Figure 5: AML cells exhibit an UPR in vivo.

(a) Experimental outline for intrafemoral injections. EVs from Molm-14-mGFP cells and healthy CD34⁺ cells were injected contralaterally into femurs of recipient mice. Femurs were harvested 48hrs later, MSCs and OPCs were sorted into RNA extraction buffer for gene expression analysis. (b) Expression analysis of UPR genes from MSCs and OPCs from Molm-14-mGFP EV injected femurs. Fold change was determined by 2^-ΔΔCt against respective cells from CD34⁺ EV injected femurs. Error bars are standard error of the mean from three animals per condition. (c) Expression analysis of UPR genes from MSCs and OPCs from CD34⁺-derived EV injected femurs. Fold change was determined by 2^-ΔΔCt against respective cells from vehicle injected femurs. Error bars are standard error of the mean from three animals per condition. (d) Expression analysis of UPR genes from in vitro cultured MSCs and OPCs exposed to Molm-14-mGFP EVs. Fold change determined by 2^-ΔΔCt against vehicle-treated cells. Error bars are standard error of the mean from three separate experiments. Significance in (b) and (c) was determined by Student’s t-test, *P<0.05, **P<0.01. (e) Timecourse of UPR gene expression in explanted Molm-14-mGFP. Molm-14-mGFP cells were sorted out of xenograft bone marrow based on human CD45 expression and cultured ex vivo. RNA was extracted from cells directly from the sort (0hr) or 48 and 72hrs in normal culture media in vitro. Fold change determined by 2^-ΔΔCt against in vitro cultured Molm-14-mGFP cells. Error bars are standard error of the mean from three separate experiments. (f) UPR status was determined by protein levels of GRP78, and phosphorylated eIF2α in AML-patient samples and healthy pediatric and adult controls. (g) Expression analysis of UPR genes from blasts from AML patient samples. Fold change determined by 2^-ΔΔCt in pairwise analysis against control samples. Error bars are standard error of the mean.