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: Nucleic Acids Research

Article Title: Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice

doi: 10.1093/nar/gkac641

Figure Lengend Snippet: FORCE–M23D structure, Fab internalization kinetics and FORCE–M23D TfR1 binding affinity in C2C12 mouse myoblasts. ( A ) Schematic representation of the FORCE–M23D conjugate. ( B ) Binding to murine TfR1 with the FORCE–M23D conjugate or with a negative control human-TfR1 specific conjugate by ELISA. ( C ) Uptake of anti-murine TfR1-Cypher5a conjugate and anti-human TfR1-Cypher5e conjugate in C2C12 myoblasts. Data represent mean ± SD. *** P < 0.001. A.U., arbitrary units; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: The remaining reagents/instruments were purchased as follows: Vivaflow 50 cassette (Sartorius, Göttingen, Germany; Cat. No. VF05P2); PES Rapidflow (Thermo Scientific Nalgene, Rochester, NY, USA; Cat. No. 09-741-03); Endosafe limulus amebocyte lysate kinetic chromogenic assay (Charles River Laboratories International, Inc., Wilmington, MA, USA; Cat. No. PTS20F); 96-well black flat bottom high bind microplates were purchased from Corning (Corning, NY; Cat. No. 3925); Recombinant mouse TfR1 protein was purchased from R&D Systems (Minneapolis, MN; Cat. No. 9706-TR-050); bovine serum albumin (BSA) was purchased from MP Biomedicals, LLC (Irvine, CA; Cat. No. 840043); phosphorothioate/DNA capture probe (Integrated DNA Technologies Inc., Coralville, IA, USA); Synergy Neo2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA, Cat. No. NEO2); Precellys Evolution homogenizer (Bertin, Montigny-le-Bretonneux, France, Cat. No. P000062-PEVO0-A); hard-tissue homogenizer (Omni International, Inc., Kennesaw, GA, USA, Cat. No. TH115); optimal cutting temperature (OCT) mounting medium (Tissue-tek, Torrance, CA, USA, Cat. No. 4583); ZEISS laser scanning 800 confocal microscope (Carl Zeiss Meditec AG, Jena, Germany); Image J software (US National Institutes of Health, Bethesda, MD, USA); M3 multimode plate reader (Molecular Devices, San Jose, CA, USA, Cat. No. M3); Mouse Activity Wheels with dual lickometers (Lafayette Instrument Co., Lafayette, IN, USA, Model 80822); plexiglass square chambers (Med Associates Inc., St Albans, VT, USA).

Techniques: Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Coxiella burnetii Infection Increases Transferrin Receptors on J774A.1 Cells †

doi:

Figure Lengend Snippet: J774A.1 cells were grown on Thermonox coverslips; TfRs were labeled with an anti-TfR monoclonal antibody and examined by confocal microscopy for TfR expression. (A) J774A.1 cells infected for 16 h with viable C. burnetii show increased TfR expression as well as a rounding up of infected cells. (B) J774A.1 cells incubated in conditioned medium from persistently infected J774A.1 cells. These cells show no upregulation of TfR but do exhibit the rounding up seen in infected cells. (C) J774A.1 cells inoculated with inactivated C. burnetii and (D) uninoculated J774A.1 cells exhibit a basal level of TfR expression and no morphological changes.

Article Snippet: The samples were then blocked and permeabilized with 0.1% gelatin–0.3% saponin in PBS for 15 min, incubated with rat anti-mouse TfR monoclonal antibody for 1 h, washed three times, and probed with a fluorescein isothiocyanate-labeled rat secondary antibody (Organon Teknika Corp., West Chester, Pa.) for 1 h. Secondary antibody was washed off once for 10 min with blocking solution and twice for 10 min with PBS.

Techniques: Labeling, Confocal Microscopy, Expressing, Infection, Incubation

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Subcellular localization of RCP. Tf-TxR (A–C, red) was bound to HeLa cells for 1 h at 4°C and then incubated at 37°C for 5 min (A) or 30 min (B and C). Cells were then fixed and stained with anti-RCP antibodies (A and C, green). In D, fixed HeLa cells were costained with anti-RCP (green) and anti-EEA1 (red) antibodies. In E and F, HeLa cells transfected with RCP-YFP and CFP-Rab11A were incubated with Tf-TxR for 1 h at 4°C and then incubated at 37°C for 30 min. Colocalization between Tf-TxR (E and F, red) and RCP–YFP/CFP–Rab11 complex was then determined using FRET (F, green). In G–J, HeLa cells were treated with either nocodazole (I and J) or brefeldin A (G and H), fixed, and stained with anti-RCP (H and J, green) or anti-TfR (G–J, red) antibodies. The yellow signal in C, F, H, and J, represents overlap. Bars, 2 μm.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Incubation, Staining, Transfection

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: RCP recruitment to endosomes is dependent on binding to Rab11. HeLa cells were incubated in the presence (A–C, E–F) or absence (D) of digitonin. Cells were then washed and incubated with either GST (C) or GST-RCP-F1 (A and B, D–F) in the presence (E and F) or absence (A–D) of soluble recombinant Rab11. Cells were fixed and stained with anti-GST (B and F, green; and C and D, gray) and anti-TfR (A and B and E and F, red) antibodies. Yellow in B and F represents the degree of overlap. Bars, 3 μm.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Binding Assay, Incubation, Recombinant, Staining

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Overexpression of RCP-F1 inhibits Tf recycling and uptake. (A and B) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated at 4°C for 30 min with 2 μg/ml anti-TfR-PE antibody. The amount of plasma membrane bound anti-TfR-PE antibody after this incubation was quantitated by FACS and is shown in A. Cells were then incubated at 37°C for varying amounts of time in the continuous presence of 2 μg/ml anti-TfR-PE antibody. The amount of internalized anti-TfR-PE antibody was quantitated by FACS analysis and is shown in B. The data shown are the means of at least three independent experiments. To better compare the rates of uptake, the plasma membrane-bound anti-TfR-PE after 4°C incubation was subtracted from the data in A. (C) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated at 4°C for 30 min with 20 μg/ml Tf-Alexa647. Cells were then incubated at 37°C for varying amounts of time in the continuous presence of 20 μg/ml Tf-Alexa647. The amount of internalized Tf-Alexa647 was quantitated by FACS analysis. The data shown are the means of at least three independent experiments. (D) HeLa cells expressing either GFP or GFP-RCP-F1 were incubated for 30 min with Tf-Alexa647 at 4°C. Cells were then incubated for an additional 20 min at 37°C in the presence of Tf-Alexa647. The amounts of internalized Tf-Alexa647 after incubation at 37°C are shown in the inset. Cells were then washed and incubated at 37°C for varying amounts of time in the presence of unlabeled Tf. The Tf-Alexa647 remaining in the cells was quantitated using FACS analysis and expressed as a percentage of total endocytosed (at time 0) Tf-Alexa647.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Over Expression, Expressing, Incubation

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Overexpression of GFP-RCP-F1 inhibits TfR recycling from endosomes to the plasma membrane. HeLa cells were mock transfected (A–C) or transfected with GFP-RCP-F1 (D–F). Cells were incubated with Tf-TxR for 1 h at 4°C; washed; incubated at 37°C for 5 min (A and D), 20 min (B and E), and 60 min (C and F); and fixed. The localization and fluorescence intensities of Tf-TxR and GFP-RCP-F1 were then imaged. The colocalization between Tf-TxR and RCP-F1-GFP are the means of five randomly picked cells. In G and H, HeLa cells were transfected with GFP-RCP-F1 (G) and stained with anti-TfR antibodies (H).

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Over Expression, Transfection, Incubation, Fluorescence, Staining

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: Effect of RCP knock-down on TfR endocytosis, lysosomal degradation and recycling. (A) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs, and Triton X-100 extracts of them were analyzed by Western blotting with anti-RCP, anti-Rip11, anti-Rab11, and anti-γ adaptin antibodies to determine the extent and specificity of the knock-down. (B) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 20 μg/ml Tf-Alexa647. Cells were then incubated for additional 30 min at 37°C in the presence of Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and amounts of internalized Tf-Alexa647 were quantitated by FACS. The data presented are means ± SE of three independent experiments. (C) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 2 μg/ml anti-TfR-PE antibody. The amount of plasma membrane bound anti-TfR-PE antibody is shown in the inset. Cells were then moved to at 37°C and incubated for varying amounts of time in the presence of 20 μg/ml Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and the time course of anti-TfR-PE antibody internalization was quantitated by FACS analysis. (D) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min with Tf-Alexa647 at 4°C followed by additional 20-min incubation at 37°C. Cells were then washed and incubated at 37°C for 20 min in the presence of unlabeled Tf. The amounts of cell associated Tf-Alexa647 was quantitated by FACS. The data presented are means ± SE of three independent experiments.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Transfection, Western Blot, Incubation

Journal:

Article Title: The RCP-Rab11 Complex Regulates Endocytic Protein Sorting D⃞

doi: 10.1091/mbc.E03-12-0918

Figure Lengend Snippet: RCP knock-down increases lysosomal degradation of TfR but not EGFR. (A and C) HeLa cells were either mock transfected or transfected with RCP or Rip11 siRNAs. After 74-h incubation, cells were either permeabilized with saponin to measure total TfR (A) or nonpermeabilized to measure cell surface TfR (C), stained with anti-TfR-PE antibodies, and quantitated by FACS analysis or Western blotting (A, inset). (B and D) Quantitation of data from A and C. The presented data are means ± SD of three independent experiments. (E and F) Cells were transfected with RCP siRNA, fixed, and stained with anti-RCP (E) and anti-TfR (F) antibodies. Arrow points to a HeLa cell with down-regulated RCP. Bars, 2 μM. (G and H) HeLa cells were either mock transfected or transfected with RCP siRNAs. After 74-h incubation, cells were either permeabilized to measure total EGFR (G) or nonpermeabilized to measure cell surface EGFR (H). The levels of EGFR were quantitated by FACS analysis.

Article Snippet: Mouse anti-transferrin receptor-phycoerythrin (TfR-PE) was purchased from BD Biosciences.

Techniques: Transfection, Incubation, Staining, Western Blot, Quantitation Assay