Effect of LCP1 I232F on G-CSF–induced granulocytic differentiation. (A) Trypan blue exclusion cell count and viability were performed during differentiation of LCP1 I232F-expressing 32D cells with G-CSF treatment (100 ng/µL) for 8 days. (B) Wright-Giemsa–stained images, obtained by using a Leica DM6B upright microscope (×40 magnification), for 32D cells expressing wtLCP1 and LCP1 I232F upon differentiation with G-CSF (left and middle) and analysis of large dysplastic cells (right). (C) LCP1 (wt and I232F) expressing 32D cells differentiated with G-CSF were stained with anti-CD11b and anti–GR-1 antibodies and analyzed with flow cytometry. (D) 32D cells expressing wtLCP1 and LCP1 I232F were differentiated with G-CSF followed by quantitative polymerase chain reaction–based gene expression analysis of indicated master transcription factors of granulopoiesis and terminal granulocytic differentiation markers and transcription factors at days 0, 2, 4, and 6. Data are shown as mean ± standard error of mean (results of n = 3 biological replicates unless otherwise specified). Statistical analysis was assessed by using the Student t test. *P < .05, **P < .01, ***P < .001, ****P < .0001. hGCSF, human G-CSF; ns, not significant.