Fig. 6: E-selectin adhesion activates AML pro-survival NF-κB signaling in vitro whereas E-selectin blockade in vivo dampens AKT/NF-κB signaling. | Nature Communications

Fig. 6: E-selectin adhesion activates AML pro-survival NF-κB signaling in vitro whereas E-selectin blockade in vivo dampens AKT/NF-κB signaling.

From: Endothelial E-selectin inhibition improves acute myeloid leukaemia therapy by disrupting vascular niche-mediated chemoresistance

Fig. 6

All analyses one-way ANOVA multiple comparison Bonferroni correction. All plots mean ± SD. ac In vitro studies. a NF-κB GFP reporter Raw264.7 cell line cultured in wells pre-coated with mouse adhesion molecules ± TNF-α as indicated (concentrations in ng mL−1). Intracellular activation of NF-κB-mediated transcription after 3 h adhesion measured by flow cytometry as percentage of viable cells. Pooled data from three independent experiments (n = 3,3,5 wells/group/experiment). b, c E-selectin adhesion-mediated chemoresistance is abrogated by small synthetic PI3K (LY294002) or NF-κB (BMS-345541) inhibitors. AML blasts were seeded into pre-coated wells for 3 days ± cytarabine. Data show percentage of surviving AML cells. b Murine AML blasts cultured with 25 ng mL−1 cytarabine ± 2.5 µM LY294002 (n = 5 wells/condition; 3 independent experiments). c Human CD34+ AML cell line KG1a cultured in the presence of 10 μg mL−1 cytarabine ± 10 µM BMS-345541 (n = 5 wells/condition). d p-AKTSer473 phosphorylation in MLL-AF9 AML blasts generated from wildtype or Fut4/Fut7/ double gene-deleted BM cells, were seeded in pre-coated wells (25 min 37 °C) then lysed for quantitative immunoblotting (Li-Cor). Cell lysate from 30,000 adherent AML blasts from WT (upper blot) and from Fut4/Fut7−/− AML KIT+ GFP+ blasts (lower blot) loaded per lane. Histogram shows fold increase in p-AKTSer473 after total loaded protein normalization. Data pooled from two independent experiments, n = 2 wells/experiment. eg In vivo studies. e Outline of experimental plan and intracellular signaling pathways investigated. f Quantitative immunoblotting of BM lysates with indicated antibodies using linear Li-COR detection. Lysates were from wildtype leukemia mice administered GMI-1271 (24 h 200 mg kg−1, BiD) or vehicle control. Each lane contains 15 µg BM lysate protein from an individual mouse (n = 6 mice/group). g Quantitative band intensity analyses by Li-COR from blots in (6f, n = 6/group). Includes additional data from same experiment with AML transplanted into (n = 4) Sele−/− hosts loaded on parallel blot (Supplementary Fig. 6). Each dot represents data from a single mouse expressed as fold-change over average for saline vehicle injected controls. Source data (including full original scans of all blots) are provided as a Source Data file.

Back to article page