Figure 3.
PU-H71 targets baseline cellular stress responses to induce cell death. (A) Isogenic TP53-WT, -KO, and -mutant (R175H and Y220C) Molm13 cells were untreated or treated with 40, 100, or 250 nM PU-H71; stained with an array of antibodies; and subjected to flow cytometry analysis. Cells from all experiments were subjected to the FlowSOM algorithm to identify clusters. Cells were then subjected to UMAP dimensional reduction and projected on 2-dimensional plots with live (blue) and dead (gray) cells. (B) Bubble plot of the FlowSOM cluster frequencies from panel A across TP53-WT, -KO, and -mutant (R175H and Y220C) Molm13 cells. (C) UMAP plots for the indicated markers. (D) The FlowSOM cluster frequencies shown in panel B were used for UMAP dimension reduction to map similarities and dissimilarities in the response of isogenic TP53-WT, -KO, and -mutant (R175H and Y220C) leukemia cells to PU-H71 treatment. Shapes indicate the cell types and colors indicate the treatment conditions. (E) Single-cell protein expression heatmap showing expression of the indicated markers (rows) across 16 different experimental conditions (TP53-WT, -KO, -R175H, and -Y220C leukemia cells that were untreated or treated with 40, 100, and 250 nM PU-H71) and the 10 FlowSOM clusters identified in panel A. (F) Volcano plot showing differentially expressed markers between CL1 and CL2 live cells. Dashed vertical lines indicate the fold change cutoff ratio of 0.5. (G) Volcano plot showing differentially expressed markers between untreated CL1 cells and CL1 cells treated with 40 or 100 nM PU-H71. Dashed vertical lines indicate the fold change cutoff ratio of 0.5. (H) Violin plots summarize expression of the indicated markers in untreated CL1 cells (bluish gray) and CL1 cells treated with 40 nM (yellow) or 100 nM (blue) PU-H71. (I) Log2-transformed CL2/CL1 ratios in TP53-WT, -KO, and -mutant leukemia cells plotted against 4 different treatment conditions. The FlowSOM CL1 and CL2 frequencies, shown in panel B, of untreated and PU-H71-treated TP53-WT, -KO and -mutant leukemia cells were used to calculate CL2/CL1 ratios. (J) Single-cell protein expression heatmap showing expression of the indicated markers (rows) across control CL2 cells and residual CL2 cells detected after treatment of TP53-KO and -mutant (R175H and Y220C) leukemia cells with 250 nM PU-71. The scale bar indicates scaled marker expression levels. (K) Violin plots summarize expression of the indicated markers in CL2 clusters from untreated TP53-KO, and -R175H and -Y220C mutant leukemia cells (bluish gray) vs residual CL2 in TP53-KO, and -R175H and -Y220C mutant leukemia cells treated with 250 nM PU-H71.

PU-H71 targets baseline cellular stress responses to induce cell death. (A) Isogenic TP53-WT, -KO, and -mutant (R175H and Y220C) Molm13 cells were untreated or treated with 40, 100, or 250 nM PU-H71; stained with an array of antibodies; and subjected to flow cytometry analysis. Cells from all experiments were subjected to the FlowSOM algorithm to identify clusters. Cells were then subjected to UMAP dimensional reduction and projected on 2-dimensional plots with live (blue) and dead (gray) cells. (B) Bubble plot of the FlowSOM cluster frequencies from panel A across TP53-WT, -KO, and -mutant (R175H and Y220C) Molm13 cells. (C) UMAP plots for the indicated markers. (D) The FlowSOM cluster frequencies shown in panel B were used for UMAP dimension reduction to map similarities and dissimilarities in the response of isogenic TP53-WT, -KO, and -mutant (R175H and Y220C) leukemia cells to PU-H71 treatment. Shapes indicate the cell types and colors indicate the treatment conditions. (E) Single-cell protein expression heatmap showing expression of the indicated markers (rows) across 16 different experimental conditions (TP53-WT, -KO, -R175H, and -Y220C leukemia cells that were untreated or treated with 40, 100, and 250 nM PU-H71) and the 10 FlowSOM clusters identified in panel A. (F) Volcano plot showing differentially expressed markers between CL1 and CL2 live cells. Dashed vertical lines indicate the fold change cutoff ratio of 0.5. (G) Volcano plot showing differentially expressed markers between untreated CL1 cells and CL1 cells treated with 40 or 100 nM PU-H71. Dashed vertical lines indicate the fold change cutoff ratio of 0.5. (H) Violin plots summarize expression of the indicated markers in untreated CL1 cells (bluish gray) and CL1 cells treated with 40 nM (yellow) or 100 nM (blue) PU-H71. (I) Log2-transformed CL2/CL1 ratios in TP53-WT, -KO, and -mutant leukemia cells plotted against 4 different treatment conditions. The FlowSOM CL1 and CL2 frequencies, shown in panel B, of untreated and PU-H71-treated TP53-WT, -KO and -mutant leukemia cells were used to calculate CL2/CL1 ratios. (J) Single-cell protein expression heatmap showing expression of the indicated markers (rows) across control CL2 cells and residual CL2 cells detected after treatment of TP53-KO and -mutant (R175H and Y220C) leukemia cells with 250 nM PU-71. The scale bar indicates scaled marker expression levels. (K) Violin plots summarize expression of the indicated markers in CL2 clusters from untreated TP53-KO, and -R175H and -Y220C mutant leukemia cells (bluish gray) vs residual CL2 in TP53-KO, and -R175H and -Y220C mutant leukemia cells treated with 250 nM PU-H71.

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