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High-risk comutations expand distinct SF3B1 -mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34 + CD38 – and CD34 + CD38 – <t>CD133</t> +/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1 -mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34 + CD38 – CD133 + (B) or CD34 + CD38 – CD133 – HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1 -mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34 + CD38 – CD133 + (left) or CD34 + CD38 – CD133 – (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1 -mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34 + CD133 + phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.
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fluorescently-labeled cd133 human fc/fitc 11-1339-41 antibody  (Thermo Fisher)
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High-risk comutations expand distinct SF3B1 -mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34 + CD38 – and CD34 + CD38 – <t>CD133</t> +/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1 -mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34 + CD38 – CD133 + (B) or CD34 + CD38 – CD133 – HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1 -mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34 + CD38 – CD133 + (left) or CD34 + CD38 – CD133 – (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1 -mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34 + CD133 + phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.
Fluorescently Labeled Cd133 Human Fc/Fitc 11 1339 41 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cd133 fitc  (Biorbyt)
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High-risk comutations expand distinct SF3B1 -mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34 + CD38 – and CD34 + CD38 – <t>CD133</t> +/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1 -mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34 + CD38 – CD133 + (B) or CD34 + CD38 – CD133 – HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1 -mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34 + CD38 – CD133 + (left) or CD34 + CD38 – CD133 – (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1 -mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34 + CD133 + phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.
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High-risk comutations expand distinct SF3B1 -mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34 + CD38 – and CD34 + CD38 – CD133 +/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1 -mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34 + CD38 – CD133 + (B) or CD34 + CD38 – CD133 – HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1 -mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34 + CD38 – CD133 + (left) or CD34 + CD38 – CD133 – (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1 -mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34 + CD133 + phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.

Journal: Blood Advances

Article Title: Distinct routes of clonal progression in SF3B1-mutant myelodysplastic syndromes ∗ †

doi: 10.1182/bloodadvances.2024014965

Figure Lengend Snippet: High-risk comutations expand distinct SF3B1 -mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34 + CD38 – and CD34 + CD38 – CD133 +/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1 -mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34 + CD38 – CD133 + (B) or CD34 + CD38 – CD133 – HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1 -mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34 + CD38 – CD133 + (left) or CD34 + CD38 – CD133 – (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1 -mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34 + CD133 + phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.

Article Snippet: HSPC populations were analyzed using markers CD34 AF700 (581; BD), CD38 PE-Cy7 (HB7; BD), CD133 APC (AC133; Miltenyi), CD133 FITC (AC133; Miltenyi), CD90 RB705 (5E10; BD), CD45RA BV605 (HI100; BD).

Techniques: Mutagenesis, In Vitro