Fig. 3: Ether lipids facilitate CD44-mediated iron endocytosis.

a Endocytic transport of fluorescently labeled transferrin as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT, AGPS KO, and AGPS addback cells. b Endocytic transport of fluorescently labeled hyaluronate probe as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT, AGPS KO, and AGPS addback cells. c Endocytic transport of fluorescently labeled transferrin as assessed by quantitative colocalization with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells +/− pretreatment with TGF-β. d Endocytic transport of fluorescently labeled hyaluronate probe as assessed by quantitative colocalization with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells +/− pretreatment with TGF-β. e Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron following treatment with either hyaluronic acid or hyaluronidase in PyMT-1099 WT or CD44 KO cells +/− pretreatment with TGF-β (n = 4 independent experiments). Box plots: interquartile range, center lines = medians and whiskers = the minimum and maximum values. f Endocytic transport of dextran as assessed by quantitative colocalization with the early endosomal marker EEA1 in pB3 WT, AGPS KO, and AGPS addback cells. g Endocytic transport of dextran as assessed by quantitative colocalization with the early endosomal marker EEA1 in PyMT-1099 WT or AGPS KO cells +/− pretreatment with TGF-β. h Endocytosis of EGFR as assessed by quantitative colocalization of internalized fluorescently labeled EGF with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells +/− pretreatment with TGF-β. Cells were treated with 2 ng/mL Alexa 555-conjugated EGF. i Endocytic transport of hyaluronate probe assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT or AGPS KO cells +/− pretreatment (16-18 hr) with polyunsaturated fatty acid (PUFA) BSA conjugate C(22:6). j Endocytic transport of hyaluronate probe assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT or AGPS KO +/− pretreatment (16-18 hr) with liposomes composed of the following: PE (18:0_20:4), PE (18:1p_20:4), and PC (18:1p_20:4). All data shown as mean +/− SEM and statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test; ns, not significant. For all endocytosis assays n = 10 fields were examined for each timepoint, and data are representative of two independent experiments with similar results. In some cases, error bars are smaller than the symbol size and not visible. Panels a, b, f: pB3 WT and AGPS KO cells were transduced with the respective vector control plasmids.