Journal: Scientific Reports

Article Title: Development of an iron overload HepG2 cell model using ferrous ammonium citrate

doi: 10.1038/s41598-023-49072-7

Figure Lengend Snippet: Effect of iron chelation in iron overload HepG2 cells. Labile iron ( A ) and total iron ( B ) were quantified using the u- ferene assay. ( C ) ROS generation was measured using a DCFDA/DCF ROS kit in at least 10,000 cells via flow cytometry. ( D ) Relative expression of TfR1, normalized to control cells, were measured using western blot and quantified using LI-COR’s Odyssey Software Application. HepG2 cells were loaded with 50 μM following the established protocol. Error bars show standard deviations for N = 3 independent replicates. One-way ANOVA with Dunnett’s correction method were performed to compare iron overloaded cells with the either control or chelator treated cells. Statistical analyses were performed using GraphPad Prism. ****Represents p < 0.0001.

Article Snippet: The membrane was blocked in 10% skim milk and incubated with both a monoclonal anti-transferrin receptor 1 (TfR1) antibody (H68.4—Thermofischer, 100 kDa), asialoglycoprotein receptor (ASGPR, 42 kDa) and GAPDH (Rabbit mAb, 37 kDa, Cell Signalling Technology,) at 3 μg/mL overnight.

Techniques: Flow Cytometry, Expressing, Control, Western Blot, Software

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: Panel A: Lysates from Hep3B cells stably expressing TfR2 were collected for Western blotting after 4 hours of 50 µM ferristatin II treatment. Ratios of band density for TfR1/Actin or TfR2/Actin are normalized to vehicle control (DMSO) in the absence of ferristatin II. The bar graph summarizes data from four separate experiments (n = 11 TfR1/Actin p<0.001, determined by two-tailed Student’s t test, n = 12 TfR2/Actin). Panel B: HeLa cells were transfected to express HFE-flag as described in . Cells were then incubated 4 h with or without 50 µM ferristatin II. Tubulin is shown as a loading control and the indicated values were normalized to control lanes in the absence of ferristatin II. The bar graph represents multiple transfections (n = 8, p<0.001 for TfR1/Actin determined by two-tailed Student’s t test).

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Two Tailed Test, Transfection, Incubation

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: Panel A: HeLa cells were treated overnight with 10 nM bafilomycin A 1 prior to 4 h treatment with or without 50 µM ferristatin II in the presence of 10 nM bafilomycin A 1 . Blot is representative of several experiments. Panel B: HeLa cells were pre-treated for 30 minutes with 25 µg/mL nystatin or left untreated. After incubation, cells were treated with 50 µM ferristatin II for 4 hours. Shown below are the density ratios for TfR1/Actin normalized to control lanes (DMSO treated) in the absence of ferristatin II. Shown is a representative blot from several similar experiments.

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Incubation, Control

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: Panel A: TRVb cells stably transfected to express WT TfR1, Y20C/F23A TfR1, or Δ3–28 TfR1 were treated for 4 hours with 50 µM ferristatin II. Density ratios for TfR1/Actin normalized to control lanes (DMSO treated) in the absence of ferristatin II are indicated for each lane. Nonconsecutive lanes are separated by a white space. Individual blots are separated by a black bar. All blots were probed using a sheep-TfR1 antibody raised against the ectodomain of TfR1. Panel B: Time course of WT TfR1, Y20C/F23A TfR1 and Δ3–28 TfR1 degradation after 0–4 hour incubation with 50 µM ferristatin II. Shown are mean TfR1/Actin ratios ± SEM from 3 separate experiments performed in duplicate.

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Stable Transfection, Transfection, Control, Incubation

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: HeLa cells were treated for 4 hours with 50 µM ferristatin II with or without 1 mg/mL Tf. Shown below are the density ratios for TfR1/Actin normalized to control lanes (DMSO treated) in the absence of ferristatin II. Shown is a representative blot with similar results observed on several separate occasions.

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Control

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: Panel A: TRVb cells were transfected with 1 µg WT TfR1 or G647A TfR1, plated on poly-L-lysine coated cover slips and fixed with 4% paraformaldehyde. Cells were immunoreacted with OKT9 (α-TfR1) followed by goat anti-mouse Alexa 488 and imaged using a Zeiss Observer Z1 Axioscope microscope. Panel B: TRVb cells were transfected as in (A) in 6-well plates and incubated for 48 hours (see Experimental Procedures for details). Cells were then chilled, washed and incubated with 500 nM 125 I-Tf in the absence or presence of 5 µM unlabeled Tf for 2 hours. After washing and lysis, cell associated radioactivity was measured by γ-counting. Absolute deviation for duplicate values for cells with 500 nM 125 I-Tf (open bars) or 500 nM 125 I-Tf +5 µM Tf (closed bars) is shown. Inset: Western blot confirms equivalent expression levels for WT and G647A TfR1. Panel C: TRVb cells, transfected with WT or G647A TfR1 were treated for 4 hours with 50 µM ferristatin II with or without 1 mg/mL Tf. Shown below are the density ratios for TfR1/Actin normalized to control lanes (DMSO treated) in the absence of ferristatin II. Nonconsecutive lanes are separated by a white space. Blot is representative of several experiments (n = 6, WT and G647A TfR1).

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Transfection, Microscopy, Incubation, Lysis, Radioactivity, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: Panel A: Liver lysates from rats injected with saline or 40 mg/kg ferristatin ll for 4 days were immunoblotted to determine TfR1 levels. Actin was used as a loading control. Bar graph shows normalized TfR1/actin ratio as the mean ± SEM (n = 7; *P<0.001 determined by two-tailed Student’s t test). Panel B: Liver RNA was isolated, reverse transcribed and qPCR performed as described under Experimental Procedures. Data were normalized to levels of 36B4. Shown are means ± SEM (n = 5–7; *P<0.001 determined by two-tailed Student’s t test). Panel C: To determine intestinal iron absorption, control and treated rats were fasted for 4 h and 59 Fe was administered by gavage. Blood samples were drawn at indicated times and radioactivity was determined by gamma counting. Shown are means ± SEM for control (open circles; n = 4–6) and ferristatin ll (closed circles; n = 7–8; *P = 0.01 and **P = 0.05 determined by two-tailed Student’s t test).

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques: Injection, Saline, Control, Two Tailed Test, Isolation, Reverse Transcription, Radioactivity

Journal: PLoS ONE

Article Title: Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

doi: 10.1371/journal.pone.0070199

Figure Lengend Snippet: The contributors to iron metabolism include TfR1, TfR2, Tf and HFE. These molecules work together to maintain systemic iron homeostasis by sensing high and low iron levels. See text for details.

Article Snippet: After electrophoresis, samples were transferred to nitrocellulose or PVDF membrane, blocked with 5% non-fat milk and immunoblotted using monoclonal mouse anti-TfR1 antibody H68.4 (1∶500 or 1∶1000, Invitrogen), sheep anti-TfR1 antibody (1∶5000, ) monoclonal mouse anti-TfR2 antibody 9F81C11 (1∶1000, Santa Cruz Biotechnology) or mouse anti-Flag (1∶1000, Sigma).

Techniques:

Journal: Nature Communications

Article Title: Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

doi: 10.1038/ncomms15835

Figure Lengend Snippet: ( a , b ) IFM on MNT-1 cells ( a ) and Normal Mouse Melanocytes (NMM) ( b ) stained for endogenous RAB6, TYRP1 and TGN46 ( a ) or Giantin ( b ), showing RAB6 distribution to the Golgi area (arrowheads) and in the vicinity of TYRP1- and melanin-positive melanosomes (white and red arrows respectively, 5X area). ( c ) Quantification of the number of endogenous RAB6 fluorescent structures associated with melanosomes in MNT-1, NMM and NHEM. ( d ) Live imaging frames of MNT-1 (n) cells (imaging plane Z-1) expressing GFP-RAB6 and mCh-VAMP7 (Pearson’s correlation coefficient; n =66, four independent experiments). ( e ) Live imaging frames of MNT-1 cells co-expressing GFP-TYRP1 and mCh-RAB6 with corresponding intensity profile of RAB6 across melanosomes measured along white line (right panel). ( f ) Representative electron micrograph of ultrathin cryosection of GFP-RAB6 expressing MNT-1 cells showed RAB6 association (anti-GFP, × 2 area) to Golgi Apparatus (GA, white arrows), vesicles (arrowheads) and limiting membranes of melanosomes (black arrows) positive for TYRP1. ( g ) Quantification of immunogold labelling for GFP-RAB6 relative to indicated cell compartments (230 gold particles; n =12 cells, 2 independent experiments). Data are normalized to the total number of gold particles and presented as means±s.e.m. ( h ) Immunogold labelling of RAB6 (PAG-10 nm) or GFP (arrows) of melanosome-enriched fractions from NHEM (left) or MNT-1 expressing GFP or GFP-RAB6 (PAG-15 nm, right). ( i ) Distribution of anti-GFP gold particles over stage-III/-IV purified melanosomes from MNT-1 expressing GFP-RAB6 presented as mean±s.e.m. ( n =209 melanosomes, with at least 1 gold particle, quantified from 2 independent experiments). ( j ) Western Blot (WB) of PNS (1%, Post-nuclear supernatant) and melanosomes-enriched fraction (25%, Mel) isolated from NHEM or MNT-1 cells expressing or not GFP-RAB6 and probed with RAB6 or GFP (*), TYRP1, TYRP2, MART-1, GM130 or GAPDH antibodies. Blots are representative of 3 independent experiments. Scale bars: 10 μm ( a , b and d , e ), 500 nm ( f – h ), M.W.in kDa, two-tailed, Unpaired t test, **** P <0.0001.

Article Snippet: Rabbit anti-GM130 (1:10,000; Abcam, ab52649), -TYRP1 (1:1,000; Santa Cruz, sc-25543), -GAPDH (1:10,000; Sigma, G9545), -β-tubulin (1:1,000; Abcam, ab6046), -RAB6 (1:1,000 Santa Cruz, sc-310), -MART-1 (1:200; Abcam, ab51061); Mouse anti-Actin (1:1000; Sigma, A5316), -MART-1 (1:200; Thermo fisher scientific, M2-7C10); -Transferrin receptor (1:1,000; Thermo fisher scientific, H68-4), -Tyrosinase (1:200; Santa Cruz, sc-20035), -TYRP2 (1:200; Santa Cruz—sc-74439); Rat anti-GFP (1:1,000; Chromoteck, 3H9); Goat anti-mouse or –rabbit coupled to horseradish peroxidase (HRP) (1:10,000; Abcam, ab6721).

Techniques: Staining, Imaging, Expressing, Purification, Western Blot, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

doi: 10.1038/ncomms15835

Figure Lengend Snippet: ( a ) WB of control or RAB6-depleted MNT-1 cell lysates probed for RAB6, TYRP2, MART-1 or Tubulin. ( b ) Quantification of the relative expression levels of MART-1 or TYRP2 in control or RAB6 siRNAs-treated cells. Data are normalized to control and presented as mean±s.e.m. ( n =7, TYRP2; n =3, MART-1). ( c ) WB of PNS (1%) and melanosomes-enriched fraction (5%, Mel) isolated from control- or RAB6-depleted MNT-1 cells and probed for RAB6 (*) and GAPDH (left panel) or TYRP1, TYRP2 and MART-1 (right panel). Note the increase in TYRP2 and MART-1 levels in PNS (black asterisk) and corresponding decrease in Mel fractions (white asterisk). ( d ) Quantification of the expression level of MART-1 and TYRP2 in melanosomes-enriched fractions of RAB6-depleted melanocytes relative to controls. Data are normalized presented as mean±s.e.m. of three independent experiments. ( e , f ) Immunogold labelling on ultrathin cryosections of GFP-RAB6 (d) or GFP-TYRP2 ( e ) expressing MNT-1 cells co-stained for GFP (PAG-15 nm) and MART-1 (PAG-10 nm) (arrows). Black arrows point co-distribution of MART-1 and TYRP2 on similar vesicles as in ( d ), and white arrows on Golgi apparatus (GA). ( g ) IFM on control- or RAB6-depleted NHEM stained for endogenous MART-1 and TGN46. ( h ) Quantification of the MART-1 fluorescent intensity enrichment in the Golgi area in control- or RAB6-depleted NHEM or MNT-1 cells (n) presented as mean±s.e.m., (NHEM, n =51–63, MNT-1, n =22–24, per respective condition, three independent experiments). ( i ) Working model illustrating the possible role of RAB6 and ELKS in the Golgi to melanosomes pathway. Note that other alternative routes cannot be excluded. Scale bars: 500 nm ( d , e ), 20 μm ( f ). M.W. in kDa, two-tailed, Unpaired t test ( b , c , g ). * P <0.05; ** P <0.01; **** P <0.0001.

Article Snippet: Rabbit anti-GM130 (1:10,000; Abcam, ab52649), -TYRP1 (1:1,000; Santa Cruz, sc-25543), -GAPDH (1:10,000; Sigma, G9545), -β-tubulin (1:1,000; Abcam, ab6046), -RAB6 (1:1,000 Santa Cruz, sc-310), -MART-1 (1:200; Abcam, ab51061); Mouse anti-Actin (1:1000; Sigma, A5316), -MART-1 (1:200; Thermo fisher scientific, M2-7C10); -Transferrin receptor (1:1,000; Thermo fisher scientific, H68-4), -Tyrosinase (1:200; Santa Cruz, sc-20035), -TYRP2 (1:200; Santa Cruz—sc-74439); Rat anti-GFP (1:1,000; Chromoteck, 3H9); Goat anti-mouse or –rabbit coupled to horseradish peroxidase (HRP) (1:10,000; Abcam, ab6721).

Techniques: Control, Expressing, Isolation, Staining, Two Tailed Test