Journal: Nature communications

Article Title: Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

doi: 10.1038/s41467-021-21938-2

Figure Lengend Snippet: Fig. 3 FTH1 as a microtubule-stabilizing factor controlled by iron and isocitrate. a Levels of major microtubule-associated proteins (MAPs) in erythroid cells cultured from human CD34+ progenitors, from proteomic data of Gautier18. Graph: mean protein copy number per cell for five most abundant MAPs at indicated stages of differentiation. Red: Stathmin 1 (STMN1); green: ferritin heavy chain (FTH1); blue: microtubule-associated protein RP/EB family member 1 (MAPRE1); grey: microtubule-associated protein RP/EB family member 2 (MAPRE2); black: regulator of microtubule dynamics protein 1 (RMDN1); purple: microtubule-associated protein 4 (MAP4). Stages: progenitor 1 (Prog1), progenitor 2 (Prog2), proerythroblast (ProE), basophilic erythroblast 1 (Baso1), basophilic erythroblast 2 (Baso2), polychromatophilic erythroblast (Poly), and orthchromatic erythroblast (Ortho). Error bars, SEM; n = 3 biologically independent replicates for Prog1 and Prog2, and four biologically independent replicates for all other stages. b FTH1 levels by immunoblot on whole cell lysates from human CD34+ progenitors cultured 1–3 days in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium ± isocitrate (IC). Red arrow: lysosomal proteolytic fragment. Graph: mean normalized FTH1 signal (day 2), relative to value with 100% TSAT. Error bars, SEM; n = 3 independent experiments; ***, ****P = 0.0008, 0.0003, one-way ANOVA with Tukey post hoc. c FTH1 levels by immunoblot on human CD34+ progenitors cultured 1–3 days in iron-replete or deficient erythroid medium ± protease inhibitor (5 µM CA074-me). Red arrow: proteolytic fragment. Graph: mean normalized FTH1 signal (day 2), relative to value with 100% TSAT. Error bars, SEM; n = 3 independent experiments; *, **, ***P = 0.028, 0.013, 0.0006, one-way ANOVA with Tukey post hoc. d FTH1 knockdown with lentiviral shRNA. Immunoblot of transduced human CD34+ progenitors cultured 3 days in iron-replete erythroid medium. e Microtubule alterations demonstrated by immunofluorescence on cells as in d. Red: β-tubulin; blue: DAPI. Graphs: microtubule (MT) density, reflected as averaged microtubule signal per cell in arbitrary units. Numbers indicate mean ± SEM; n = 4 independent experiments for assessment of FTH1#1 and three independent experiments for assessment of FTH1#2; *, ***P = 0.029, 0.0014, unpaired two-sided Student’s t test. Note: most experiments separately assessed FTH1#1 and FTH1#2. See also Supplementary Figs. 5–7 and Supplementary Data Table 1. Source data are provided as a Source data file.

Article Snippet: The primary antibody for FTH1 consisted of a rabbit monoclonal IgG (Cell Signaling Technology, 4393) used at 1/1000 in TBST with 2.5% BSA at 4 °C for 16 h; rabbit polyclonal anti-FTL (Abcam, ab69090) was used under the same conditions.

Techniques: Cell Culture, Western Blot, Protease Inhibitor, Knockdown, shRNA

Journal: Nature communications

Article Title: Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

doi: 10.1038/s41467-021-21938-2

Figure Lengend Snippet: Fig. 4 Implication of FTH1 in the erythroid iron restriction response. a, b Influence of FTH1 levels on viability and proliferation. Graphs: mean % viable cells and fold increases in cell number for human CD34+ progenitors transduced with lentiviral shRNA vectors targeting either green fluorescent protein (GFP) or ferritin heavy chain (FTH1), and cultured 4 days in iron-replete erythroid medium. Error bars, SEM; n = 3 biologically independent experiments; *, **P = 0.011, 0.009, one-way ANOVA with Tukey post hoc. NS not significant. GPA glycophorin A. c Influence of FTH1 levels on erythroid differentiation as determined by flow cytometry on cells treated as in a. d Mean fold change in GPA+ cells associated with lentiviral transductions and 4 days culture in iron- replete erythroid medium. Error bars, SEM; n = 3 independent experiments; *, **P = 0.019, 0.013, unpaired two-sided Student’s t test. Note: most experiments separately assessed FTH1#1 and FTH1#2. e Distinct consequences of ferritin enforcement in iron-replete versus iron-restricted progenitors. Flow cytometry analysis of CD34+ progenitors transduced with lentiviral (LV) expression vectors for ferritin heavy (FTH1) and light (FTL) chains followed by 4 days culture in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium. f Mean fold change in GPA+ cells associated with ferritin enforcement in cells cultured as in e. Error bars, SEM; n = 3 independent experiments; *P = 0.02, unpaired two-sided Student’s t test. g Ferritin levels in cells transduced and cultured as in e and f, as assessed by immunoblot. LV-OE lentiviral overexpression; Vec vector. Representative results from three independent experiments. See also Supplementary Fig. 8. Source data are provided as a Source data file.

Article Snippet: The primary antibody for FTH1 consisted of a rabbit monoclonal IgG (Cell Signaling Technology, 4393) used at 1/1000 in TBST with 2.5% BSA at 4 °C for 16 h; rabbit polyclonal anti-FTL (Abcam, ab69090) was used under the same conditions.

Techniques: Transduction, shRNA, Cell Culture, Cytometry, Flow Cytometry, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Nature communications

Article Title: Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

doi: 10.1038/s41467-021-21938-2

Figure Lengend Snippet: Fig. 5 Cooperative restoration of FTH1 by isocitrate and fumarate. a FTH1 levels by immunoblot on whole cell lysates from human CD34+ progenitors cultured 3 days in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium ± 1–5 mM isocitrate (IC) and ±1 mM fumarate (Fum or F). Colored boxes highlight synergy of 3 mM IC with 1 mM Fum. Red: iron restriction with no or single metabolite treatment; green: iron restriction with combined metabolite treatment. Representative results from three independent experiments. b Graph: mean normalized FTH1 signal (±3 mM IC and ±1 mM Fum), relative to value with 100% TSAT. Error bars, SEM; n = 3 biologically independent experiments; *, **, ***P = 0.02, 0.009, 0.0009, one-way ANOVA with Tukey post hoc. NS not significant. See also Supplementary Fig. 9. Source data are provided as a Source data file.

Article Snippet: The primary antibody for FTH1 consisted of a rabbit monoclonal IgG (Cell Signaling Technology, 4393) used at 1/1000 in TBST with 2.5% BSA at 4 °C for 16 h; rabbit polyclonal anti-FTL (Abcam, ab69090) was used under the same conditions.

Techniques: Western Blot, Cell Culture