![Figure 3. Relationship of FLNA cleavage and TIF-90 expression to pre-rRNA synthesis in AML cells. (A) Relative expression of phosphorylated Akt (p-Akt) and FLNA in primary AML cells. Cell lysate (30 μg) from 4 normal bone marrows and 21 AML samples was separated on sodium dodecyl sulfate gels and immunoblotted with the indicated antibodies (FL, full length; CP, cleavage product). (B and C) Correlation of FLNA cleavage and TIF-90 expression with rRNA synthesis and cell survival. Twenty-one AML patient samples were divided into 4 groups based on the expression of TIF-90 and presence of the FLNA cleavage product. Measurement of TIF-90 was performed by qPCR (supplemental Figure 2D), and measurements of FLNA cleavage were carried out using densitometry on western blots (Figure 3A). Samples are divided into low and high expression relative to the average value for all samples. The value of 5′ETS pre-rRNA in supplemental Figure 2E and Pol I recruited to rDNA promoter in supplemental Figure 2F for each sample was used for graphic representation. (B) Level of pre-rRNA synthesis and RNA labeling with [32P] was performed with the mixture of AML cells from each group; level of Pol I recruited to rDNA (C, left). The bar indicates the average value and the ends of the whiskers represent minimum and maximum values. Significance was determined using the Student t test; 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay (C, right). (D and E) Effects of FLNA cleavage on TIF-IA–Pol I binding, pre-rRNA synthesis, and cell survival in primary AML cells. Fourteen samples from AML patients were divided into low and high FNLA cleavage expression groups (each group, n = 7), and the level of FLNA cleavage was measured based on the densitometry results from panel A. (D) TIF-IA protein was immunoprecipitated and immunoblotted with anti-Pol I antibody (left); effects of knockdown of FLNA on TIF-IA and Pol I binding in primary AML cells (right). A mixture of AML cells (n = 7) that have FLNA cleavage levels above the average value was transfected with siSCR or siFLNA for 36 hours. TIF-IA protein was immunoprecipitated and immunoblotted with anti-Pol I antibody. (E) The 5′ETS pre-rRNA was measured by qPCR (left), ChIP assay (middle), and MTT assay (right).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/4/10.1182_blood-2013-12-544726/4/579f3.jpeg?Expires=1735500395&Signature=Ptg59oDnAB8lCM5-FO4xsqGtGDCyyEYR6xoPb8DImV9BkYDdmtDK3yzho~EsCU6E8eo4ZfdmbZJk3cg11bsTUX~IgIrRO5aHVtd2jL3rW4V-36WN8OkNI4RB16IueFxuQG6jaCiTODdHNI0MH1wy~r-t~4bGyuKli4A2wouZJP5gJhxt7yyUBIlkW1sY9y6yx1AgNoQJibRbkWfRB5YFAryaW21pPfQXBjwMtdz5sNKM9lViiTosctuh7Eq8SVjk21gOIUHhdjeVUNkr-WNjm7a6E3MCYoMIVTpHkF5jtVT~9FrW1u6pSqdCUT-ybAMoijDTcnwTpzKXEDElKtg2Pg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Relationship of FLNA cleavage and TIF-90 expression to pre-rRNA synthesis in AML cells. (A) Relative expression of phosphorylated Akt (p-Akt) and FLNA in primary AML cells. Cell lysate (30 μg) from 4 normal bone marrows and 21 AML samples was separated on sodium dodecyl sulfate gels and immunoblotted with the indicated antibodies (FL, full length; CP, cleavage product). (B and C) Correlation of FLNA cleavage and TIF-90 expression with rRNA synthesis and cell survival. Twenty-one AML patient samples were divided into 4 groups based on the expression of TIF-90 and presence of the FLNA cleavage product. Measurement of TIF-90 was performed by qPCR (supplemental Figure 2D), and measurements of FLNA cleavage were carried out using densitometry on western blots (Figure 3A). Samples are divided into low and high expression relative to the average value for all samples. The value of 5′ETS pre-rRNA in supplemental Figure 2E and Pol I recruited to rDNA promoter in supplemental Figure 2F for each sample was used for graphic representation. (B) Level of pre-rRNA synthesis and RNA labeling with [32P] was performed with the mixture of AML cells from each group; level of Pol I recruited to rDNA (C, left). The bar indicates the average value and the ends of the whiskers represent minimum and maximum values. Significance was determined using the Student t test; 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay (C, right). (D and E) Effects of FLNA cleavage on TIF-IA–Pol I binding, pre-rRNA synthesis, and cell survival in primary AML cells. Fourteen samples from AML patients were divided into low and high FNLA cleavage expression groups (each group, n = 7), and the level of FLNA cleavage was measured based on the densitometry results from panel A. (D) TIF-IA protein was immunoprecipitated and immunoblotted with anti-Pol I antibody (left); effects of knockdown of FLNA on TIF-IA and Pol I binding in primary AML cells (right). A mixture of AML cells (n = 7) that have FLNA cleavage levels above the average value was transfected with siSCR or siFLNA for 36 hours. TIF-IA protein was immunoprecipitated and immunoblotted with anti-Pol I antibody. (E) The 5′ETS pre-rRNA was measured by qPCR (left), ChIP assay (middle), and MTT assay (right).
