Figure 4.
HyperD-ALL blasts show chromosome hypocondensation, loss of centromere stiffness, and defects in the condensin complex. (A) Analysis of spindle abnormalities in B-ALL primary blasts. Left, representative DNA-Kinetochore-spindle IF staining of mitotic cells with bipolar, multipolar, and disorganized spindles. Right, frequency of mitotic cells displaying spindle defects; n = 3 non-HyperD-ALLs (n = 251 mitosis) and n = 3 HyperD-ALLs (n = 251 mitosis) PDX-expanded samples. (B) Frequency of metaphases with hypocondensed chromosomes in primary B-ALL blasts; n = 200 metaphases from 4 non-HyperD and n = 250 metaphases from 5 HyperD-ALLs primary samples. Left, representative images of normal and hypocondensed metaphase chromosomes. Insets represent ×3 magnifications. (C) Chromosome structure of formaldehyde-crosslinked PDX-expanded B-ALL samples. Left, Representative images of metaphase cells with hypocondensed chromosomes. Anti-ACA staining is shown in green. Right, frequency of formaldehyde-crosslinked metaphases showing hypocondensed or hypocondensed with unstructured chromosomes in B-ALL primary samples; n = 60 metaphases from 3 non-HyperD and n = 57 metaphases from 3 HyperD-ALLs. (D) Chromosome arm width using PDX-expanded B-ALL samples from panel C; n = 191 chromosomes from 3 non-HyperD and n = 143 chromosomes from 3 HyperD-ALLs. (E) Representative IF images of metaphase PDX-expanded B-ALL blasts stained with DAPI, anti-SMC2, and anti-ACA. (F) Quantification of the SMC2 total volume in metaphase chromosomes from panel E; n = 30 metaphases from 3 non-HyperD and 3 HyperD-ALLs. (G) Schematic cartoon of the 2 human condensin complexes. (H) WB analysis of the indicated condensin members in whole-cell lysates from PDX-expanded B-ALL samples. (I) Quantification of WB bands from panel H normalized to actin. (J) Representative HPLC-ESI-MS chromatograms of the indicated peptides for HyperD and non-HyperD PDX-expanded B-ALLs. (K) Acetylation levels of SMC2 peptide SQAASILTK (m/z = 480.8). (L) Phosphorylation levels of CAPD2 peptide GPAASTQEK (m/z = 524.7). Results depict the average of the peak areas from independent MS experiments from 2 non-HyperD and 2 HyperD-ALL PDX-expanded blasts. (M) Representative line-scan measurements of individual centromeres in the indicated B-ALL primary samples. DAPI and ACA are depicted as a blue and red lines, respectively. Yellow arrowheads point to the analyzed chromosome. (N) Intercentromeric distance from MG132-treated PDX-expanded B-ALL blasts; n = 155 centromeres from 3 non-HyperD and n = 119 centromeres from 3 HyperD-ALLs. (O) Intercentromeric distance from colcemid-treated PDX-expanded B-ALL blasts; n = 130 centromeres from 3 non-HyperD and n = 111 centromeres from 3 HyperD-ALLs. Graphs represent the mean, and error bars represent the standard error of the mean. *P < .05, **P < .01; ***P < .001; ****P < .0001. Two-way ANOVA (A-C) or Student t test (D, F, I, N, O). Scale bars, 10 µm.

HyperD-ALL blasts show chromosome hypocondensation, loss of centromere stiffness, and defects in the condensin complex. (A) Analysis of spindle abnormalities in B-ALL primary blasts. Left, representative DNA-Kinetochore-spindle IF staining of mitotic cells with bipolar, multipolar, and disorganized spindles. Right, frequency of mitotic cells displaying spindle defects; n = 3 non-HyperD-ALLs (n = 251 mitosis) and n = 3 HyperD-ALLs (n = 251 mitosis) PDX-expanded samples. (B) Frequency of metaphases with hypocondensed chromosomes in primary B-ALL blasts; n = 200 metaphases from 4 non-HyperD and n = 250 metaphases from 5 HyperD-ALLs primary samples. Left, representative images of normal and hypocondensed metaphase chromosomes. Insets represent ×3 magnifications. (C) Chromosome structure of formaldehyde-crosslinked PDX-expanded B-ALL samples. Left, Representative images of metaphase cells with hypocondensed chromosomes. Anti-ACA staining is shown in green. Right, frequency of formaldehyde-crosslinked metaphases showing hypocondensed or hypocondensed with unstructured chromosomes in B-ALL primary samples; n = 60 metaphases from 3 non-HyperD and n = 57 metaphases from 3 HyperD-ALLs. (D) Chromosome arm width using PDX-expanded B-ALL samples from panel C; n = 191 chromosomes from 3 non-HyperD and n = 143 chromosomes from 3 HyperD-ALLs. (E) Representative IF images of metaphase PDX-expanded B-ALL blasts stained with DAPI, anti-SMC2, and anti-ACA. (F) Quantification of the SMC2 total volume in metaphase chromosomes from panel E; n = 30 metaphases from 3 non-HyperD and 3 HyperD-ALLs. (G) Schematic cartoon of the 2 human condensin complexes. (H) WB analysis of the indicated condensin members in whole-cell lysates from PDX-expanded B-ALL samples. (I) Quantification of WB bands from panel H normalized to actin. (J) Representative HPLC-ESI-MS chromatograms of the indicated peptides for HyperD and non-HyperD PDX-expanded B-ALLs. (K) Acetylation levels of SMC2 peptide SQAASILTK (m/z = 480.8). (L) Phosphorylation levels of CAPD2 peptide GPAASTQEK (m/z = 524.7). Results depict the average of the peak areas from independent MS experiments from 2 non-HyperD and 2 HyperD-ALL PDX-expanded blasts. (M) Representative line-scan measurements of individual centromeres in the indicated B-ALL primary samples. DAPI and ACA are depicted as a blue and red lines, respectively. Yellow arrowheads point to the analyzed chromosome. (N) Intercentromeric distance from MG132-treated PDX-expanded B-ALL blasts; n = 155 centromeres from 3 non-HyperD and n = 119 centromeres from 3 HyperD-ALLs. (O) Intercentromeric distance from colcemid-treated PDX-expanded B-ALL blasts; n = 130 centromeres from 3 non-HyperD and n = 111 centromeres from 3 HyperD-ALLs. Graphs represent the mean, and error bars represent the standard error of the mean. *P < .05, **P < .01; ***P < .001; ****P < .0001. Two-way ANOVA (A-C) or Student t test (D, F, I, N, O). Scale bars, 10 µm.

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