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Blood, Vol. 111, Issue 2, 924-931, January 15, 2008
Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis
Blood Silvestri et al.
111: 924
Supplemental Materials for: Silvestri et al
Files in this Data Supplement:
- Figure S1. The polyclonal anti-HJV antibody recognizes the native protein (PDF, 120 KB) -
HeLa cells were transfected with 1,6 µg of plasmid DNA and 4 µl of the liposomal transfection reagent Lipofectamine 2000, according to the manufacturer’s instructions. After 12 hours, the medium was replaced and, 24 hours later, cells were processed for immunfluorescence analysis. Rabbit anti-HJV was used 1:1000. Cells were visualized at a 63× magnification under confocal microscope BioRad Leica TCS SP2 AOBS (Leica Microsystems, Bannockburn, IL).
Mock- (A, A′, C, C′) and HJV-transfected cells (B, B′, D, D′) were analyzed for HJV expression under unpermeabilized (A, A′, B, B′) or permeabilized conditions (C, C′, D, D′). Nuclei were stained with DAPI (A′, B′, C′, D′). Scale bars: 10 µm.
- Figure S2. Apoptosis analysis after DFO treatment (PDF, 38 KB) -
Transfected HeLa cells were incubated with 300 µM DFO for 4, 8 and 24 hours and total cell lysates were analyzed by western blot for the presence of the apoptotic PARP fragment of 85 kDa using the anti-PARP1 antibody (Santa Cruz, CA). The uncleaved form of 116 kDa was used as an internal control. Relative molecular mass, in kilodaltons, is indicated on the left.
- Figure S3. H Ferritin (FTH) dosage after DFO, FAC and CoCl2 treatments (PDF, 47 KB) -
Transfected HeLa cells were treated with 300 µDFO for 8 hours and with 100 µM FAC or 500 µM CoCl2 for 24 hours. Total cellular extracts were analyzed for H ferritin (FTH) content by using an ELISA assay based on monoclonal antibodies specific for the human FTH, calibrated on the corresponding homopolymers. Proteins were quantified using Bio-Rad Protein Assay. Total FTH amount was expressed as ng/mg proteins.
- Figure S4. Hypoxia and iron modulation regulate s-HJV release in HepG2 cells (PDF, 72 KB) -
Transiently transfected HepG2 cells were incubated with 300 µM DFO (24 hours), 250 µM CoCl2 (24 hours) or 100 µM FAC (24 hours). Soluble and cell associated HJV were analyzed by western blot using the anti-HJV.  -tubulin was used as a loading control.
- Figure S5. Analyses of HIF-1α stabilization and its nuclear translocation in CoCl2 treated HeLa cells (PDF, 92 KB) -
Upper panel. To induce chemical hypoxia, HeLa cells were incubated with different CoCl2 concentrations (10-500µM) in serum-free media. After 18 hours, nuclei were isolated according to standard procedures, and analyzed for HIF-1 stabilization by Western Blot. The lower CoCl2 concentration inducing HIF-1 translocation to the nucleus was used for s-HJV analysis. *: unspecific band was used as an internal loading control. Lower panel. Untreated (A, A′) or treated (500 µM CoCl2, B and B′) Hela cells were fixed and Triton X-100 permeabilized for immunofluorescence analysis on HIF-1 stabilization, using the anti- HIF-1 1:50. Nuclei were stained with DAPI (A′, B′). Scale bar refers to 10 µm.
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