ferric ammonium citrate  (Valiant Co Ltd)

Journal: Nature Communications

Article Title: Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy

doi: 10.1038/s41467-021-24333-z

Figure Lengend Snippet: a – d Maternal systemic inflammation was induced in iron-adequate (gray circles) and iron-loaded (blue circles) dams on E15.5 by a single subcutaneous injection of 0.5 μg/g LPS for 24 h. Western blot (left) and quantitation (right) of a whole placenta and b embryo liver for apoptotic marker cleaved caspase-3 normalized to β-actin ( n = 3-9/group). c TUNEL stain of placental sections. Representative images of n = 3 sections/group. d Immunohistochemistry for cleaved caspase-3 (brown) and endothelial marker CD31 (red) in paraffin-embedded placenta ( n = 3), embryo lung ( n = 5), heart ( n = 5), and liver ( n = 5) sections. Scale bar= 50 μm. a – d Embryo and placentas were randomly selected for analysis. e Primary HUVECs were treated with 100 μM ferric ammonium citrate (FAC) for 24 h prior to being stimulated with IL6, IFNγ, IL1α, IL1β, or TNFα (all 50 ng/ml) in solvent or FAC-supplemented media for 16 h. Western blot (left) and quantitation (right) for cleaved caspase-3 normalized to β-actin, representative image of n = 4 independent experiments. Ferritin-H is a marker of cellular iron loading. a , b , e Error bars represent mean ± s.e.m. Statistical differences were determined by two-way ANOVA (denoted by &) or one-way ANOVA (denoted by #) followed by Holm-Sidak method for multiple comparisons. P -values are indicated in each figure panel. Source data are provided as a Source data file.

Article Snippet: For iron loading experiments, HUVECs were plated in regular media or media supplemented with 100 μM ferric ammonium citrate (FAC) (MP biomedicals #158040), or cupric chloride (Sigma #C-6917), or zinc sulfate (Fisher #Z-58) for 24 h. For iron depletion studies, HUVECs were treated with 100 µM FAC with and without 100 µM deferoxamine (Sigma D9533) for 24 h. The optimal time of culture was based on preliminary concentration and time-dependence studies.

Techniques: Injection, Western Blot, Quantitation Assay, Marker, TUNEL Assay, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy

doi: 10.1038/s41467-021-24333-z

Figure Lengend Snippet: a Iron-adequate and iron-loaded dams were treated with solvent or LPS (0.5 µg/g) on E15.5 for 6 h. Placentas from solvent and LPS-treated mice were collected for endothelial cell isolation by magnetic separation using Cd31 beads and total RNA was analyzed by RNA-Seq. b Cd31 expression relative to Rpl4 in the Cd31-unbound (non-endothelial cells) and Cd31-bound (endothelial cells) fractions. c Endothelial cell expression of iron importer Tfrc relative to Rpl4 from iron-adequate (light gray circles) and iron-loaded (dark blue circles) placentas. b , c Placenta cells isolated from n = 8 dams. d , e RNA-Seq Ingenuity Pathway Analysis of significantly downregulated (white bars) and enriched (red bars) genes ( Z -score < and > than ±1.5) from placental endothelial cells comparing hepcidin KO and WT mice after d solvent and e LPS injection. f – h Primary HUVECs were treated with solvent (gray circles), or different forms of iron (light blue circles) including ferric ammonium citrate (100 µM, FAC), hemin (20 µM), or holo-ferritin (Ft, 2 mg/ml) for 40 h and analyzed for f TFRC g NQO1 and h HMOX1 expression. n = 3 independent experiments. b , c , f – h Error bars represent mean ± s.e.m. Statistical differences were determined by two-tailed Mann–Whitney U , two-tailed Student’s t -test (denoted by *, P -values are indicated in each figure panel), or one-way ANOVA followed by Holm-Sidak method for multiple comparisons (denoted by # P < 0.05, ### P < 0.001, #### P < 0.0001). Source data are provided as a Source data file.

Article Snippet: For iron loading experiments, HUVECs were plated in regular media or media supplemented with 100 μM ferric ammonium citrate (FAC) (MP biomedicals #158040), or cupric chloride (Sigma #C-6917), or zinc sulfate (Fisher #Z-58) for 24 h. For iron depletion studies, HUVECs were treated with 100 µM FAC with and without 100 µM deferoxamine (Sigma D9533) for 24 h. The optimal time of culture was based on preliminary concentration and time-dependence studies.

Techniques: Cell Isolation, RNA Sequencing Assay, Expressing, Isolation, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy

doi: 10.1038/s41467-021-24333-z

Figure Lengend Snippet: a Primary HUVECs were treated with solvent (gray gircles), 100 μM ferric ammonium citrate (FAC) (light blue circles), or 100 μM FAC with vitamin E analog Trolox (300 μM, white circles) for 24 h prior to being stimulated with TNFα (50 ng/ml) for 16 h. Western blot (left) and quantitation (right) of cleaved caspase-3 normalized to β-actin (representative image of n = 3 independent experiments). b – i Hepcidin KO dams were treated with subcutaneous solvent (dark blue circles) or αTocopherol (αToc: vitamin E, white circles) 14 and 2 h prior to LPS treatment on E15.5 for 24 h: c embryo gross morphology and incidence of resorbing embryos; d maternal liver Il6 expression relative to Hprt from n = 6 dams; e – g placental Il1b , Cxcl2 , and Il6 expression relative to Rpl4 from n = 10 placentas. h Western blot (left) and quantitation (right) of cleaved caspase-3 in whole placentas normalized to β-actin. n = 6 placentas/group. i Immunohistochemistry for cleaved caspase-3 (brown) and CD31 (red) in paraffin-embedded placenta, embryo lung and liver (representative image of n = 3 sections/group). Scale bar = 50 μm. a , c – h Error bars represent mean ± s.e.m. e – i Embryo and placentas were randomly selected for analysis. Statistical differences were determined by two-tailed Mann–Whitney U , two-tailed Student’s t -test (denoted by *) or one-way ANOVA followed by Holm-Sidak method for multiple comparisons (denoted by #). P -values are indicated in each figure panel. Source data are provided as a Source data file.

Article Snippet: For iron loading experiments, HUVECs were plated in regular media or media supplemented with 100 μM ferric ammonium citrate (FAC) (MP biomedicals #158040), or cupric chloride (Sigma #C-6917), or zinc sulfate (Fisher #Z-58) for 24 h. For iron depletion studies, HUVECs were treated with 100 µM FAC with and without 100 µM deferoxamine (Sigma D9533) for 24 h. The optimal time of culture was based on preliminary concentration and time-dependence studies.

Techniques: Western Blot, Quantitation Assay, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY