Figure 5.
Validation of predicted CC progression differences. (A) Experimental workflow. (B) PCNAVENUS nuclear patterns identify CC phase transitions. Scale bar = 5 µm. (C) Correlation of CC phase durations and lysosomal inheritance between paired HSC daughters. (D) G1 symmetry (indicated by red line) of 152 symmetric (lysosome ratio ≤1.3×) and 96 ACD (lysosome ratio ≥1.6×, throughout figure) HSC daughter pairs. Sorted by LysoHigh G1 length (5 replicates). (E) Agreement between scRNA-Seq transcriptome-inferred and PCNAVENUS-measured CC state assignment for individual cells. S/G2M transition occurs earlier in scRNA-Seq compared with PCNAVENUS data. Percentage of cells in each CC phase (G1, S, and G2M) for every hour of TSD plotted for scRNA-Seq and PCNAVENUS derived data. (F) ACD correlates with CC elongation in LysoLow daughter due to G1 and to lesser extent S-phase expansion. P values from Wilcoxon matched-pairs signed-rank tests. Error bars: Tukey. (G) CC phase duration expansions between paired daughter cells (% relative to longer sister) sorted by relative elongation per pair. Some symmetric cell division outliers trimmed to maximize displayed dynamic range (CC, 1; G1, 1; S, 3; G2M, 1). (H-I) The systematic CC difference after ACD (asymmetric lysosome inheritance) is neutralized by inhibiting trackSeq candidates. (H) We identified trackSeq candidates that are known G1/S regulators with available small molecule inhibitors. (I) Inhibiting candidates neutralizes the systematic CC difference after ACD while unspecific inhibition of lysosomes and CC does not. Green line indicates lack of ACD CC effect. None (n = 82 cells [3 replicates]); dimethyl sulfoxide (DMSO) (n = 56 [5 replicates]); NH4Cl (1 μM, n = 38; 10 μM, n = 46 [2 replicates]); bafilomycin (0.5 nM, n = 42; 1 nM, n = 32 [2 replicates]); Compound 5 (0.2 μM, n = 54 [2 replicates]; 2 μM, n = 122 [4 replicates]); and NSC15520 (5 μM, n = 120; 50 μM, n = 106 [4 replicates]); Pyrcoumin (0.5 μM: n = 74; 5 μM: n = 122 [4 replicates]); NSC663246 (34 nM, n = 20 [1 replicate]; 340 nM, n = 102 [4 replicates]). Outliers (>100% elongation) omitted, full range shown in supplemental Figure 6. P values from Wilcoxon signed-rank test of LysoHigh vs LysoLow daughter CC. SCF, stem cell factor; TPO, thrombopoietin.

Validation of predicted CC progression differences. (A) Experimental workflow. (B) PCNAVENUS nuclear patterns identify CC phase transitions. Scale bar = 5 µm. (C) Correlation of CC phase durations and lysosomal inheritance between paired HSC daughters. (D) G1 symmetry (indicated by red line) of 152 symmetric (lysosome ratio ≤1.3×) and 96 ACD (lysosome ratio ≥1.6×, throughout figure) HSC daughter pairs. Sorted by LysoHigh G1 length (5 replicates). (E) Agreement between scRNA-Seq transcriptome-inferred and PCNAVENUS-measured CC state assignment for individual cells. S/G2M transition occurs earlier in scRNA-Seq compared with PCNAVENUS data. Percentage of cells in each CC phase (G1, S, and G2M) for every hour of TSD plotted for scRNA-Seq and PCNAVENUS derived data. (F) ACD correlates with CC elongation in LysoLow daughter due to G1 and to lesser extent S-phase expansion. P values from Wilcoxon matched-pairs signed-rank tests. Error bars: Tukey. (G) CC phase duration expansions between paired daughter cells (% relative to longer sister) sorted by relative elongation per pair. Some symmetric cell division outliers trimmed to maximize displayed dynamic range (CC, 1; G1, 1; S, 3; G2M, 1). (H-I) The systematic CC difference after ACD (asymmetric lysosome inheritance) is neutralized by inhibiting trackSeq candidates. (H) We identified trackSeq candidates that are known G1/S regulators with available small molecule inhibitors. (I) Inhibiting candidates neutralizes the systematic CC difference after ACD while unspecific inhibition of lysosomes and CC does not. Green line indicates lack of ACD CC effect. None (n = 82 cells [3 replicates]); dimethyl sulfoxide (DMSO) (n = 56 [5 replicates]); NH4Cl (1 μM, n = 38; 10 μM, n = 46 [2 replicates]); bafilomycin (0.5 nM, n = 42; 1 nM, n = 32 [2 replicates]); Compound 5 (0.2 μM, n = 54 [2 replicates]; 2 μM, n = 122 [4 replicates]); and NSC15520 (5 μM, n = 120; 50 μM, n = 106 [4 replicates]); Pyrcoumin (0.5 μM: n = 74; 5 μM: n = 122 [4 replicates]); NSC663246 (34 nM, n = 20 [1 replicate]; 340 nM, n = 102 [4 replicates]). Outliers (>100% elongation) omitted, full range shown in supplemental Figure 6. P values from Wilcoxon signed-rank test of LysoHigh vs LysoLow daughter CC. SCF, stem cell factor; TPO, thrombopoietin.

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