Fig. 3.
Fig. 3. Transcriptional down-regulation of MBN by ATRA requires Myb down-regulation. / (A) ATRA-induced down-regulation of MBN mRNA in PLB-985 and NB4 cells correlates with growth arrest and down-regulation of c-Myb. Growth (i) and morphologic (ii) assessments of PLB-985 (left) and NB4 (right) at 2, 4, and 6 days of treatment with 10−6 M ATRA. (iii) Regulation of MBN and c-Myb mRNAs in PLB-985 cells untreated or treated for 0.5, 1, 2, 4, and 6 days with 10−6 M ATRA. For RNA blots, 3 μg total RNA was loaded in each lane. The lower part is a control hybridization of the same blots with GAPDH probe for assessment of RNA quantities. Panels Ai-iii are for PLB-985 and NB4 cells on left and right column, respectively. (B) Overexpression of c-Myb in PLB-985 cells abolished transcriptional down-regulation of MBN by ATRA. (i) Twenty micrograms of the pMBN-658 (−658) or pMBN-658Mybmut (−658 Mybmut) constructs were individually transfected (−) or cotransfected with 1, 2, or 5 μg CMV-Myb (c-Myb) into PLB-985 cells and cultured with (▪) or without (░) ATRA (10−6 M) for 40 hours. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities were calculated relative to the wt (−658). The means and standard errors of 3 determinations are shown. (i) Autoradiogram of MBN mRNA expression in PLB-985 cells transfected with 5 μg of either the empty (−) or CMV-Myb (c-Myb) vector. The lower part is an autoradiogram of GAPDH mRNA for assessment of RNA quantities. Fold increase is indicated. (C) Overexpression of C/EBP and PU.1 in PLB-985 cells did not affect transcriptional down-regulation of MBN by ATRA. (i,ii) Induced PLB-985 growth arrest correlates with increased PU.1- and C/EBP-binding activity. Nuclear extracts from PLB-985 cells untreated or treated with 10−6M ATRA for the indicated days were assayed for PU.1 binding and C/EBP binding by EMSA performed with the PU.1 and C/EBP probes in the presence of 5 μg and 0.2 μg poly(dIdC), respectively (left panels). Competition experiments were carried out with a 400-fold molar excess of the indicated wt or mut competitor, and supershift experiments were performed with 1 μL PU.1 Ab; C/EBPα, C/EBPβ, C/EBPδ Abs; and unrelated control Abs (right). Arrows indicate the specific PU.1 or C/EBP complexes. Arrowheads indicate specific supershifted complexes. (iii) Twenty micrograms of the pMBN-658 (−658) was transfected (−) or cotransfected with 5 μg of the expression vectors for C/EBPα (MSV-C/EBPα), C/EBPβ (MSV-C/EBPβ), C/EBPδ (MSV-C/EBPδ), and PU.1 (pECE-PU.1) into PLB-985 cells and cultured with (▪) or without (░) ATRA (10−6 M) for 40 hours. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities calculated relative to the wt (−658) as a reference are presented. This is representative of 3 independently performed experiments.

Transcriptional down-regulation of MBN by ATRA requires Myb down-regulation.

(A) ATRA-induced down-regulation of MBN mRNA in PLB-985 and NB4 cells correlates with growth arrest and down-regulation of c-Myb. Growth (i) and morphologic (ii) assessments of PLB-985 (left) and NB4 (right) at 2, 4, and 6 days of treatment with 10−6 M ATRA. (iii) Regulation of MBN and c-Myb mRNAs in PLB-985 cells untreated or treated for 0.5, 1, 2, 4, and 6 days with 10−6 M ATRA. For RNA blots, 3 μg total RNA was loaded in each lane. The lower part is a control hybridization of the same blots with GAPDH probe for assessment of RNA quantities. Panels Ai-iii are for PLB-985 and NB4 cells on left and right column, respectively. (B) Overexpression of c-Myb in PLB-985 cells abolished transcriptional down-regulation of MBN by ATRA. (i) Twenty micrograms of the pMBN-658 (−658) or pMBN-658Mybmut (−658 Mybmut) constructs were individually transfected (−) or cotransfected with 1, 2, or 5 μg CMV-Myb (c-Myb) into PLB-985 cells and cultured with (▪) or without (░) ATRA (10−6 M) for 40 hours. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities were calculated relative to the wt (−658). The means and standard errors of 3 determinations are shown. (i) Autoradiogram of MBN mRNA expression in PLB-985 cells transfected with 5 μg of either the empty (−) or CMV-Myb (c-Myb) vector. The lower part is an autoradiogram of GAPDH mRNA for assessment of RNA quantities. Fold increase is indicated. (C) Overexpression of C/EBP and PU.1 in PLB-985 cells did not affect transcriptional down-regulation of MBN by ATRA. (i,ii) Induced PLB-985 growth arrest correlates with increased PU.1- and C/EBP-binding activity. Nuclear extracts from PLB-985 cells untreated or treated with 10−6M ATRA for the indicated days were assayed for PU.1 binding and C/EBP binding by EMSA performed with the PU.1 and C/EBP probes in the presence of 5 μg and 0.2 μg poly(dIdC), respectively (left panels). Competition experiments were carried out with a 400-fold molar excess of the indicated wt or mut competitor, and supershift experiments were performed with 1 μL PU.1 Ab; C/EBPα, C/EBPβ, C/EBPδ Abs; and unrelated control Abs (right). Arrows indicate the specific PU.1 or C/EBP complexes. Arrowheads indicate specific supershifted complexes. (iii) Twenty micrograms of the pMBN-658 (−658) was transfected (−) or cotransfected with 5 μg of the expression vectors for C/EBPα (MSV-C/EBPα), C/EBPβ (MSV-C/EBPβ), C/EBPδ (MSV-C/EBPδ), and PU.1 (pECE-PU.1) into PLB-985 cells and cultured with (▪) or without (░) ATRA (10−6 M) for 40 hours. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities calculated relative to the wt (−658) as a reference are presented. This is representative of 3 independently performed experiments.

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