Journal: iScience

Article Title: Pathological frataxin deficiency in mice causes tissue-specific alterations in iron homeostasis

doi: 10.1016/j.isci.2025.114625

Figure Lengend Snippet: Consequences of frataxin deficiency in the cerebellum For proteins assessed by western blot, representative images are shown, while histograms represent the relative protein content calculated from the western blot signal normalized to the CBB or Ponceau stain. Data are presented as the mean ± SD, symbols correspond to biological replicates. Significant p values are indicated as <0.05(∗), <0.01(∗∗), <0.001(∗∗∗), <0.0001(∗∗∗∗). Differences between groups in iron content were assessed by two-way ANOVA with Tukey’s multiple comparison test. Two-tailed Student’s t test was used to assess the significance between WT and I151F mice in all other data presented. (A) Frataxin levels in 10-week-old mice. (B) Non-heme iron levels from WT and FXNI151F mice at indicated ages. (C) ACO1/IRP1 and IRP2 protein content measured by western blot. (D) H-FT and L-FT ferritins measured by western blot. (E) Tfrc mRNA content measured by quantitative reverse-transcription PCR (RT-qPCR). (F) Aconitase 2 to citrate synthase ratios (ACO2/CS). Relative protein ratio was calculated by SRM-proteomics, while activity ratio was calculated spectrophotometrically.

Article Snippet: Assays were performed in a CFX96 Real-Time System (Bio-Rad) using TaqMan 2X Universal PCR Master Mix (Applied Biosystems, 4304437) mixed with TaqMan probes: Tfrc1 (Mm00441941_m1), Aco1 (Mm00801417_m1) and GAPDH (Mm99999915_g1) or Rpl19 (Mm02601633_g1) as a loading control.

Techniques: Western Blot, Staining, Comparison, Two Tailed Test, Reverse Transcription, Quantitative RT-PCR, Activity Assay

Journal: iScience

Article Title: Pathological frataxin deficiency in mice causes tissue-specific alterations in iron homeostasis

doi: 10.1016/j.isci.2025.114625

Figure Lengend Snippet: Consequences of frataxin deficiency in the heart Data are presented as the mean ± SD, symbols correspond to biological replicates. Significant p values are indicated as <0.05(∗), <0.01(∗∗), <0.0001(∗∗∗∗). Differences between groups in iron content were assessed by two-way ANOVA with the Tukey’s multiple comparison test. Two-tailed Student’s t test was used to assess the significance between WT and I151F mice in all other data presented. (A) Frataxin levels measured by western blot in 10-week-old mice. (B) Non-heme iron levels from WT and FXNI151F mice at indicated ages. (C) ACO1/IRP1 and IRP2 protein content measured by western blot. (D) FT-H and FT-L ferritins measured by western blot. (E) Tfrc mRNA content measured by RT-qPCR. (F) Aconitase 2 to citrate synthase ratios (ACO2/CS). The relative protein ratio was calculated by SRM-proteomics, while the activity ratio was calculated spectrophotometrically.

Article Snippet: Assays were performed in a CFX96 Real-Time System (Bio-Rad) using TaqMan 2X Universal PCR Master Mix (Applied Biosystems, 4304437) mixed with TaqMan probes: Tfrc1 (Mm00441941_m1), Aco1 (Mm00801417_m1) and GAPDH (Mm99999915_g1) or Rpl19 (Mm02601633_g1) as a loading control.

Techniques: Comparison, Two Tailed Test, Western Blot, Quantitative RT-PCR, Activity Assay

Journal: iScience

Article Title: Pathological frataxin deficiency in mice causes tissue-specific alterations in iron homeostasis

doi: 10.1016/j.isci.2025.114625

Figure Lengend Snippet: Consequences of frataxin deficiency in liver Data are presented as the mean ± SD, symbols correspond to biological replicates (squares to males, triangles to females). Significant p values are indicated as <0.05(∗), <0.01(∗∗), <0.001(∗∗∗). Differences between groups in iron content were assessed by two-way ANOVA with the Tukey’s multiple comparison test. Two-tailed Student’s t test was used to assess the significance between WT and I151F mice in all other data presented. (A) Non-heme iron levels from WT and FXNI151F mice, females, and males, at indicated ages. (B) Frataxin levels measured by western blot in 10-week-old mice, in both females and males. (C) ACO1/IRP1 and IRP2 protein content measured by western blot in females. (D) FT-H and FT-L ferritins measured by western blot in females. (E) Tfrc mRNA content measured by qPCR in females. (F) ACO2 content measured by western blot in females. (G) Aconitase 1 and 2 activities measured by in-gel activity assay in females. (H) Aconitase to citrate synthase ratio calculated spectrophotometrically in livers from 21-week-old females.

Article Snippet: Assays were performed in a CFX96 Real-Time System (Bio-Rad) using TaqMan 2X Universal PCR Master Mix (Applied Biosystems, 4304437) mixed with TaqMan probes: Tfrc1 (Mm00441941_m1), Aco1 (Mm00801417_m1) and GAPDH (Mm99999915_g1) or Rpl19 (Mm02601633_g1) as a loading control.

Techniques: Comparison, Two Tailed Test, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Generation of humanized Tf and Tfrc rats . A , The rat Tfrc gene consists of 19 exons, with the ATG start codon located in exon 2 and the TAA stop codon in exon 19. In our KI model, the last 32 base pairs (3′) of rat Tfrc gene exon 2, counting from the ATG start codon, were replaced with the human TFRC CDS (Transcript: 201-ENST00000360110), followed by the rat 3′ untranslated region (3′UTR) and the SV40 polyadenylation sequence. B , the rat Tf gene ( NM_001013110.1 ) is comprised of 17 exons, with the ATG start codon located in exon 1 and the TAA stop codon in exon 17. In the KI model, a portion of exon 2 coding sequence (140 base pairs) of the rat Tf gene was substituted with the Human TF CDS (amino acids 20–698, Transcript: 201-ENST00000402696), followed by the rat 3′UTR and the SV40 polyadenylation sequence. Importantly, the murine signal peptide (amino acids 1–19), encoded by exon 1 and the 5′ sequences of rat exon 2, was retained. Consequently, this modified allele results in a TF protein encompassing the rat leader sequence and the human TF protein.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Sequencing, Modification

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Tissue-specific mRNA and protein expression of TF and TfR1 in heterozygous KI rats . qRT-PCR analysis of human TFRC and TF ( left panels ) and rat Tfrc and Tf ( right panels ) was performed on brain, heart, kidney, liver, and spleen tissues from male and female Tf h/w : Tfrc h/w rats (n = 4 per sex). Human TF expression was largely restricted to the liver, similar to rat Tf , which was also liver-enriched ( upper panels ). Human TFRC mRNA was most abundant in the spleen, mirroring the expression pattern of endogenous rat Tfrc ( lower panels ). No significant sex differences were observed in TFRC expression, while both human and rat TF expressions were higher in males than in females in the liver. KI, knock-in; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing, Quantitative RT-PCR, Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Expression of human transferrin (TF), rat transferrin (Tf), and soluble TfR1 (sTfR1) in serum and CSF of KI rats . Human- and rat-specific ELISA assays were used to measure concentrations of TF and sTfR1 in serum and CSF from rats carrying various combinations of wildtype and humanized TF and TFRC alleles. A , human TF was detected only in animals carrying human Tf alleles, showing gene dosage-dependent expression, with the highest levels observed in Tf h/h rats. B , rat Tf levels decreased as the human TF allele dosage increased, indicating efficient replacement of rat Tf by human TF. C , human sTfR1 was present only in animals carrying humanized TFRC alleles, with levels corresponding to gene dosage: Tfrc h/h > Tfrc h/w ; undetectable in Tfrc w/w rats. D , E , human TF and sTfR1 were also detected in CSF at lower concentrations than in serum, confirming secretion of the human proteins into the CNS. Data represent mean ± SEM. Statistical comparisons were performed using two-way ANOVA with Tukey’s post hoc test; exact p -values are reported in . CNS, central nervous system; KI, knock-in; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Protein-level validation of human and rat TF/TfR1 expression in brain by Western blot and immunoprecipitation . A , from top to bottom, immunoblots show detection of human TF, rat Tf, monomeric human TfR1, monomeric rat Tfr1 and GAPDH, which was used as loading control. Human TF and TfR1 were detected only in animals carrying one or two humanized alleles at the respective loci, whereas rat Tf and Tfr1 were present only in animals carrying one or two endogenous (wildtype) alleles. These results confirm species-specific expression of each transgene and absence of antibody cross-reactivity. B , Western blot under nonreducing conditions using the same cortical samples as in A , prepared without β-mercaptoethanol. A ∼190 kDa band corresponding to dimeric TfR1 was observed. C , immunoprecipitation from cortical lysates using anti-rat Tfr1 antibody followed by Western blot with human-specific TfR1 antibody revealed chimeric dimers composed of human and rat TfR1 in heterozygous Tfrc h/w rats. All blots were loaded with equal protein amounts. Reducing (with β-mercaptoethanol) and nonreducing conditions were used to assess monomeric and dimeric TfR1 forms, respectively. Asterisks indicate nonspecific background bands. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human and rat TfR1 in vessel-like structures of the frontal cortex . A , species-specific immunofluorescence confirms that the anti-human TfR1 antibody ( green ) detects only human TfR1, while the anti-rat Tfr1 antibody ( green ) recognizes only the endogenous rat protein. B , dual labeling with anti-human TfR1 ( magenta ) and anti-rat Tfr1 ( green ) in brains of heterozygous Tfrc h/w rats reveals colocalization in vessel-like structures. No staining was observed with the anti-human TfR1 antibody in Tfrc w/w rats, confirming its specificity. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Immunofluorescence, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the duodenum . Sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were costained with antibodies specific for human TfR1 ( magenta ) and rat Tfr1( green ). Colocalization was observed only in heterozygous Tfrc h/w rats, while Tfrc w/w and Tfrc h/h rats showed exclusive expression of rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the kidney . Kidney sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the liver . Liver sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h genotypes were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization of human TfR1 and rat Tfr1 was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the lung . Lung sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization of human TfR1 and rat Tfr1 was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: CBC analysis across all nine genotypic combinations of humanized Tf and Tfrc knock-in rats . A , Leukocyte parameters—white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), and basophils (BAS)—are shown by genotype and sex, with normal physiological ranges indicated for reference. Two-way ANOVA revealed significant effects of genotype and sex, with a significant genotype × sex interaction specifically for NEU. Post hoc comparisons are reported in Table 2. B , erythrocyte parameters—including red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW)—are plotted across genotypes and sexes. Significant genotype effects were detected for all parameters (see Table 3 for full statistical details). C , platelet parameters—platelet count (PLT) and mean platelet volume (MPV)—were unaffected by genotype, sex, or their interaction, as determined by two-way ANOVA (see ). All data are presented as mean ± SEM. Normal reference ranges are indicated by shaded areas in each plot, where applicable.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Knock-In, Cell Characterization, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Altered systemic iron distribution in humanized Tf and Tfrc knock-in rats . Nonheme iron levels in spleen, liver, serum, heart, and hippocampus are shown across genotypes and sexes, along with spleen-to-liver iron ratios and liver–spleen iron regression plots. Two-way ANOVA revealed significant genotype effects in liver and spleen, with altered iron distribution patterns in humanized KI models. Data are represented as mean ± SEM; multiple comparisons are detailed in , and regression analysis results are summarized in .

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Prussian blue staining of liver sections reveals altered hepatic iron accumulation in Tf h/w : Tfrc h/h rats . Representative images of liver sections stained with Prussian blue to visualize nonheme iron deposition in male and female rats of the following genotypes: Tf w/w : Tfrc w/w , Tf h/w : Tfrc h/w , Tf h/w : Tfrc h/h . Increased staining intensity was observed in Tf h/w : Tfrc h/h animals of both sexes, indicating elevated hepatic iron content. White square indicate zoomed regions, and white arrows denote Prusian blue-positive iron deposits.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Altered hepatic hepcidin levels in a genotype and sex dependent manner . Hepatic hepcidin concentration were measured by ELISA in liver lysates from Tf w/w : Tfrc w/w , Tf h/w : Tfrc h/w , and Tf h/w : Tfrc h/h rats of both sexes (n = 5–10 per group). Male Tf h/w : Tfrc h/w and Tf h/w : Tfrc h/h rats exhibited decreased hepcidin levels compared with wildtype controls, whereas females showed overall lower hepcidin levels across genotypes. Data are presented as mean ± SEM. Statistical comparisons were performed using two-way ANOVA followed by Tukey’s multiple-comparison test, refer to text for detailed p -values. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: The BBB remains intact in humanized TfR1 rats . A , SDS-PAGE followed by Coomassie Blue staining of equal volumes of serum and CSF from rats with humanized alleles at the Tfrc and Tf gene loci. The protein content and pattern in CSF are similar between control rats ( Tf w/w : Tfrc w/w ) and those carrying heterozygous or homozygous humanized TfR1 alleles ( Tf w/w : Tfrc h/w and Tf w/w : Tfrc h/h ), as well as rats homozygous for both human Tf and Tfrc ( Tf h/h : Tfrc h/h ), indicating that BBB integrity is preserved following humanization. A ∼15 kDa band of unknown identity is visible in the CSF of one control ( Tf w/w : Tfrc w/w ) and one double-humanized rat( Tf h/h : Tfrc h/h ). Serum samples (diluted 1:10 in PBS prior to loading) show comparable protein patterns across genotypes. B , additional Tfrc h/h rat samples confirm the findings. While serum lanes display abundant protein bands, only a faint ∼70 kDa band is detected in CSF, consistent with the low protein content of normal CSF and exclusion of serum proteins by an intact BBB. C , quantification of BBB integrity by ELISA for albumin, a ∼66 kDa serum protein. CSF/serum albumin ratios were consistently low and showed no significant differences between wildtype, heterozygous, and homozygous humanized rats, confirming that the BBB remains functionally intact across all genotypes tested. BBB, blood–brain barrier; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: SDS Page, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Humanized TfR1 mediates receptor-dependent transcytosis of nanobody cargo NN-843 across the BBB . To assess functional activity of human TfR1, a bispecific nanobody, NN-843, composed of TfR1 binding carrier (Newrobus) fused to anti-TNFα inhibitor nanobody was administered subcutaneously to Tf w/w : Tfrc w/w and Tf w/w : Tfrc h/w rats. Brain, CSF, and serum samples were collected 24 h postdose and analyzed by ELISA. A , NN-843 demonstrated robust CNS penetration with a CSF/serum ratio ∼0.6 in Tf w/w : Tfrc h/w rats, whereas it showed no detectable penetration in Tf w/w : Tfrc w/w rats. B , NN-843 was enriched ∼4-fold in the parenchymal fraction (P.F.) than in the vascular fraction (V.F.), indicating preferential distribution to brain parenchyma. C , representative Western blots confirming fractionation quality and genotype specificity. GAPDH was detected in both V.F. and P.F. fractions, while GLUT1 was absent from P.F., confirming effective separation. Human TfR1 was detected only in Tf w/w : Tfrc h/w rats, whereas rat Tfr1 was present in all animals, validating correct genotyping. Data represent mean ± SEM (n = 4). Statistical analysis was performed using the Mann–Whitney test; ∗ p < 0.05. BBB, blood–brain barrier; CNS, central nervous system; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Functional Assay, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fractionation, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Generation of humanized Tf and Tfrc rats . A , The rat Tfrc gene consists of 19 exons, with the ATG start codon located in exon 2 and the TAA stop codon in exon 19. In our KI model, the last 32 base pairs (3′) of rat Tfrc gene exon 2, counting from the ATG start codon, were replaced with the human TFRC CDS (Transcript: 201-ENST00000360110), followed by the rat 3′ untranslated region (3′UTR) and the SV40 polyadenylation sequence. B , the rat Tf gene ( NM_001013110.1 ) is comprised of 17 exons, with the ATG start codon located in exon 1 and the TAA stop codon in exon 17. In the KI model, a portion of exon 2 coding sequence (140 base pairs) of the rat Tf gene was substituted with the Human TF CDS (amino acids 20–698, Transcript: 201-ENST00000402696), followed by the rat 3′UTR and the SV40 polyadenylation sequence. Importantly, the murine signal peptide (amino acids 1–19), encoded by exon 1 and the 5′ sequences of rat exon 2, was retained. Consequently, this modified allele results in a TF protein encompassing the rat leader sequence and the human TF protein.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Sequencing, Modification

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Tissue-specific mRNA and protein expression of TF and TfR1 in heterozygous KI rats . qRT-PCR analysis of human TFRC and TF ( left panels ) and rat Tfrc and Tf ( right panels ) was performed on brain, heart, kidney, liver, and spleen tissues from male and female Tf h/w : Tfrc h/w rats (n = 4 per sex). Human TF expression was largely restricted to the liver, similar to rat Tf , which was also liver-enriched ( upper panels ). Human TFRC mRNA was most abundant in the spleen, mirroring the expression pattern of endogenous rat Tfrc ( lower panels ). No significant sex differences were observed in TFRC expression, while both human and rat TF expressions were higher in males than in females in the liver. KI, knock-in; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing, Quantitative RT-PCR, Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Expression of human transferrin (TF), rat transferrin (Tf), and soluble TfR1 (sTfR1) in serum and CSF of KI rats . Human- and rat-specific ELISA assays were used to measure concentrations of TF and sTfR1 in serum and CSF from rats carrying various combinations of wildtype and humanized TF and TFRC alleles. A , human TF was detected only in animals carrying human Tf alleles, showing gene dosage-dependent expression, with the highest levels observed in Tf h/h rats. B , rat Tf levels decreased as the human TF allele dosage increased, indicating efficient replacement of rat Tf by human TF. C , human sTfR1 was present only in animals carrying humanized TFRC alleles, with levels corresponding to gene dosage: Tfrc h/h > Tfrc h/w ; undetectable in Tfrc w/w rats. D , E , human TF and sTfR1 were also detected in CSF at lower concentrations than in serum, confirming secretion of the human proteins into the CNS. Data represent mean ± SEM. Statistical comparisons were performed using two-way ANOVA with Tukey’s post hoc test; exact p -values are reported in . CNS, central nervous system; KI, knock-in; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Protein-level validation of human and rat TF/TfR1 expression in brain by Western blot and immunoprecipitation . A , from top to bottom, immunoblots show detection of human TF, rat Tf, monomeric human TfR1, monomeric rat Tfr1 and GAPDH, which was used as loading control. Human TF and TfR1 were detected only in animals carrying one or two humanized alleles at the respective loci, whereas rat Tf and Tfr1 were present only in animals carrying one or two endogenous (wildtype) alleles. These results confirm species-specific expression of each transgene and absence of antibody cross-reactivity. B , Western blot under nonreducing conditions using the same cortical samples as in A , prepared without β-mercaptoethanol. A ∼190 kDa band corresponding to dimeric TfR1 was observed. C , immunoprecipitation from cortical lysates using anti-rat Tfr1 antibody followed by Western blot with human-specific TfR1 antibody revealed chimeric dimers composed of human and rat TfR1 in heterozygous Tfrc h/w rats. All blots were loaded with equal protein amounts. Reducing (with β-mercaptoethanol) and nonreducing conditions were used to assess monomeric and dimeric TfR1 forms, respectively. Asterisks indicate nonspecific background bands. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human and rat TfR1 in vessel-like structures of the frontal cortex . A , species-specific immunofluorescence confirms that the anti-human TfR1 antibody ( green ) detects only human TfR1, while the anti-rat Tfr1 antibody ( green ) recognizes only the endogenous rat protein. B , dual labeling with anti-human TfR1 ( magenta ) and anti-rat Tfr1 ( green ) in brains of heterozygous Tfrc h/w rats reveals colocalization in vessel-like structures. No staining was observed with the anti-human TfR1 antibody in Tfrc w/w rats, confirming its specificity. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Immunofluorescence, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the duodenum . Sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were costained with antibodies specific for human TfR1 ( magenta ) and rat Tfr1( green ). Colocalization was observed only in heterozygous Tfrc h/w rats, while Tfrc w/w and Tfrc h/h rats showed exclusive expression of rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the kidney . Kidney sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the liver . Liver sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h genotypes were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization of human TfR1 and rat Tfr1 was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Colocalization of human TfR1 and rat Tfr1 in the lung . Lung sections from Tfrc w/w , Tfrc h/w , and Tfrc h/h rats were stained for both human TfR1 ( magenta ) and rat Tfr1 ( green ). Colocalization of human TfR1 and rat Tfr1 was observed only in Tfrc h/w rats, while Tfrc w/w and Tfrc h/h showed signal only for rat Tfr1 or human TfR1, respectively. Yellow arrows in the zoomed-in panel indicate colocalization in white spots within the region highlighted by the yellow square in the overlay. TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: CBC analysis across all nine genotypic combinations of humanized Tf and Tfrc knock-in rats . A , Leukocyte parameters—white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), and basophils (BAS)—are shown by genotype and sex, with normal physiological ranges indicated for reference. Two-way ANOVA revealed significant effects of genotype and sex, with a significant genotype × sex interaction specifically for NEU. Post hoc comparisons are reported in Table 2. B , erythrocyte parameters—including red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW)—are plotted across genotypes and sexes. Significant genotype effects were detected for all parameters (see Table 3 for full statistical details). C , platelet parameters—platelet count (PLT) and mean platelet volume (MPV)—were unaffected by genotype, sex, or their interaction, as determined by two-way ANOVA (see ). All data are presented as mean ± SEM. Normal reference ranges are indicated by shaded areas in each plot, where applicable.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Knock-In, Cell Characterization, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Altered systemic iron distribution in humanized Tf and Tfrc knock-in rats . Nonheme iron levels in spleen, liver, serum, heart, and hippocampus are shown across genotypes and sexes, along with spleen-to-liver iron ratios and liver–spleen iron regression plots. Two-way ANOVA revealed significant genotype effects in liver and spleen, with altered iron distribution patterns in humanized KI models. Data are represented as mean ± SEM; multiple comparisons are detailed in , and regression analysis results are summarized in .

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Knock-In

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Prussian blue staining of liver sections reveals altered hepatic iron accumulation in Tf h/w : Tfrc h/h rats . Representative images of liver sections stained with Prussian blue to visualize nonheme iron deposition in male and female rats of the following genotypes: Tf w/w : Tfrc w/w , Tf h/w : Tfrc h/w , Tf h/w : Tfrc h/h . Increased staining intensity was observed in Tf h/w : Tfrc h/h animals of both sexes, indicating elevated hepatic iron content. White square indicate zoomed regions, and white arrows denote Prusian blue-positive iron deposits.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Altered hepatic hepcidin levels in a genotype and sex dependent manner . Hepatic hepcidin concentration were measured by ELISA in liver lysates from Tf w/w : Tfrc w/w , Tf h/w : Tfrc h/w , and Tf h/w : Tfrc h/h rats of both sexes (n = 5–10 per group). Male Tf h/w : Tfrc h/w and Tf h/w : Tfrc h/h rats exhibited decreased hepcidin levels compared with wildtype controls, whereas females showed overall lower hepcidin levels across genotypes. Data are presented as mean ± SEM. Statistical comparisons were performed using two-way ANOVA followed by Tukey’s multiple-comparison test, refer to text for detailed p -values. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: The BBB remains intact in humanized TfR1 rats . A , SDS-PAGE followed by Coomassie Blue staining of equal volumes of serum and CSF from rats with humanized alleles at the Tfrc and Tf gene loci. The protein content and pattern in CSF are similar between control rats ( Tf w/w : Tfrc w/w ) and those carrying heterozygous or homozygous humanized TfR1 alleles ( Tf w/w : Tfrc h/w and Tf w/w : Tfrc h/h ), as well as rats homozygous for both human Tf and Tfrc ( Tf h/h : Tfrc h/h ), indicating that BBB integrity is preserved following humanization. A ∼15 kDa band of unknown identity is visible in the CSF of one control ( Tf w/w : Tfrc w/w ) and one double-humanized rat( Tf h/h : Tfrc h/h ). Serum samples (diluted 1:10 in PBS prior to loading) show comparable protein patterns across genotypes. B , additional Tfrc h/h rat samples confirm the findings. While serum lanes display abundant protein bands, only a faint ∼70 kDa band is detected in CSF, consistent with the low protein content of normal CSF and exclusion of serum proteins by an intact BBB. C , quantification of BBB integrity by ELISA for albumin, a ∼66 kDa serum protein. CSF/serum albumin ratios were consistently low and showed no significant differences between wildtype, heterozygous, and homozygous humanized rats, confirming that the BBB remains functionally intact across all genotypes tested. BBB, blood–brain barrier; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: SDS Page, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110995

Figure Lengend Snippet: Humanized TfR1 mediates receptor-dependent transcytosis of nanobody cargo NN-843 across the BBB . To assess functional activity of human TfR1, a bispecific nanobody, NN-843, composed of TfR1 binding carrier (Newrobus) fused to anti-TNFα inhibitor nanobody was administered subcutaneously to Tf w/w : Tfrc w/w and Tf w/w : Tfrc h/w rats. Brain, CSF, and serum samples were collected 24 h postdose and analyzed by ELISA. A , NN-843 demonstrated robust CNS penetration with a CSF/serum ratio ∼0.6 in Tf w/w : Tfrc h/w rats, whereas it showed no detectable penetration in Tf w/w : Tfrc w/w rats. B , NN-843 was enriched ∼4-fold in the parenchymal fraction (P.F.) than in the vascular fraction (V.F.), indicating preferential distribution to brain parenchyma. C , representative Western blots confirming fractionation quality and genotype specificity. GAPDH was detected in both V.F. and P.F. fractions, while GLUT1 was absent from P.F., confirming effective separation. Human TfR1 was detected only in Tf w/w : Tfrc h/w rats, whereas rat Tfr1 was present in all animals, validating correct genotyping. Data represent mean ± SEM (n = 4). Statistical analysis was performed using the Mann–Whitney test; ∗ p < 0.05. BBB, blood–brain barrier; CNS, central nervous system; TfR1, transferrin receptor 1.

Article Snippet: Reference gene Gapdh was detected with probe Rn01775763_g1, and target genes were detected with Hs00169070_m1 ( TF , human Transferrin), Rn01445482_m1 ( Tf , rat Transferrin), Hs00951083_m1 ( TFRC , human TfR1), and Rn01474701_m1 ( Tfrc , rat TfR1).

Techniques: Functional Assay, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fractionation, MANN-WHITNEY