Figure 5.
Mislocalized AURKB and Survivin from the inner centromere and loss of chromatid cohesion and SAC impairment in HyperD-ALL blasts. (A-B) Representative IF staining for CENP-A and AURKB (A) or Survivin (B) in PDX-expanded B-ALL blasts. (C-D) Quantification of the AURKB (C) and Survivin (D) fluorescence signal at the inner centromere; n = 30 metaphases from 3 non-HyperD and n = 30 metaphases from 3 HyperD-ALL. (E) Representative IF showing either centromeric and scattered localization of AURKB, Survivin, and CENP-A. (F-G) Frequency of PDX-expanded non-HyperD (n = 3) and HyperD-ALL (n = 3) blasts showing centromeric vs scattered localization of AURKB (n = 797 chromosomes from non-HyperD and n = 676 chromosomes from HyperD) (F), and Survivin (n = 964 chromosomes from non-HyperD and n = 814 chromosomes from HyperD) (G). (H) Quantification of total AURKB fluorescence signal from samples in panel C. AURKB levels are expressed relative to non-HyperD blasts, which are arbitrarily set to 100. (I) Left, representative FACS staining of H3S10P. Right, MFI of H3S10P in 3 non-HyperD and 3 HyperD-ALL samples. (J) Left, representative images of normal or railroad-shaped chromosomes. Right, frequency of metaphases showing the indicated number of chromosomes with PCS; n = 200 metaphases from 4 non-HyperD, n = 250 metaphases from 5 HyperD-ALLs samples. (K) Schematic depicting the workflow for functional analysis of the SAC. (L) Representative FACS of mitotic PDX-expanded B-ALL blasts (H3S10P+CD19+ cells, green) in the presence or absence of nocodazole. (M) Fold-change of mitotic (H3S10P+) blasts in nocodazol-treated (relative to DMSO-treated) non-HyperD (n = 3) and HyperD-ALL (n = 3) primary blasts. (N) Representative FACS cell cycle distribution of nocodazol- vs DMSO-treated PDX-expanded B-ALL blasts. (O) Quantification of the cell cycle phases in nocodazol- vs DMSO-treated PDX-expanded B-ALL blasts; n = 3 non-HyperD and n = 3 HyperD-ALL. (P) Quantitative reverse transcription–PCR analysis of SAC proteins in B-ALL primary samples; n = 9 non-HyperD and n = 11 HyperD. Graphs represent the mean, and error bars represent the standard error of the mean. *P < .05, **P < .01; ***P < .001; **** P < .0001 (2-way ANOVA or Student t test). Scale bars, 10 µm.

Mislocalized AURKB and Survivin from the inner centromere and loss of chromatid cohesion and SAC impairment in HyperD-ALL blasts. (A-B) Representative IF staining for CENP-A and AURKB (A) or Survivin (B) in PDX-expanded B-ALL blasts. (C-D) Quantification of the AURKB (C) and Survivin (D) fluorescence signal at the inner centromere; n = 30 metaphases from 3 non-HyperD and n = 30 metaphases from 3 HyperD-ALL. (E) Representative IF showing either centromeric and scattered localization of AURKB, Survivin, and CENP-A. (F-G) Frequency of PDX-expanded non-HyperD (n = 3) and HyperD-ALL (n = 3) blasts showing centromeric vs scattered localization of AURKB (n = 797 chromosomes from non-HyperD and n = 676 chromosomes from HyperD) (F), and Survivin (n = 964 chromosomes from non-HyperD and n = 814 chromosomes from HyperD) (G). (H) Quantification of total AURKB fluorescence signal from samples in panel C. AURKB levels are expressed relative to non-HyperD blasts, which are arbitrarily set to 100. (I) Left, representative FACS staining of H3S10P. Right, MFI of H3S10P in 3 non-HyperD and 3 HyperD-ALL samples. (J) Left, representative images of normal or railroad-shaped chromosomes. Right, frequency of metaphases showing the indicated number of chromosomes with PCS; n = 200 metaphases from 4 non-HyperD, n = 250 metaphases from 5 HyperD-ALLs samples. (K) Schematic depicting the workflow for functional analysis of the SAC. (L) Representative FACS of mitotic PDX-expanded B-ALL blasts (H3S10P+CD19+ cells, green) in the presence or absence of nocodazole. (M) Fold-change of mitotic (H3S10P+) blasts in nocodazol-treated (relative to DMSO-treated) non-HyperD (n = 3) and HyperD-ALL (n = 3) primary blasts. (N) Representative FACS cell cycle distribution of nocodazol- vs DMSO-treated PDX-expanded B-ALL blasts. (O) Quantification of the cell cycle phases in nocodazol- vs DMSO-treated PDX-expanded B-ALL blasts; n = 3 non-HyperD and n = 3 HyperD-ALL. (P) Quantitative reverse transcription–PCR analysis of SAC proteins in B-ALL primary samples; n = 9 non-HyperD and n = 11 HyperD. Graphs represent the mean, and error bars represent the standard error of the mean. *P < .05, **P < .01; ***P < .001; **** P < .0001 (2-way ANOVA or Student t test). Scale bars, 10 µm.

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