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: 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

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: Conjugates components with cognate antigens and ASO targets

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques: Virus

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: Affinity and selectivity of FAB02 for TfR1 are maintained in DYNE-101

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques:

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: Representative images of WT and TfR1 −/− HeLa cells ( a ) treated for 4 h with FAB02 labeled with AlexaFluor 647 (pseudocolored to green). Cells were stained with wheat germ agglutinin labeled with AlexaFlour 488 (membrane, pseudocolored to red,) and DAPI (nuclei). White bars within images indicate size. b WT (blue bars) and TfR1 −/− (black bars) HeLa cells were treated for 24 h with FAB02 labeled with a pH-sensitive dye (CypHer5E). Cells were harvested and analyzed by flow cytometry for median fluorescent intensity normalized to the degree of labeling (Dye/Ab). Data are medians + SD. Each circle represents a technical replicate from two independent experiments. c Representative images of human cardiomyocytes treated for 30 min with FAB02 labeled with AlexaFluor 647 (pseudocolored to green), fixed, stained with DAPI (nuclei) and RAB5 (30 min sample, red). The image with merged AlexaFluor 647, RAB5, DAPI signals is an enlargement of the white boxed area. White bars within images indicate size.

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques: Labeling, Staining, Membrane, Flow Cytometry

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: TfR1 hu/mu mice were injected via tail vein on days 0 and 7 with vehicle (gray bars), 10 mg/kg ASO1 (red bars), or FDC1 delivering 10 mg/kg ASO-equivalent of ASO1 (blue bars) and sacrificed 14 days later for analysis of gene expression. Total RNA extracted from the indicated muscles was reverse-transcribed and Dmpk RNA expression was measured by qPCR using Ppib as the reference gene. Expression in each muscle from mice treated with FDC1 or ASO1 was normalized to Dmpk expression in the corresponding muscle of mice treated with vehicle. Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle or between ASO1 and FDC1 are indicated with exact P values. Each circle represents an individual mouse.

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques: Injection, Gene Expression, Muscles, Reverse Transcription, RNA Expression, Expressing

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: a TfR1 ; DMSXL mice enable assessment of DYNE-101 ability to target mutant human DMPK RNA via TfR1-mediated uptake. b DMPK , Malat1 , Birc5 , and Gapdh expression measured by RT-qPCR in total RNA from nuclear (N) and cytoplasmic (C) fractions from gastrocnemius of TfR1 hu/mu ; DMSXL WT/Tg mice. Data are means + SD. Each circle represents independent measurements except for analysis of Birc5 and GAPDH expression, where n = 1. c DMPK expression in fractions from gastrocnemius of TfR1 hu/mu ; DMSXL WT/Tg mice administered with 10 mg/kg ASO-equivalent of DYNE-101 (blue bars) relative to mice injected with vehicle (black bars). Data are means + SD. Each circle represents independent measurements. d DMPK expression measured by RT-qPCR in total RNA from muscles of TfR1 hu/mu ; DMSXL Tg/Tg female mice administered weekly with 10 mg/kg ASO-equivalent of DYNE-101 (blue bars), 28 days after initial injection, relative to expression in corresponding muscles of mice injected with vehicle (black bars). Data are means + SD; data were analyzed by unpaired t test with Welch’s correction, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse. e Representative images of nuclei (blue) and DMPK foci (red) in cardiomyocytes used to calculate foci area. Insets display myofibrils (gray) in greyscale. Total DMPK foci area in heart of TfR1 hu/mu ; DMSXL Tg/Tg mice 28 days after initial injection. Data are means + SD, analyzed by unpaired t test: * P < 0.05. Each circle represents an individual mouse. f Composite splicing index in muscle of TfR1 hu/mu mice injected with vehicle (gray circle) or TfR1 hu/mu ; DMSXL Tg/Tg mice injected with vehicle (black circle) or DYNE-101 (blue circle), 28 days after initial injection. Horizontal lines are means ± SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from TfR1 hu/mu mice injected vehicle or between TfR1 hu/mu ; DMSXL Tg/Tg mice treated with vehicle and DYNE-101 are indicated with exact P values. Each circle represents an individual mouse.

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Injection, Muscles

Journal: Communications Medicine

Article Title: FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

doi: 10.1038/s43856-025-00733-w

Figure Lengend Snippet: Mutant human DMPK expression measured by RT-qPCR in muscle of TfR1 hu/mu ; DMSXL WT/Tg male and female mice a 1 month after a single IV injection with DYNE−101 (blue bars) or b 1 month following the last of 4 monthly doses at the indicated ASO-equivalent doses (blue bars), relative to expression in the same muscles of mice injected with vehicle (black bars). Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle or between DYNE-101 dose levels are indicated with exact P values. Each circle represents an individual mouse.

Article Snippet: The latter consists of MAB01 a commercially available anti-TfR1 antibody (BE0175, BioXCell) conjugated to ASO1.

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, IV Injection, Muscles, Injection

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 interacts with mGluR2. (A) Schematic representation of the AP-MS approach used to identify mGluR2-protein interactions in HEK293 cells. (B and C) HEK293 cells were co-transfected with mGluR2-Flag and TfR1-Myc, then cells were solubilized by NP-40 lysis buffer (B) or RIPA lysis buffer (C), and then subjected to immunoprecipitation (IP) by using anti-Flag agarose beads. Representative Western blots of whole cell lysates and eluates after IP are shown. (D) Co-immunoprecipitation of mGluR2-Flag with endogenous TfR1 in HEK293 cells. (E and F) Purified mGluR2-GST protein (E) or TfR1-GST protein (F) was pooled with the lysate from TfR1-Myc- (E) or mGluR2-Flag- (F) transfected HEK293 cells and then pulled down by using anti-GST beads. The GST protein was used as the negative control.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Purification, Negative Control

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: mGluR2, RABV, and TfR1 internalize together. (A) Increasing amounts of TfR1-GST were respectively mixed with mGluR2-Flag-conjugated beads, then the lysate from RABV G-Myc-transfected HEK293 cells was added for use in the protein interaction competition assay. (B to D) N2a cells were infected with ERA-N-mCherry for 10 min at 37°C, and then multiplex immunofluorescence was performed. The arrows indicate the colocalization of TfR1 (green), ERA-N-mCherry (red), clathrin (yellow), and mGluR2 (purple). Two representative co-localizations (scale bar, 3 μm) (yellow arrows) are shown in three dimensions (B); the dashed box is magnified at the indicated location of the same image (scale bar, 0.7 μm) (C and D).

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Competitive Binding Assay, Infection, Multiplex Assay, Immunofluorescence

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 and TTP are required for agonist-triggered mGluR2 endocytosis. (A) HEK293 cells were treated with or without LY354740 for 30 min at 37°C, and then were stained with an antibody against mGluR2 (red). Cell nuclei were stained with Hoechst 33342. Representative images are shown. The fluorescence signal of mGluR2 was quantified by using ZEN software. The relative fluorescence of mGluR2 was quantified by normalization to control cells. The circles represent individual datapoints. At least 100 cells per sample were quantified. (B and C) TfR1-silenced (B) or TTP-silenced (C) HEK293 cells were treated with LY354740 as described in (A). Representative images are shown. The data represent the sum of three independent experiments. The two-tailed unpaired Student's t test was used for the statistical analysis. *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Staining, Fluorescence, Software, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 is important for the internalization of SARS-CoV-2. (A) The TfR1 mRNA level in the indicated siRNA-transfected Vero-E6 cells or Caco2 cells was measured by qPCR. (B) TfR1-silenced Vero-E6 cells or Caco2 cells were infected with SARS-CoV-2 HRB25 strain. At 24 h postinfection, virus in the culture supernatant was detected by use of plaque assays. (C and D) Co-immunoprecipitation of TfR1-Flag and SARS-CoV-2 S-Myc (C), SARS-CoV-2 S1-Myc, or SARS-CoV-2 RBD-Myc (D) in plasmid-transfected HEK293 cells. (E) Co-immunoprecipitation of TfR1-Flag and SARS-CoV-2 S-Myc in plasmid-transfected Vero-E6 cells. (F) Co-immunoprecipitation of TfR1-Flag and ACE2-Myc in plasmid-transfected HEK293 cells. (G) Cells were incubated with SARS-CoV-2 for 1 h at 4°C, then washed with acid buffer/trypsin, and lysed for qPCR to detect virus bound to the cell surface. (H and I) SARS-CoV-2 binding (H) and internalization (I) assays were performed in TfR1-silenced Vero-E6 cells or Caco2 cells. (J) Increasing amounts of mGluR2-GST were respectively mixed with TfR1-Flag-conjugated beads, and then with the lysate from SARS-CoV-2 S-Myc-transfected HEK293 cells for the protein interaction competition assay. In panels (A), (B), and (G to I), error bars represent the means ± SDs of 3 independent experiments or replicates. The two-tailed unpaired Student's t test was used for the statistical analysis. ns, not significant, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Infection, Immunoprecipitation, Plasmid Preparation, Incubation, Binding Assay, Competitive Binding Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 is important for SARS-CoV S- and MERS-CoV S-mediated internalization. (A and B) Co-immunoprecipitation of TfR1-Flag and SARS-CoV S-Myc (A), or MERS-CoV S-Myc (B) in plasmid-transfected HEK293 cells. (C and D) Co-immunoprecipitation of TfR1-Flag and SARS-CoV S-Myc (C), or MERS-CoV S-Myc (D) in plasmid-transfected Vero-E6 cells. (E) Cells were incubated with rVSV-SARS-CoV-S, or rVSV-MERS-CoV-S for 1 h at 4°C, then washed with acid buffer/trypsin, and lysed for qPCR to detect virus bound to the cell surface. (F and G) rVSV-SARS-CoV-S, or rVSV-MERS-CoV-S binding (F) and internalization (G) assays were performed in TfR1-silenced Vero-E6 cells. (H and I) Increasing amounts of mGluR2-GST were respectively mixed with TfR1-Flag-conjugated beads, and then with the lysate from SARS-CoV S-Myc- (H) or MERS-CoV S-Myc- (I) transfected HEK293 cells for the protein interaction competition assay. In panels (E to G), error bars represent the means ± SDs of 3 independent experiments or replicates. The two-tailed unpaired Student's t test was used for the statistical analysis. ns, not significant, ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Immunoprecipitation, Plasmid Preparation, Transfection, Incubation, Binding Assay, Competitive Binding Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: Proposed model for the role of mGluR2 and TfR1 in RABV or SARS-CoV-2 entry. The virus first binds to a specific receptor(s) at the cell surface, then the virus interacts with and activates mGluR2. The virus-mGluR2 complex migrates to a TfR1-containing pre-existing CCP, where it interacts with TfR1 and hijacks the endocytic signaling of TfR1 to enter the cell.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques:

Journal: Cells

Article Title: Upregulation of Transferrin Receptor 1 (TfR1) but Not Glucose Transporter 1 (GLUT1) or CD98hc at the Blood–Brain Barrier in Response to Valproic Acid

doi: 10.3390/cells13141181

Figure Lengend Snippet: TfR1 protein in cultured brain endothelial cells (BCECs) after valproic acid (VPA) treatment. Zonula occludens (ZO-1; green) and TfR1 (red) detection using immunocytochemistry in BCECs co-cultured with mixed glial cells and treated with PBS ( A – D ) or 2 mM VPA ( E – H ). The tight junction protein ZO-1 remains highly expressed despite treatment with VPA (compare ( A ) with ( E )). Morphologically, the increased expression of Tfrc translates to increased TfR1 protein, compare ( B ) with ( F ). When shown in high magnification ( D , H ), it is evident that the TfR1 protein is highly expressed after VPA treatment, revealing a perinuclear distribution probably reflecting a higher abundance in the granular endoplasmic reticulum. The nuclei are stained with DAPI (blue). Scale bar: 20 μm. White box in ( C , G ) represents the zoomed in image ( D , H ).

Article Snippet: Mouse TfR1 ELISA kit (Cat. no. EKM2800) was purchased from Nordic BioSite (Copenhagen, Denmark).

Techniques: Cell Culture, Immunocytochemistry, Expressing, Staining

Journal: Cells

Article Title: Upregulation of Transferrin Receptor 1 (TfR1) but Not Glucose Transporter 1 (GLUT1) or CD98hc at the Blood–Brain Barrier in Response to Valproic Acid

doi: 10.3390/cells13141181

Figure Lengend Snippet: Tfrc gene expression ( A , B ) and TfR1 protein levels ( C , D ) after valproic acid (VPA) treatment in vivo; uptake of gold-labeled TfR1-targeted antibodies ( E – H ). ( A , B ) Tfrc gene expression in capillary-depleted brain extracts from mice treated with PBS, 100 mg/kg VPA, or 400 mg/kg VPA for 24 h. ( A ) Total cohort. ( B ) Separation by sex. (Males, n = 6; Females, n = 6 for each treatment group). Gene expression is analyzed using one-way ANOVA with Dunnett’s multiple comparisons and when investigating the difference in mRNA expression between male and female mice, a two-way ANOVA with the Holm–Sidak multiple comparisons test is used. Data ( n = 6–12) are shown mean ± SEM. No differences are observed in Tfrc mRNA expression in the group of all animals ( A ) or after division by sex ( B ). ( C ) When analyzed for TfR1 protein in isolated capillaries, both 100 mg ( n = 12) and 400 mg ( n = 12) VPA treatment for 24 h significantly increased TfR1 protein, with upregulation in both male and female mice ( D ). One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis. ** p = 0.001–0.01. Data ( n = 11–12) are depicted as mean ± SEM. ( E – H ) Uptake of gold-labeled TfR1-targeted antibodies. Uptake of TfR1-conjugated gold 1 nm nanoparticles in the brains, livers, spleens, and lungs of untreated mice and mice treated with 100 mg/kg VPA. The uptake of TfR1-antibody-conjugated gold nanoparticles, determined based on the content of gold in the isolated capillaries and brain fraction using ICP-MS, is similar in the brains ( E ) and hepatocytes of the liver ( F ) in control and VPA-injected mice. The uptake in the spleen ( G ) and lung ( H ) seems to increase slightly, although the differences are insignificant. The statistical difference between control and VPA-treated animals is analyzed with an unpaired t -test, each point represents data from a single mouse ( n = 6), and the data are depicted as mean ± SEM.

Article Snippet: Mouse TfR1 ELISA kit (Cat. no. EKM2800) was purchased from Nordic BioSite (Copenhagen, Denmark).

Techniques: Expressing, In Vivo, Labeling, Isolation, Control, Injection