Figure 2. Nuclear relocalization of NPM1c induces cell growth arrest and differentiation in AML cell lines.

(A) Viable cell counts by trypan blue exclusion of OCI-AML3 (O3; NPM1c⁺) and OCI-AML2 (O2; NPM1 WT) cells transfected with Cas9 only, a guide targeting CD19 (sgCD19), or sgNPM1c. Equal numbers of cells were plated 5 days after electroporation. Results are reported as ratio to Cas9 only control. (OCI-AML3 n=3, OCI-AML2 n=2; mean ± SEM).

(B) Indel frequencies at targeted loci (i.e. CD19, CD45 and NPM1c) calculated by highthroughput amplicon sequencing at days 3 and 12 after electroporation with indicated sgRNAs (n=3, mean ± SEM).

(C) May-Grünwald Giemsa staining of OCI-AML3 and IMS-M2 cells 9 and 6 days, respectively, after transfection with the indicated sgRNA. Arrows indicate azurophilic granules. Scale bar: 20 μm.

(D) Flow cytometry analysis of CD11b expression in OCI-AML3 cells 9 days after electroporation. Left panel shows distribution of CD11b signal in cells transfected with sgRNAs against the indicated genes (CD19 and CD45 serving as controls). Right panel shows the percentage of CD11b⁺ cells (n=6; mean ± SEM). The cut-off point for CD11b positivity was set at the last one percentile of the gaussian curve of Cas9 only controls.

(E) Flow cytometry analysis of myeloperoxidase (MPO) expression in IMS-M2 cells 6 days after electroporation. Left panel shows distribution of MPO signal for indicated sgRNA. Right panel shows the percentage of MPO⁺ cells (n=6; mean ± SEM). The cut-off point for MPO positivity was set at the last one percentile of the gaussian curve of Cas9 only controls.

(F) Cell cycle analysis by BrdU incorporation and 7-AAD 5 and 9 days following electroporation with sgNPM1c and controls (n=3, mean ± SEM). * p<0.05, ** p<0.01, *** p<0.001; unpaired t-test with Welch’s correction. See also Figure S2 and Table S2.