0.05; **p < 0.005. g Expression analysis of CYBB and ITGAM in untreated OCI-AML3 and in ATRA-treated (1 μM) KD#1 without or with doxycycline induction. Gene expression was normalized to two housekeeping genes (PGK1 and PPIA). Data represent three independent experiments. Statistical significance was estimated using T-test (one-tailed p value). h U937 cells were stably infected by a doxycycline inducible shCtrl (U-shCtrl) or shH1-3 (U-KD#1 and U-KD#2). Left panel: percentage of CD11b-CD11c cells upon 72 h of ATRA-treatment (0.1 μM). Level of H1d in the different conditions was measured by immunoblot. Right panel: May–Grünwald Giemsa staining of KD#1 clone with or without doxycycline upon 72 h hours of ATRA-treatment. Data represent two independent experiments. Statistical significance was estimated using unpaired Two-tailed t test * p < 0.05; ***p < 0.001

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