Mechanisms underlying the inhibitory effects of LCP4 treatment on MF CD34⁺ cells. (A-C) pSTAT3 and pSTAT5 levels in MF and normal BM CD34⁺ cells measured using phospho-flow cytometric analysis. (A) Representative flow cytometric plots showing pSTAT3 (left) and pSTAT5 (right) levels in MF splenic (SP14) and normal BM20 CD34⁺ cells. (B-C) Fold change in MFI of pSTAT3 (B) and pSTAT5 (C) for CD34⁺ cells from each MF spleen or normal BM, which was calculated using the equation: MFI_{Cytokines Alone} / MFI_{No Cytokines} or MFI_{Cytokines + LCP4} / MFI_{No Cytokines}. Cytokines alone vs cytokines + LCP4: pSTAT3: MF and N BM: P for both > .05; pSTAT5: MF: P < .05; N BM: P > .05. (D) pMPL, pJAK2, pSTAT3, and pSTAT5 levels measured using western blotting in splenic MF CD34⁺ cells from 1 JAK2V617F⁻ and CALR mutation⁻ (SP14), 1 JAK2V617F⁻ but CALR mutation⁺ (SP15), and 2 JAK2V617F⁺ (SP9 and SP22) patients following treatment with cytokines alone or cytokines + LCP4. As indicated by arrows, LCP4 treatment resulted in the inhibition of pMPL, and/or pJAK2, pSTAT3/5 levels to varying degrees in both JAK2V617F⁺ and CALR mutation⁺ MF CD34⁺ cells, whereas only limited inhibition of TPO/MPL and JAK-STAT activity was observed with normal BM CD34⁺ cells. (E-F) Both the percentage (E) and absolute number (F) of CD34⁺ cells that were Annexin V⁺ and PI⁻ were greater in cultures of splenic MF CD34⁺ cells treated with cytokines plus LCP4 as compared with cells treated with cytokines alone. *P < .05. Splenic MF: n = 8; N BM: n = 6. N BM, normal BM.