American Society of Hematology

Figure 1

$Figure 1. Cell-specific differences in HES1 effects, complexes, and interaction with PARP1. (A) Differential effects of HES1 on growth in B-ALL and T-ALL cell lines. Line graphs represent the cell counts of GFP+ cells up to 7 days after transfection. A panel of 3 B-ALL and 3 T-ALL cell lines were transfected with a HES1/GFP construct (solid lines, black symbols) or control GFP-only vector (dashed lines, open symbols). Numbers of GFP+ cells were calculated by combining the percentage of GFP+ cells measured by flow cytometry and Trypan blue exclusion cell counts performed daily. The results are presented as mean ± SD of 3 independent experiments. (P < .02). (B) Different HES1 complexes in B-ALL and T-ALL. Size-exclusion chromatography was performed to assess the relative size of HES1 complexes in B-ALL (JM1) and T-ALL (SupT1) cells. Freshly prepared lysates from B-ALL (JM1) and T-ALL (SupT1) cells transfected with FLAG-HES1 constructs were run through a Superdex 200 column, and 70 fractions were collected. Even fractions were used for immunoblotting to detect HES1-containing fractions with the use of anti-FLAG antibody. No signal was seen before fraction 30 or after fraction 50 in either sample. Protein mass standards were run on the same column to provide approximate molecular sizes (in kilodaltons) of complexes. Differences in fractions with FLAG-HES1 in B-ALL and T-ALL suggest that there are cell type–specific differences in HES1 complexes. Lack of FLAG-HES1 above fraction 50 suggests that no free monomers (35 kDa) are present in either cell line. (C) PARP1 binds to endogenous HES1 in B-ALL cells. IP of PARP1 in nuclear extracts from JM-1 (B-ALL) cells shows binding to endogenous HES1. (D) HES1 interacts with both full-length and cleaved PARP1. B-ALL (JM1) cells transfected with FLAG-HES1 mRNA, after 48 hours nuclear extracts were immunoprecipitated with PARP1 or FLAG beads and immunoblotted with anti-PARP1 and anti-FLAG antibodies. FLAG-IP of exogenous HES1 is associated with PARP1 cleavage and binds to both full-length and cleaved PARP1. IgG indicates immunoglobulin G.$

Cell-specific differences in HES1 effects, complexes, and interaction with PARP1. (A) Differential effects of HES1 on growth in B-ALL and T-ALL cell lines. Line graphs represent the cell counts of GFP⁺ cells up to 7 days after transfection. A panel of 3 B-ALL and 3 T-ALL cell lines were transfected with a HES1/GFP construct (solid lines, black symbols) or control GFP-only vector (dashed lines, open symbols). Numbers of GFP⁺ cells were calculated by combining the percentage of GFP⁺ cells measured by flow cytometry and Trypan blue exclusion cell counts performed daily. The results are presented as mean ± SD of 3 independent experiments. (P < .02). (B) Different HES1 complexes in B-ALL and T-ALL. Size-exclusion chromatography was performed to assess the relative size of HES1 complexes in B-ALL (JM1) and T-ALL (SupT1) cells. Freshly prepared lysates from B-ALL (JM1) and T-ALL (SupT1) cells transfected with FLAG-HES1 constructs were run through a Superdex 200 column, and 70 fractions were collected. Even fractions were used for immunoblotting to detect HES1-containing fractions with the use of anti-FLAG antibody. No signal was seen before fraction 30 or after fraction 50 in either sample. Protein mass standards were run on the same column to provide approximate molecular sizes (in kilodaltons) of complexes. Differences in fractions with FLAG-HES1 in B-ALL and T-ALL suggest that there are cell type–specific differences in HES1 complexes. Lack of FLAG-HES1 above fraction 50 suggests that no free monomers (35 kDa) are present in either cell line. (C) PARP1 binds to endogenous HES1 in B-ALL cells. IP of PARP1 in nuclear extracts from JM-1 (B-ALL) cells shows binding to endogenous HES1. (D) HES1 interacts with both full-length and cleaved PARP1. B-ALL (JM1) cells transfected with FLAG-HES1 mRNA, after 48 hours nuclear extracts were immunoprecipitated with PARP1 or FLAG beads and immunoblotted with anti-PARP1 and anti-FLAG antibodies. FLAG-IP of exogenous HES1 is associated with PARP1 cleavage and binds to both full-length and cleaved PARP1. IgG indicates immunoglobulin G.

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