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: Nature Communications

Article Title: Hypoxia-tropic delivery of nanozymes targeting transferrin receptor 1 for nasopharyngeal carcinoma radiotherapy sensitization

doi: 10.1038/s41467-025-56134-z

Figure Lengend Snippet: a Schematic representation of CSPs upregulated in hypoxic NPC identified through DGE analysis among a total of 1442 CSPs. b Left: Venn diagram illustrating the intersection of CSPs upregulated in tumor samples of the three cohorts: GSE12452 (log 2 (fold change) >1 and P < 0.01), GSE53819 (log 2 (fold change) >1 and P < 0.01), and GSE61218 (log 2 (fold change) >1 and P < 0.01). Right: Venn diagram illustrating the intersection of CSPs that are upregulated in tumor samples with high hypoxia scores of the GSE102349 cohort (log 2 (fold change) >10 and P < 0.01). c Distribution of TfR1 mRNA abundance in tumor and normal samples from different cohorts: GSE12452 (normal, n = 10; tumor, n = 31), GSE53819 (normal, n = 18; tumor, n = 18), and GSE61218 (normal, n = 6; tumor, n = 10). d Distribution of TFRC mRNA abundance in tumor samples from the GSE102349 cohort ( n = 113) with high or low hypoxia scores. e Representative images of IHC staining showing TfR1 protein expression in clinical primary NPC tissues and NNE tissues collected from multiple patients. Scale bar, 50 μm. f Distribution of TfR1 IHC staining scores in NPC ( n = 174) and NNE ( n = 98) tissues. g Distribution of TfR1 IHC staining scores in NPC and matched adjacent non-cancerous tissues ( n = 117). h IF staining analysis in NPC tissues depicting the co-expression of HIF-1α and TfR1. Scale bar, 50 μm. i Spearman correlation between HIF-1α expression and TfR1 expression based on h ( n = 40 random regions from different tissues). The box plots in c , d , and f show the median ±1 quartile, with whiskers extending to the minimum or maximum values within 1.5 times the interquartile range from the box boundaries. Mann–Whitney test in c , d , and f and Wilcoxon matched-pairs signed rank test in g were used to calculate P values. Source data are provided as a Source Data file.

Article Snippet: Subsequently, 300 µL of 5% skimmed milk was added to each well and incubated at 37 °C for 2 h. After further washing, each well was incubated with 100 µL of TfR1 solution (SinoBiological, catalog no. 11020-H07H, 2 µg/mL) at 37 °C for 2 h. After another round of washing, each well was incubated with 100 µL of anti-TfR1 antibody (SinoBiological, catalog no. 11020-MM02, 1 µg/mL) at 37 °C for 2 h. Following additional washes, 100 µL of HRP-conjugated anti-mouse IgG (GE Healthcare, catalog no. NA931, 1:5000) was added and incubated at 37 °C for 1 h. Finally, 100 µL of one-component TMB chromogenic substrate (InnoReagents, catalog no. TMB-S-004) was added to each well.

Techniques: Immunohistochemistry, Expressing, Staining, MANN-WHITNEY

Journal: PLoS ONE

Article Title: SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes

doi: 10.1371/journal.pone.0199829

Figure Lengend Snippet: (A) IMS32 cells transfected with control or SIMPLE siRNA were treated with sulfo-NHS-SS-biotin at 4°C, after which the cells were incubated at 37°C for the indicated amount of time. Cells were then treated with reducing buffer containing GSH to remove remaining biotin from the plasma membrane, and cell lysates were precipitated with neutravidin-agarose beads. Precipitates were analyzed using Western blot with an anti-TfnR mAb (biotinylated TfnR), and cell lysates used for precipitation (10% input) were probed with anti-TfnR mAb (TfnR), anti-β-actin mAb and anti-SIMPLE pAb. (B) After cell-surface biotinylation using sulfo-NHS-SS-biotin, IMS32 cells transfected with control or SIMPLE siRNA were incubated for 15 min. Remaining surface biotin was stripped using reducing buffer, and cells were incubated for the indicated periods of time. Biotinylated proteins that recycled back to the cell surface were again treated with reducing buffer, and cell lysates were precipitated with neutravidin-agarose beads. Precipitates were analyzed by Western blot with an anti-TfnR antibody, and cell lysates (10% input) were probed with the indicated antibodies. (C) The amount of biotinylated TfnR in (B) was quantified using Image J software. The results shown are means ± S.D. of the ratio between biotinylated TfnR at each time point and biotinylated TfnR at time zero from three independent experiments. Values at time zero are set to 100%. P-values (control cells vs. SIMPLE knockdown cells at 10, 20, 30 min) are determined using a t-test. *P<0.05, **P<0.001.

Article Snippet: The antibodies used in this study were as follows: anti-Rab11 mAb (47, 1:500 dilution; BD Biosciences, Franklin Lakes, NJ, USA), anti-Syntaxin-6 mAb (1:500 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-EEA1 pAb (1:500 dilution; Cell Signaling Technology, Inc.), anti-TSG101 mAb (4A10, 1:500 dilution; Genetex, San Antonio, TX, USA), anti-NEDD4 pAb (1:500 dilution; Cell Signaling Technology, Inc.), anti-TfnR mAb (H68.4, 1:1000 dilution; Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Control, Incubation, Clinical Proteomics, Membrane, Western Blot, Software, Knockdown

Journal: PLoS ONE

Article Title: SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes

doi: 10.1371/journal.pone.0199829

Figure Lengend Snippet: (A) Co-localization of immunofluorescence within Hela cells imaged using confocal microscopy. Left-side panels show endogenous SIMPLE (Green). Middle panels show PI4P in the same cells labeled with specific antibodies (Red). Merged images are shown on the right panels with DAPI-stained nuclei (blue). Scale bar, 20 μm. (B) Subcellular fractionation of Hela cell lysates separated into 12 fractions through discontinuous sucrose density-gradient centrifugation. Equal volumes of each fraction were subjected to SDS-PAGE followed by immunoblot analysis. Previous studies suggested fraction 5 (Red) is enriched in PI4P . (C) Hela cells were transfected with control or SIMPLE siRNA and cultured for 48 h. After serum starvation, Alexa-594-Tfn treatment and incubation for the indicated periods of time, cell lysates were prepared and subjected to SDS-PAGE (30 μg of total protein/lane). Immunoblots were probed with anti-SIMPLE mAb or anti-β-actin mAb. (D) Hela cells transfected with control or SIMPLE siRNA were serum starved for 3 h and then incubated with Alexa-594-Tfn at 37°C for 1 h. After washing out unbound Alexa-594-Tfn, cells were incubated at 37°C for the indicated amount of time and processed for immunostaining with anti-TfnR mAb. Fluorescent images of Alexa-594-Tfn (red) and TfnR (green) are shown along with merged images. Scale bar, 20 μm. (E) The intensity of Alexa-594-Tfn signal in Hela cells was expressed as signal intensity per unit area. At each time point, the signal intensity of at least 20 cells was measured in three independent experiments. The results shown are means ± S.D. of Alexa-594-Tfn signal at each time normalized to signal at time zero, which are assigned a value of 100% (n = 3). P-values (control cells vs. SIMPLE knockdown cells at 10, 20, 30 min) are determined using a t-test. *P<0.05.

Article Snippet: The antibodies used in this study were as follows: anti-Rab11 mAb (47, 1:500 dilution; BD Biosciences, Franklin Lakes, NJ, USA), anti-Syntaxin-6 mAb (1:500 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-EEA1 pAb (1:500 dilution; Cell Signaling Technology, Inc.), anti-TSG101 mAb (4A10, 1:500 dilution; Genetex, San Antonio, TX, USA), anti-NEDD4 pAb (1:500 dilution; Cell Signaling Technology, Inc.), anti-TfnR mAb (H68.4, 1:1000 dilution; Thermo Fisher Scientific, Inc.).

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Staining, Fractionation, Gradient Centrifugation, SDS Page, Western Blot, Transfection, Control, Cell Culture, Incubation, Immunostaining, Knockdown

Journal: Molecular Biology of the Cell

Article Title: The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization

doi: 10.1091/mbc.E20-04-0225

Figure Lengend Snippet: Endosomal localization of the N-terminus–swapped ATP10 chimeras. (A–C) HeLa cells transiently coexpressing C-terminally HA-tagged ATP10B, B-ATP10A, or B-ATP10D, and N-terminally FLAG-tagged CDC50A, were fixed and stained for HA and EEA1 (a marker for early endosome), TfnR (transferrin receptor, a marker for early/recycling endosomes), or Lamp-1 (a marker for late endosomes), followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488–conjugated anti-mouse secondary antibodies. Images were taken by confocal microscopy. Scale bars, 20 μm. Scale bars in enlarged images, 5 μm.

Article Snippet: Sources of antibodies used in this study were as follows: monoclonal rabbit anti-ATP1A1 (EP1845Y; Abcam); monoclonal mouse anti-EEA1 and anti-Lamp-1 (BD Biosciences); monoclonal mouse anti-TfnR (Zymed); monoclonal mouse anti-β-tubulin (KMX-1; Millipore); monoclonal mouse anti-DYKDDDDK(1E6) (Wako Chemicals); monoclonal rat anti-HA (3F10; Roche Applied Science); Alexa Fluor–conjugated secondary antibodies (Invitrogen/ThermoFisher Scientific); Cy3-, DyLight-, and horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Staining, Marker, Incubation, Confocal Microscopy

Journal: Molecular Biology of the Cell

Article Title: The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization

doi: 10.1091/mbc.E20-04-0225

Figure Lengend Snippet: Endosomal localization of the C-terminus–swapped ATP11 chimeras. (A–C) HeLa cells transiently coexpressing N-terminally FLAG-tagged CDC50A and C-terminally HA-tagged ATP11B, ATP11A-B, or ATP11C-B were fixed and stained for HA and EEA1 (a marker for early endosomes), TfnR (transferrin receptor, a marker for early/recycling endosomes), or Lamp-1 (marker for late endosomes) followed by incubation with Cy3-conjugated anti-rat antibody and Alexa Fluor 488–conjugated anti-mouse secondary antibody. The images were taken by confocal microscopy. Scale bars, 20 μm. Scale bars in enlarged images, 5 μm.

Article Snippet: Sources of antibodies used in this study were as follows: monoclonal rabbit anti-ATP1A1 (EP1845Y; Abcam); monoclonal mouse anti-EEA1 and anti-Lamp-1 (BD Biosciences); monoclonal mouse anti-TfnR (Zymed); monoclonal mouse anti-β-tubulin (KMX-1; Millipore); monoclonal mouse anti-DYKDDDDK(1E6) (Wako Chemicals); monoclonal rat anti-HA (3F10; Roche Applied Science); Alexa Fluor–conjugated secondary antibodies (Invitrogen/ThermoFisher Scientific); Cy3-, DyLight-, and horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Staining, Marker, Incubation, Confocal Microscopy

Journal: Molecular Biology of the Cell

Article Title: The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization

doi: 10.1091/mbc.E20-04-0225

Figure Lengend Snippet: The NT regions of ATP10 proteins and the C-terminal regions of ATP11 proteins are sufficient to determine their cellular localization. (A) Schematic representation of chimeric proteins. An EGFP-labeled plasma membrane–targeting signal comprised of the N-terminal 11 amino acids of Lyn (Lyn 11 ) was fused to the N-terminal cytoplasmic region (NT) of each ATP10 protein or the C-terminal cytoplasmic region (CT) of each ATP11 protein. (B–F) HeLa cells were transiently transfected with expression vectors for Lyn 11 -EGFP–fused ATP10A-NT, ATP10B-NT, ATP10D-NT, ATP11A-CT, ATP11B-CT, or ATP11C-CT (C–F), or the original vector encoding Lyn 11 -EGFP–fused FRB (FKBP-rapamycin–binding domain) (B). The cells were stained for ATP1A1 (B–D), EEA1, TfnR, or Lamp-1 (E, F) followed by incubation with AlexaFluor 555–conjugated anti-rabbit or anti-mouse secondary antibodies (E, F). Images were taken by confocal microscopy. Scale bars, 20 μm. Scale bars in enlarged images, 5 μm.

Article Snippet: Sources of antibodies used in this study were as follows: monoclonal rabbit anti-ATP1A1 (EP1845Y; Abcam); monoclonal mouse anti-EEA1 and anti-Lamp-1 (BD Biosciences); monoclonal mouse anti-TfnR (Zymed); monoclonal mouse anti-β-tubulin (KMX-1; Millipore); monoclonal mouse anti-DYKDDDDK(1E6) (Wako Chemicals); monoclonal rat anti-HA (3F10; Roche Applied Science); Alexa Fluor–conjugated secondary antibodies (Invitrogen/ThermoFisher Scientific); Cy3-, DyLight-, and horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Labeling, Transfection, Expressing, Plasmid Preparation, Binding Assay, Staining, Incubation, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: c-erbB-3

doi: 10.1083/jcb.200109033

Figure Lengend Snippet: Immunochemical demonstration of c-erbB-3 in MCF-7 cells. (A) 400 μg protein from untreated (−) or LMB-treated (+) C or N MCF-7 fractions were immunoprecipitated (IP) with SGP1, which detects an extracellular epitope of c-erbB-3, or with mIgG 1 and blotted (IB) with C17, which binds to the cytoplasmic part of c-erbB-3. (B) 20 μg total protein from each fraction (C or N) were blotted with anti–c-erbB-3 (C17), antipyruvate kinase, anti–histone H1, anticalnexin, or antitransferrin receptor. Note calnexin has an apparent molecular mass of 90 kD (arrow). The bottom band seen in N represents a nonspecifically stained bulk nuclear protein. (C) Cytoplasmic-enriched fractions were prepared with or without the addition of 0.2% Triton X-100 and blotted for c-erbB-3 (C17). After centrifugation at 100,000 g , an arbitrary amount of the membrane pellet (M) from the fraction prepared without Triton X-100 was loaded as a control.

Article Snippet: Proteins were blotted onto polyvinylidene difluoride membranes (New England Nuclear), which were then blocked for 1 h in 5% BSA/PBS at room temperature (RT), washed with PBS containing 0.1% Tween 20 (PBST), and immunostained for 3 h at RT in 1% BSA/PBS containing the primary antibody: polyclonal anti–c-erbB-3 (0.2 μg/ml, C17; Santa Cruz Biotechnology, Inc.), polyclonal anti-CPK (cross-reactive with human pyruvate kinase) (1:4,000; Convance), monoclonal anti–histone H1 (0.2 μg/ml; Neomarkers), monoclonal anti-calnexin (1:400; Affinity BioReagents, Inc.), or monoclonal antitransferrin receptor (1 μg/ml; Neomarkers).

Techniques: Immunoprecipitation, Staining, Centrifugation