AHR null mice exhibit platelet defects and decreased steady-state Mk polyploidization. Peripheral blood from wild type (WT), AHR^+/− (Het) and AHR-null (KO) mice was collected and examined for (A) platelet counts and (B) percentage of reticulated platelets. WT (black triangles), AHR^+/− (open squares) and AHR-null mice (open circles) were subjected to bleeding time assays (n = 8 for WT and AHR^+/−; n = 7 for AHR-null); shown is (C) the duration of the primary bleed and (D) average volume of blood lost during these experiments. A horizontal line indicates the average bleeding time for each genotype. (E) Bone marrow mononuclear cells were isolated and DNA content of CD41⁺ cells from WT, AHR^+/− and AHR-null mice was examined by flow cytometry. Error bars for all experiments in this figure represent the SEM (n = 8). Asterisks denote statistically significant differences compared to WT mice; a single asterisk (*) represents P < 0·05 and two asterisks (**) represent P < 0·01.

TCDD induces polyploidization of ex vivo cultured primary murine Mks; AHR protein levels increase during ex vivo megakaryopoiesis and is predominantly localized in the nucleus in differentiated Mks. (A) Murine bone marrow progenitors were ex vivo expanded in the presence of either dimethyl sulfoxide (DMSO), 10 nmol/l tetrachlorodibenzodioxin (TCDD), or thrombopoietin (TPO) and DNA content was assessed by flow cytometry; shown is the percentage of very high ploidy (≥16n) cells. (B) Q-RT-PCR analysis (n = 3) of AHR mRNA expression in differentiating CHRF cells. Values were normalized to housekeeping genes GUSB and RPLP0 mRNA expression. (C) Western blot analysis (n = 3) of AHR protein expression during CHRF megakaryopoiesis. Densitometry values were normalized to housekeeping protein expression (beta-actin) (D) In separate experiments (n = 3), nuclear and cytoplasmic fractions were obtained from differentiating CHRF cells and used in Western blot analysis. Error bars represent the SEM for all experiments in this figure. Asterisks denote statistically significant differences as compared to undifferentiated CHRF cultures; a single asterisk (*) represents P < 0·05 two asterisks (**) represent P < 0·01.

AHR knockdown effectively decreases AHR expression and activity in CHRF cells. (A) Q-RT-PCR experiments (n = 3) investigated AHR expression in NegA, AHR-A and AHR-B cell lines and were normalized to housekeeping genes. (B) A representative Western Blot of AHR protein expression in stable AHR-A and AHR-B CHRF cell lines, as compared to scrambled control (n = 3). Densitometry values used in this figure were normalized to GAPDH expression using ImageJ software. (C) Nuclear extracts from either undifferentiated or PMA-differentiated knockdown CHRF cell lines were used in EMSA experiments (n = 3) and were incubated with biotinylated oligonucleotides corresponding to the putative AHR binding sequence within the HES1 promoter. Sequence specificity was determined during competition assays using unlabeled oligonucleotides; protein specificity was determined by pre-incubation with antibodies to AHR. The regions marked with an asterisk (*) were used in subsequent densitometry analysis and normalized to free probe, indicated by a plus sign (+). (D) HES1 mRNA expression was determined by Q-RT-PCR analyses (n = 3) in NegA, AHR-A and AHR-B cell lines. Densitometry values were normalized to housekeeping gene expression using ImageJ software. Error bars represent the SEM. Asterisks denote statistically significant differences compared to NegA cells from the same day of PMA-induced differentiation; a single asterisk (*) represents P < 0·05 and two asterisks (**) represent P < 0·01.

AHR knockdown results in increased DNA synthesis of low ploidy cells without influencing apoptosis of differentiating CHRF cells. BrdU incorporation was used to investigate the role of AHR down regulation on the cell cycle of PMA-differentiated NegA, AHR-A and AHR-B cells (n = 3 for each cell line). (A) Representative experiments after 5 d of PMA-induced differentiation and their (B) corresponding ploidy histograms. Dashed lines indicate high ploidy (≥8n) cells; percent polyploidization (P₂ + P₄) is indicated. (C) BrdU incorporation (P₁ + P₂) was analyzed during PMA-induced differentiation of NegA (black squares), AHR-A (open triangles) and AHR-B (open circles) cells. (D) A further analysis of the data investigated the percentage of BrdU⁺ cells that were polyploid [P₂/(P₁ + P₂)]. (E) Caspase-3 activity was assessed as a measure of apoptosis in differentiating CHRF cell lines. Error bars represent the SEM.

AHR mRNA increases during ex vivo megakaryopoiesis of primary human CD34⁺ cells undergoing Tpo-stimulated megakaryopoiesis. Q-RT-PCR analysis (n = 4) of (A) AHR and (B) HES1 mRNA expression in differentiating primary megakaryocytes, granulocytes and erythrocytes deriving from human CD34⁺ cells from two separate donors. Values were normalized to the housekeeping genes GUSB and RPLP0. Comparative analysis of (C) AHR and (D) HES1 mRNA expression in megakaryocytes (Mk) relative to isogenic granulocytic (G) cultures from the same day. Error bars represent the SEM for all experiments in this figure. Asterisks denote statistically significant differences (by students t-test) as compared to Mk cultures; a single asterisk (*) represents P < 0·05 two asterisks (**) represent P < 0·01.

AHR DNA binding and HES1 expression increases during CHRF megakaryocytic differentiation. (A) A representative EMSA experiment (n = 4) used nuclear extracts from differentiating CHRF cells and was incubated with biotinylated AHR consensus binding sequence oligonucleotides. During competition assays, nuclear extracts were pre-incubated with unlabeled AHR consensus binding sequence oligonucleotides to ascertain band specificity. The region marked with an asterisk (*) was used in subsequent densitometry analysis using imageJ software and free probe is indicated by a plus sign (+). (B) Q-RT-PCR experiments (n = 3) examined HES1 mRNA expression (a known AHR transcriptional target) after PMA-induced Mk differentiation of CHRF cells. Values were normalized to housekeeping gene expression (GUSB and RPLP0) to minimize loading differences and compared to undifferentiated (d0) cells. (C) Western blots using total cell lysates from differentiating CHRF cells examined protein expression of HES1 (n = 3); a representative Western blot of PMA differentiated CHRF whole cell lysates is shown. Densitometry values were normalized to housekeeping gene expression using ImageJ software. Error bars represent the SEM. Asterisks denote statistically significant differences as compared to undifferentiated CHRF cultures; a single asterisk (*) represents P < 0·05 two asterisks (**) represent P < 0·01. (D) CHRF cells were analyzed by chromatin immunoprecipitation (IP) for in vivo AHR binding to the HES1 promoter. Total input (non-precipitated) chromatin was used as a positive control, and chromatin coprecipitating with antibodies toward Immunoglobin G was used as a negative control in these experiments.

AHR knockdown leads to decreased polyploidization. (A) DNA content was assessed by flow cytometry on the days indicated and the percentage of high-ploidy (≥8N) cells among all cells was reported for NegA (black squares), AHR-A (open triangles) and AHR-B (open circles) CHRF cells. Error bars represent the SEM (n = 3 for each cell line). (B) DNA histograms from a representative experiment at the designated time-points. Dashed lines indicate high ploidy (≥8n) cells and the percent polyploidization is indicated. (C) DNA content and (D) BrdU incorporation was assessed in undifferentiated NegA, AHR-A and AHR-B CHRF cell lines by flow cytometry.

HES1 knockdown leads to decreased polyploidization. (A) Q-RT-PCR experiments (n = 3) investigated HES1 mRNA expression in NegA, HES1-A and HES1-B cell lines and were normalized to housekeeping genes (GUSB and RPL0). (B) A representative Western Blot of HES1 protein expression in stable HES1-A and HES1-B CHRF cell lines, as compared to scrambled control (n = 3). (C) DNA content was assessed by flow cytometry on the days indicated and the percentage of high-ploidy (≥8N) cells was reported for NegA (black squares), HES1-A (open triangles) and HES1-B (open circles) cells. Error bars represent the SEM (n = 4 for each cell line). (D) DNA histograms from a representative experiment at the designated time-points after PMA-induced Mk differentiation. Dashed lines indicate high ploidy (≥8n) cells and the percentage of polyploidization is indicated. Error bars represent the SEM. Asterisks denote statistically significant differences compared to NegA cells from the same day of PMA-induced differentiation; a single asterisk (*) represents P < 0·05 and two asterisks (**) represent P < 0·01.