Journal: bioRxiv

Article Title: Identification of Human Transferrin Receptor as an Entry Co-receptor for Parvovirus B19 Infection of Human Erythroid Progenitor Cells

doi: 10.64898/2026.04.02.715920

Figure Lengend Snippet: (A) Protein diagram. VP1u-APEX2 consists of APEX2 fused to the C-terminus of the unique region of B19V VP1 (VP1u) via a seven-residue glycine-serine linker (GGSGGSG), followed by a Flag tag and a 6 × Histidine (His) tag. APEX2 has a linker-Flag-His tag fused at the C-terminus. (B) Analysis of purified proteins. VP1u-APEX2 and APEX2 proteins were expressed in bacteria and purified. Approximately (∼) 1 µg of each protein was separated by SDS-PAGE, followed by Coomassie blue staining. M, molecular weight marker. (C) Confocal microscopy of VP1u-APEX2 entry. 1 × 10 6 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C for 2 h. The cells were then immunostained with α-Flag to visualize internalized proteins under a Leica STED confocal microscope. Scale bar = 10 μm. Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole). (D) Western blotting of APEX2-biotinylated proteins. 1 × 10 7 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C. After 2 h, APEX2-mediated biotinylation was then performed as described in the Materials and Methods and Figure S1 . Biotinylated host proteins were purified with streptavidin-conjugated magnetic beads. The supernatant was collected as the flow-through (FT), and the beads were further washed several times and eluted as the pull-down (PD). Both FT and PD samples were analyzed by SDS-PAGE and immunoblotting using Alexa Fluor 680-conjugated streptavidin. (E) Analysis of VP1u-APEX2-biotinylated/associated proteins using quantitative mass spectrometry (qMS). Three independent PD samples prepared from VP1u-APEX2 and APEX2 (control) treated cells were analyzed by on-bead digestion and qMS. MS data were processed and analyzed as described in the Materials and Methods. The bubble plot shows protein enrichment (log 2 fold change) in the VP1u-APEX2 group relative to the APEX control, with color indicating subcellular localization based on Gene Ontology (GO) annotation. TFRC denotes human transferrin receptor 1 (hTfR).

Article Snippet: Purified proteins: Recombinant hTfR ECD protein tagged with a His-tag at the C-terminus (#11020-H07H) and recombinant human ferritin heavy chain 1/FTH1 (#13217-HNAE) were purchased from SinoBiological (Paoli, PA).

Techniques: Residue, FLAG-tag, Purification, Bacteria, SDS Page, Staining, Molecular Weight, Marker, Confocal Microscopy, Incubation, Microscopy, Western Blot, Magnetic Beads, Mass Spectrometry, Control, Protein Enrichment

Journal: Nature Aging

Article Title: Iron homeostasis and cell clonality drive cancer-associated intestinal DNA methylation drift in aging

doi: 10.1038/s43587-025-01021-x

Figure Lengend Snippet: a , Schematic representation of a single-crypt DNAm analysis coupled with RNA sequencing. b , Scatter-plot showing the average DNAm level on the Dkk1 , Dkk2 , Sfrp1 and Sfrp2 gene promoters in single crypts isolated from aged mice. The most methylated crypts (the top 10%, high 5mC crypts) and the least-methylated crypts (the bottom 10%, low 5mC crypts) were selected and analyzed by RNA-seq. n = 4 mice were used. c , PCA of single crypt RNA-seq datasets generated in crypts of the top 10% (high 5mC crypts, red dots) and the bottom 10% (low 5mC crypts, blue dots) as in b . To assess the statistical significance of the separation between two groups, a MANOVA test using Pillai’s trace was performed, yielding a highly significant result ( P = 4.235 × 10 −5 ). d , Transferrin receptor ( Tfrc ) and ferroportin ( Slc40a1 ) expression levels in high ( n = 16) and low ( n = 12) 5mC crypts as in b . P value was calculated by a two-tailed Welch’s t -test. Each dot represents a single crypt. n = 4 mice were used. e , Western blot analysis of the protein levels of TfR1 and ferroportin (FPN1) in small intestinal crypts isolated from young and aged mice. Actin was used as a loading control. n = 5 mice per group were analyzed. f , Ferrous (Fe 2+ ) iron levels in small intestinal crypts isolated from young and aged mice. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t -test. g , Schematic representation of our hypothesis. h , Quantification of TET hydroxymethylase enzymatic activity in intestinal crypts isolated from young ( n = 5) and aged ( n = 6) mice. P value was calculated by a two-tailed Welch’s t -test. i , Quantification of DNMT enzymatic activity in the intestinal crypts isolated from young ( n = 4) and aged ( n = 5) mice. P value was calculated by a two-tailed Welch’s t -test. j , Bar chart showing DNAm level of the Dkk2 gene promoter analyzed by BS pyrosequencing at the indicated time points upon C35 TET inhibitor treatment of organoids derived from young intestinal crypts. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t -tests. k , Bar charts showing DNAm level of the Dkk1 and Dkk2 gene promoters in intestinal crypts isolated from wild-type (WT) and Tet2/3 -dKO mice. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t -test. l , Bar chart showing DNAm level of the Dkk2 gene promoter analyzed by BS pyrosequencing in organoids treated with the iron chelator, DFO for 1 and 2 months. n = 3 mice per group were analyzed. P value was calculated by a two-tailed paired t -test. Error bars in the figure bar charts represent the s.d.

Article Snippet: The complementary DNA of murine transferrin receptor ( Tfrc ) was purchased from Origene (plasmid MC221416 ) and cloned into the CSII-EF Venus-P2A-3F-MCS vector using the BamHI and NotI restriction sites.

Techniques: RNA Sequencing, Isolation, Methylation, Generated, Expressing, Two Tailed Test, Western Blot, Control, Activity Assay, Derivative Assay

Journal: Nature Aging

Article Title: Iron homeostasis and cell clonality drive cancer-associated intestinal DNA methylation drift in aging

doi: 10.1038/s43587-025-01021-x

Figure Lengend Snippet: a , The expression level of the Dnmt enzymes in high (red dots; n = 16) and low (blue dots; n = 12) 5mC crypts based on RNAseq as in Fig. . b , The expression level of the Tet enzymes in high (red dots; n = 16) and low (blue dots; n = 12) 5mC crypts based on RNAseq as in Fig. . c , Gene ontology analysis of the differentially expressed genes (DEGs) between the crypts with high (the top 10%) and low (the bottom 10%) DNAm as in Fig. . p-value was calculated by one-sided Fisher’s Exact test. d , Transferrin receptor ( Tfrc ) and ferroportin ( Slc40a1 ) expression levels in the Lgr5 hi cells isolated from young and old mice. n = 3 mice per group were analyzed. p-value is calculated by DESEQ2. e , Quantification of the western blot analysis as in Fig. . n = 5 mice per group were analyzed. p-value was calculated by Welch’s t-test, 2-tails. f , ChIP-qRT-PCR analysis of the ChIP of TET1, TET2 and TET3 on the Dkk2 gene promoter in the intestinal crypts isolated from young and old mice. n = 3 mice per group were analyzed. IgG was used as a control. p-value was calculated by Welch’s t-test, 2-tails. g , ChIP-qRT-PCR analysis of the ChIP of TET1, TET2 and TET3 on the Sfrp1 gene promoter in the intestinal crypts isolated from young and old mice. n = 3 mice per group were analyzed. IgG was used as a control. p-value was calculated by Welch’s t-test, 2-tails. Error bars in the figure bar charts represent the SD.

Article Snippet: The complementary DNA of murine transferrin receptor ( Tfrc ) was purchased from Origene (plasmid MC221416 ) and cloned into the CSII-EF Venus-P2A-3F-MCS vector using the BamHI and NotI restriction sites.

Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR, Control

Journal: Nature Aging

Article Title: Iron homeostasis and cell clonality drive cancer-associated intestinal DNA methylation drift in aging

doi: 10.1038/s43587-025-01021-x

Figure Lengend Snippet: a , Western blot analysis of the protein levels of the transferrin receptor (TfR1) in intestinal organoids derived from aged mice and analyzed 40 days after transduction with a 3xFlag-Tfrc- expressing lentivirus. Mock (parental vector) was used as a negative control. Actin was used as a loading control. n = 3 mice per group were analyzed. b , Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in a . n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t -test. c , Bar chart showing the DNAm level of the Dkk2 gene promoter analyzed by BS pyrosequencing in intestinal organoids as in a . Parental organoids, untransduced organoids at day 0. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t -test. d , Representative images of the western blot analysis of the protein levels of TfR1 and FPN1 in intestinal organoids cultured in different media as indicated. ENRW (EGF, Noggin, R-spondin1, Wnt3a); ENRC (EGF, Noggin, R-spondin1, CHIR99021). GAPDH was used as a loading control. n = 3 mice per group were analyzed. For details, see the main text. e , Ferrous (Fe 2+) iron levels in intestinal organoids as in d . n = 3 mice per group were analyzed. P value was calculated by one-way analysis of variance (ANOVA). f , Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in d . n = 3 mice per group were analyzed. P value was calculated by one-way ANOVA. g , Representative images of the western blot analysis of the protein levels of the TfR1 and FPN1 in intestinal organoids treated with IFNγ (0.2 ng ml −1 ) or with both IFNγ (0.2 ng ml −1 ) and Wnt3a (100 ng ml −1 ) for 5 days as indicated. GAPDH was used as a loading control. n = 3 mice per group were analyzed. For details, see the main text. h , Ferrous (Fe 2+ ) iron levels in intestinal organoids as in g . n = 3 mice per group were analyzed. P value was calculated by one-way ANOVA. i , Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in g . n = 3 mice per group were analyzed. P value was calculated by ordinary one-way ANOVA. Error bars in bar charts represent the s.d.

Article Snippet: The complementary DNA of murine transferrin receptor ( Tfrc ) was purchased from Origene (plasmid MC221416 ) and cloned into the CSII-EF Venus-P2A-3F-MCS vector using the BamHI and NotI restriction sites.

Techniques: Western Blot, Derivative Assay, Transduction, Expressing, Plasmid Preparation, Negative Control, Control, Activity Assay, Two Tailed Test, Cell Culture

Journal: Chinese Medicine

Article Title: Targeting the ferritinophagy axis: multi-target regulation of TFRC/FTH1/NCOA4 by artesunate ameliorates salivary gland dysfunction in Sjögren’s Disease

doi: 10.1186/s13020-026-01354-8

Figure Lengend Snippet: Artesunate inhibits iron-autophagy axis to suppress ferroptosis. A Molecular docking of human FTH1-NCOA4 protein–protein complex and ART with FTH1, NCOA4, TFRC (protein–ligand complexes). Upper: Best docking conformations. Middle: PLIP-identified interactions (blue: hydrogen bond; gray dashed: hydrophobic interaction; yellow: salt bridge). Lower: 2D interaction diagrams (Discovery Studio). B Key docking metrics: binding affinity (ΔG, kcal/mol), log inhibition constant (logKi, M), efficiency indices (LE, SILE, LLE, LELP), and interaction types (HB, HI, SB; calculated via AutoDock Vina). C – F Western blot analysis and quantification of ACSL4, GPX4, TFRC, FTH1, NCOA4, and LC3A/B. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: anti-LC3A/B antibody (1:800, Cell Signaling Technology, #4108), anti-xCT antibody (1:1000, Abcam, ab307601), anti-GPX4 antibody (1:4000, Abclonal, A25009), anti-TFRC antibody (1:1000, Abclonal, A25900), anti-NCOA4 antibody (1:1000, Abclonal, A5695), anti-AQP2 antibody (1:1000, Abclonal, A16209), anti-ACSL4 antibody (1:20,000, Abclonal, A20414), and anti-FTH1 antibody (1:1000, Abclonal, A25458-PM).

Techniques: Binding Assay, Inhibition, Western Blot

Journal: Chinese Medicine

Article Title: Targeting the ferritinophagy axis: multi-target regulation of TFRC/FTH1/NCOA4 by artesunate ameliorates salivary gland dysfunction in Sjögren’s Disease

doi: 10.1186/s13020-026-01354-8

Figure Lengend Snippet: Artesunate restores iron metabolic homeostasis and suppresses ferritinophagy in vivo. A , B Western blot analysis and quantification of TFRC, FTH1, NCOA4, and LC3A/B. C , D Confocal microscopy of AQP5 and TFRC localization in submandibular glands across treatment groups (semi-quantification: ImageJ, 20 images from 3 mice). Scale = 100 μm. E , F Confocal microscopy of NCOA4, FTH1, and LC3A/B localization in submandibular glands across treatment groups (semi-quantification: ImageJ, 20 images from 3 mice/group, n = 3) with 2D intensity histograms showing Pearson’s coefficient (PR), red Manders’ coefficient (M1), and green Manders’ coefficient (M2). Data are mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: The membranes were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: anti-LC3A/B antibody (1:800, Cell Signaling Technology, #4108), anti-xCT antibody (1:1000, Abcam, ab307601), anti-GPX4 antibody (1:4000, Abclonal, A25009), anti-TFRC antibody (1:1000, Abclonal, A25900), anti-NCOA4 antibody (1:1000, Abclonal, A5695), anti-AQP2 antibody (1:1000, Abclonal, A16209), anti-ACSL4 antibody (1:20,000, Abclonal, A20414), and anti-FTH1 antibody (1:1000, Abclonal, A25458-PM).

Techniques: In Vivo, Western Blot, Confocal Microscopy

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: Protein sequencing results.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: A . Huh7 cells were transiently transfected with DDK tagged TFR1 cDNA. Lysate of transfected or non-transfected cells was immunoprecipitated with DDK or AF20 mAb, followed by sequential detection of the blot with DDK and TFR1 antibodies. B . Lysate of nontransfected Huh7 cells was incubated with AF20 mAb immobilized on protein G beads, or protein G beads alone to serve as a negative control, followed by Western blot detection of beads-associated proteins by antibodies against AF20, TFR1, HSP and N + /K + ATPase, respectively. Note that a minigel was used for electrophoresis, which could not resolve proteins of similar sizes.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Incubation, Negative Control, Western Blot, Electrophoresis

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: NIH 3T3 cells grown in 24-well plate with cover slips were transfected with expression constructs of DDK tagged TFR1, DDK tagged Na + /K + ATPase, or untagged HSP90. Cells were fixed with acid alcohol followed by staining with AF20, TFR1, DDK, and HSP90 antibodies, respectively. Note that AF20 mAb only revealed signals in TFR1 transfected cells, although all three proteins were successfully expressed in NIH 3T3 cells. In addition, cell death or unusual morphology (elongated) was observed in cells expressing the exogenous proteins (dead cells are indicated by arrows). All images were taken at 20x magnification.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Transfection, Expressing, Construct, Staining

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: TFR1 transfected NIH 3T3 cells were fixed and stained with either AF20 mAb (panel A) or TFR1 mAb (panel B), followed by confocal microscopy to reveal subcellular localization and 3D pattern of the protein. (C) Stacks of X axis for panels A and B, respectively.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Transfection, Staining, Confocal Microscopy

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: Huh7 cell lysate was subject to immunoprecipitation with AF20 mAb and retained proteins on protein G beads were treated with PNGase F at 37°C for 1hr. Samples were directly loaded onto 10% SDS-PAGE (minigel) in duplicate followed by Western blot detection using AF20 (left) and TFR1 antibodies (right), respectively. Note that an additional protein band above the 75-kd size marker was detected by TFR1 antibody from PNGase F treated sample, most likely corresponding to deglycosylated form of TFR1 (79kDd).

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Marker

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: Three pairs of adjacent tissue sections of normal colon ( A ) and colon cancer ( B ) were stained with TFR1 (upper panels, 1:50 dilution) and AF20 mAb (lower panels, 1:500 dilution), respectively. Images were taken at 1000x magnification. Note that overexpression of both TFR1 and AF20 antigen was only observed in colon cancer but not in normal colon. In addition, the expression pattern and localization as revealed by TFR1 and AF20 Ab are indistinguishable.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Staining, Over Expression, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 5. In vivo HU treatment of Nos2–/– mice impairs HU inhibition of proliferation in erythroid progenitors. a) Schematic representation of experimental setup: Nos2–/– or wild-type (WT) mice were treated orally with 200 mg/kg HU or drinking water for 2 weeks. WT mice were injected with 20 mg/kg of 1400W twice daily for 3 consecutive days. Mouse erythroid progenitors (mERP) were isolated from bone marrow by immunomagnetic cell separation using anti-CD71-PE and anti- Ter119-FITC antibodies. b) Immunocytochemistry for Nos2 protein in mERP isolated from WT mice treated or not with HU. Quantification of Nos2-positive cells. c) Citrulline concentration in the bone marrow of WT and Nos2–/– mice treated or not with HU. d) Colony formation assay showing the number of late erythroid (CFU-E), early erythroid (BFU-E), or granulocyte/macrophage progenitors (CFU-GM) in the bone marrow of WT or Nos2–/– mice treated or not with HU. e) Immunocytochemistry for Ki67 in mERP cells isolated from WT and Nos2–/– mice treated or not with HU. f) Quantification of Ki67-positive cells. g) Cell cycle distribution by flow cytometry showing the percentage of cells in G0/G1, S, or G2/M phases of the cell cycle. c) n = 3, f) n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT.

Article Snippet: Bone marrow cells were flushed out of the femurs and tibias of WT or Nos2–/– mice treated with HU or 1400W and resuspended in 100 μL of buffer (1 % PBS, 0.5 % BSA, 2 mM EDTA) and stained with 7 μL of anti-human CD71-PE REAfinityTM antibody (Miltenyi Biotec, 130–120–809, Bergisch Gladbach, Germany) for 15 min at 4◦C.

Techniques: In Vivo, Inhibition, Injection, Isolation, Immunocytochemistry, Concentration Assay, Colony Assay, Flow Cytometry