Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton

  1. Bhaba K Das
  2. Lei Wang
  3. Toshifumi Fujiwara
  4. Jian Zhou
  5. Nukhet Aykin-Burns
  6. Kimberly J Krager
  7. Renny Lan
  8. Samuel G Mackintosh
  9. Ricky Edmondson
  10. Michael L Jennings
  11. Xiaofang Wang
  12. Jian Q Feng
  13. Tomasa Barrientos
  14. Jyoti Gogoi
  15. Aarthi Kannan
  16. Ling Gao
  17. Weirong Xing
  18. Subburaman Mohan  Is a corresponding author
  19. Haibo Zhao  Is a corresponding author
  1. Southern California Institute for Research and Education, United States
  2. Department of Orthopedics, The Third People’s Hospital of Hefei, Third Clinical College, Anhui Medical University, China
  3. Center for Osteoporosis and Metabolic Bone Diseases, Division of Endocrinology, Department of Internal Medicine, University of Arkansas for Medical Sciences, United States
  4. Department of Orthopedic Surgery, Kyushu University Hospital, Japan
  5. Department of Orthopedics, First Affiliated Hospital, Anhui Medical University, China
  6. Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, United States
  7. Department of Pediatrics, University of Arkansas for Medical Sciences, United States
  8. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, United States
  9. Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, United States
  10. Department of Biomedical Sciences, Texas A&M University, United States
  11. Department of Orthopedics, Duke University, United States
  12. Division of Dermatology, Department of medicine, Long Beach VA Healthcare System, United States
  13. Musculoskeletal Disease Center, VA Loma Linda Healthcare System, United States
9 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
Tfr1 is a major transferrin transporter and regulates cellular iron homeostasis in osteoclasts.

(A) Detection of mRNA expression of genes involved in iron uptake and export pathways in bone marrow monocytes (BMM), mononuclear pre-osteoclasts (pOC), and mature osteoclasts (OC) by real-time …

Figure 1—source data 1

Tfr1 is a major transferrin transporter and regulates cellular iron homeostasis in osteoclasts.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Graphic diagrams of iron in energy metabolism in mammalian cells (A) and cellular iron-uptake pathways (B).

C-I to C-V: mitochondrial respiration chain complex-I to complex-V; DMT1: divalent metal ion transporter 1; GLUT1: glucose transporter 1; ROS: reactive oxygen species; Slc1a5: glutamine transporter; …

Figure 2 with 9 supplements
Tfr1 myeloid conditional knockout female mice develop normally and display increased trabecular bone mass in femurs at 10-week-old and 6-month-old ages.

(A) Representative µCT images of distal femurs of 10-week-old female control (con) and Tfr1ΔLysM mice. scale bar = 1 mm. (B– F) µCT analysis of trabecular and cortical bone mass and structure of …

Figure 2—source data 1

Tfr1 myeloid conditional knockout female mice develop normally and display increased trabecular bone mass in femurs at 10-week-old and 6-month-old ages.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
The trabecular bone mass and structure among three different genotypes of control mice are similar.

μCT analysis of distal femurs of three different genotypes of 10-week-old male and female control mice. Tb: trabecular bone; BV/TV: bone volume/tissue volume; Tb.N: trabecular number; Tb.Th: …

Figure 2—figure supplement 1—source data 1

The trabecular bone mass and structure among three different genotypes of control mice are similar.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Partial deletion of Tfr1 by one-allele of LysM-Cre has no effects on bone mass and structure in male and female mice on C57BL6/J background.

µCT analysis of trabecular bone mass and structure of distal femurs from 10-week-old C57BL6/J background mice. Tb: trabecular bone; BV/TV: bone volume/tissue volume; Tb.N: trabecular number; Tb.Th: …

Figure 2—figure supplement 2—source data 1

Partial deletion of Tfr1 by one-allele of LysM-Cre has no effects on bone mass and structure in male and female mice on C57BL6/J background.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Loss of Tfr1 in myeloid osteoclast precursor cells did not alter the serum iron and hepcidin levels and had no effects on erythropoiesis in 10-week-old male and female mice.

The serum total iron concentration of 10-week-old control (con) and Tfr1ΔLysM mice was measured by a colorimetric based iron detection kit from Fisherscientic Inc. The serum hepcidin level was …

Figure 2—figure supplement 3—source data 1

Loss of Tfr1 in myeloid osteoclast precursor cells did not alter the serum iron and hepcidin levels and had no effects on erythropoiesis in 10-week-old male and female mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp3-data1-v2.xlsx
Figure 2—figure supplement 4
Tfr1 myeloid conditional knockout male mice develop normally and exhibit no significant changes in trabecular and cortical bone.

(A) Representative µCT images of distal femurs of 10-week-old male control (con) and Tfr1ΔLysM mice. scale bar = 1 mm. (B–F) µCT analysis of trabecular and cortical bone mass and structure of distal …

Figure 2—figure supplement 4—source data 1

Tfr1 myeloid conditional knockout male mice develop normally and exhibit no significant changes in trabecular and cortical bone.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp4-data1-v2.xlsx
Figure 2—figure supplement 5
Loss of Tfr1 in myeloid osteoclast precursor cells had less effects on trabecular bone mass in lumber vertebrae than femurs.

µCT analysis of lumber vertebrae of 10-week-old and 6-month-old control (con) and Tfr1ΔLysM mice. Tb: trabecular bone; BV/TV: bone volume/tissue volume; Tb.N: trabecular number; Tb.Th: trabecular …

Figure 2—figure supplement 5—source data 1

Loss of Tfr1 in myeloid osteoclast precursor cells had less effects on trabecular bone mass in lumber vertebrae than femurs.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp5-data1-v2.xlsx
Figure 2—figure supplement 6
Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts results in increased trabecular bone mass in distal femurs of 10-week-old male and female mice.

(A) The representative µCT images of distal femurs of male and female control (con) and Tfr1ΔCTSK mice on the 129×C57BL6J mixed background. scale bar = 1 mm. (B–F) µCT analysis of trabecular and …

Figure 2—figure supplement 6—source data 1

Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts results in increased trabecular bone mass in distal femurs of 10-week-old male and female mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp6-data1-v2.xlsx
Figure 2—figure supplement 7
Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts slightly increases trabecular thickness of lumber vertebrae of 10-week-old male and female mice.

(A) Representative µCT images of L4 lumber vertebra of male and female control (con) and conditional knockout (Tfr1ΔCTSK) mice on the 129×C57BL6J mixed background. scale bar = 1 mm. (B–E) µCT …

Figure 2—figure supplement 7—source data 1

Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts slightly increases trabecular thickness of lumber vertebrae of 10-week-old male and female mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp7-data1-v2.xlsx
Figure 2—figure supplement 8
Estrogen but not androgen synergistically with Tfr1-deficiency inhibits the mRNA expression of mitochondrial cytochrome c oxidase subunit I in osteoclast lineage cells.

Quantitative PCR detection of the mRNA levels of Tfrc, Fpn, and mt-Co1 in vehicle (v), estrogen (E2), and androgen (T) treated control (con) and Tfr1ΔLysM osteoclast lineage cells. The data are …

Figure 2—figure supplement 8—source data 1

Estrogen but not androgen synergistically with Tfr1-deficiency inhibits the mRNA expression of mitochondrial cytochrome c oxidase subunit I in osteoclast lineage cells.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp8-data1-v2.xlsx
Figure 2—figure supplement 9
The deletion efficiency of Tfr1 by LysM-Cre is more potent in the distal femur than in the lumber vertebra as examined by quantitative PCR.

The data are presented as mean ± SD. n=3. ** p<0.01 and *** p<0.001 vs control (con) or vertebra analyzed by one-way ANOVA.

Figure 2—figure supplement 9—source data 1

The deletion efficiency of Tfr1 by LysM-Cre is more potent in the distal femur than in the lumber vertebra as examined by quantitative PCR.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig2-figsupp9-data1-v2.xlsx
Figure 3 with 3 supplements
Loss of Tfr1 in myeloid osteoclast precursors has no impacts on osteoclast number and bone formation in 10-week-old female mice.

(A) Images of fast green and TRAP staining (scale bar = 200 μm) and (B–G) histomorphometric analysis of the metaphysis of decalcified distal femur histological sections of 10-week-old female control …

Figure 3—source data 1

Loss of Tfr1 in myeloid osteoclast precursors has no impacts on osteoclast number and bone formation in 10-week-old female mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Loss of Tfr1 in myeloid osteoclast precursors has no impacts on osteoclast number and bone formation in 10-week-old male mice.

(A) Images of fast green and TRAP staining (scale bar = 200 μm) and (B–G) histomorphometric analysis of the metaphysis of decalcified distal femur histological sections of 10-week-old male control …

Figure 3—figure supplement 1—source data 1

Loss of Tfr1 in myeloid osteoclast precursors has no impacts on osteoclast number and bone formation in 10-week-old male mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
The mRNA levels of osteoclast marker gene Acp5 and osteoblast marker gene Runx2 increase in the distal femurs of female Tfr1ΔLysM mice.

Quantitative PCR detect of the mRNA expression of Acp5 and Runx2 in the distal femurs of control (con) and Tfr1ΔLysM 10-week-old female mice. The data are presented as mean ± SD. n=3. * p<0.05 and …

Figure 3—figure supplement 2—source data 1

The mRNA levels of osteoclast marker gene Acp5 and osteoblast marker gene Runx2 increase in the distal femurs of female Tfr1ΔLysM mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts increases trabecular bone mass without influence on osteoclastogenesis in vivo.

(A) The low (12.5×) and high (100×) magnification of images of TRAP staining of histological sections of decalcified distal femurs of 10-week-old control (con) and Tfr1 conditional knockout (Tfr1ΔCTS…

Figure 3—figure supplement 3—source data 1

Deletion of Tfr1 in cathepsin K-Cre expressing osteoclasts increases trabecular bone mass without influence on osteoclastogenesis in vivo.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig3-figsupp3-data1-v2.xlsx
Figure 4 with 1 supplement
Deletion of Tfr1 in osteoclast myeloid precursors has no effects on osteoclast differentiation but attenuates cytoskeleton organization and bone resorption in mature osteoclasts cultured from female mice.

(A) TRAP staining (scale bar = 10 μm) and quantification of numbers of total and spreading osteoclasts (OCs) cultured on plastic 24-well dishes. n=6. (B) Quantitative PCR detection of the mRNA …

Figure 4—source data 1

Deletion of Tfr1 in osteoclast myeloid precursors has no effects on osteoclast differentiation but attenuates cytoskeleton organization and bone resorption in mature osteoclasts cultured from female mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig4-data1-v2.xlsx
Figure 4—source data 2

Immunoblotting for Figure 4C.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig4-data2-v2.tif
Figure 4—figure supplement 1
Deletion of Tfr1 in osteoclast myeloid precursors has no effects on osteoclast differentiation but attenuates cytoskeleton organization and bone resorption in mature osteoclasts cultured from male mice.

(A) TRAP staining (scale bar = 10 μm) and quantification of numbers of total and spreading osteoclasts (OCs) cultured on plastic 24-well dishes. n=6. (B) The protein level of Tfr1 and osteoclast …

Figure 4—figure supplement 1—source data 1

Deletion of Tfr1 in osteoclast myeloid precursors has no effects on osteoclast differentiation but attenuates cytoskeleton organization and bone resorption in mature osteoclasts cultured from male mice.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 1—source data 2

Immunoblotting for Figure 4—figure supplement 1B.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig4-figsupp1-data2-v2.tif
High dose of hemin but not ferric ammonium citrate rescues the phenotypes of Tfr1-deficient osteoclasts.

(A) and (B) TRAP staining and quantification of the number of spreading osteoclasts in control (con) and Tfr1ΔLysM osteoclast cultures. n=6. scale bar = 10 μm. (C) and (D) Staining of actin …

Figure 5—source data 1

High dose of hemin but not ferric ammonium citrate rescues the phenotypes of Tfr1-deficient osteoclasts.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
The mitochondrial oxidative phosphorylation is mostly affected by Tfr1-deficiency in mature osteoclasts.

(A) Heatmaps of proteins that are differentially regulated in control (con) and Tfr1ΔLysM bone marrow monocytes (BMM), mononuclear pre-osteoclasts (pOC), and mature osteoclasts (OC) identified by …

Figure 6—figure supplement 1
Quantification of immunoblotting bands in Figure 6D.

Each band in immunoblots was quantified by densitometry using the NIH ImageJ software. n=3 from three immunoblots. * p<0.05 and *** p<0.001 vs the corresponding vehicle-treated cells of the same …

Figure 6—figure supplement 1—source data 1

Quantification of immunoblotting bands in Figure 6D.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig6-figsupp1-data1-v2.xlsx
Loss of Tfr1 inhibits mitochondrial mass, reactive oxygen species (ROS) production, and membrane potential in mature osteoclasts.

(A) Quantification of mitochondrial mass by Mito Tracker green staining in control (con) and Tfr1ΔLysM bone marrow monocytes (BMM), mononuclear pre-osteoclasts (pOC), and mature osteoclasts (OC). (B)…

Figure 7—source data 1

Loss of Tfr1 inhibits mitochondrial mass, reactive oxygen species (ROS) production, and membrane potential in mature osteoclasts.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig7-data1-v2.xlsx
Tfr1-deletion in osteoclast lineage cells impairs mitochondrial and non-mitochondrial respirations in mature osteoclasts.

(A) A graphic illustration of oxygen consumption measured by a Seahorse Extracellular Flux analyzer. FCCP: carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone, a synthetic mitochondrial uncoupler. …

Figure 8—source data 1

Tfr1-deletion in osteoclast lineage cells impairs mitochondrial and non-mitochondrial respirations in mature osteoclasts.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig8-data1-v2.xlsx
Figure 9 with 1 supplement
Loss of Tfr1 in osteoclasts inhibits the macrophage colony-stimulating factor (M-CSF) stimulated activation of c-Src and Rac1, and overexpression of Hem1 partially recuses the cytoskeletal organization defect in Tfr1-null osteoclasts.

(A) A schematic map of cytoskeletal pathways generated by Ingenuity pathway analysis (IPA). The downregulated proteins are shown in green and upregulated proteins are marked in red orange. (B) …

Figure 9—source data 1

Loss of Tfr1 in osteoclasts inhibits the macrophage colony-stimulating factor (M-CSF) stimulated activation of c-Src and Rac1 and overexpression of Hem1 partially recuses the cytoskeletal organization defect in Tfr1-null osteoclasts.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig9-data1-v2.xlsx
Figure 9—source data 2

Immunoblotting for Figure 9B.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig9-data2-v2.tif
Figure 9—figure supplement 1
Quantification of immunoblotting bands in Figure 9B.

Each band in immunoblots was quantified by densitometry using the NIH ImageJ software. n=3 from three immunoblots. * p<0.05, ** p<0.01, and p<0.001 vs control (con) of the same time point; The data …

Figure 9—figure supplement 1—source data 1

Quantification of immunoblotting bands in Figure 9B.

https://cdn.elifesciences.org/articles/73539/elife-73539-fig9-figsupp1-data1-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain and strain background
(Mus musculus)
Tfrc-floxDr. Nancy C Andrews129Sv
Strain and strain background
(Mus musculus)
Tfrc-floxZhao LabC57BL6/J
Strain and strain background
(Mus musculus)
Lyz2-CreThe Jackson Laboratorycat#004781C57BL6/J
Strain and strain background
(Mus musculus)
Ctsk-CreDr. Takashi NakamuraC57BL6/J
Otheralpha-MEMMilliporeSigmaM0644cell culture medium
OtherBlasticidinMilliporeSigma203,350Antibiotics (2 μg/ml)
OtherDMEMMilliporeSigmaD-5648cell culture medium
OtherFetal bovine serum (FBS)Hyclonecell culture (10%)
Other10× penicillin-streptomycin- l-glutamineMilliporeSigmaG1146Antibiotics (1×)
Other10× trypsin/EDTAThermo-Fisher Scientific15400–054Antibiotics (1×)
Chemical compound and drugMouse apo-transferrinMilliporeSigmaT0523
Chemical compound and drugDihydrotestosteroneMilliporeSigmaD-07310–8 M
Chemical compound and drugEstrodiolMilliporeSigmaE225710–8 M
Chemical compound and drugferric ammonium citrate (FAC)MilliporeSigmaF5879
Chemical compound and drugFe59 ferric chloridePerkinElmer Inc
Chemical compound and drugHeminMilliporeSigmaH9039
Chemical compound and drugSodium bicarbonateMilliporeSigmaS5761
OtherAlbuminMilliporeSigmaA7906Blocking reagent (0.20% in PBS)
OtherAlexa Fluro-488 PhalloidinThermo-Fisher ScientificA12379Filament actin staining (1:400)
OtherGlycerolMilliporeSigmaG5516mounting reagent (80% in PBS)
OtherHoechst 33,342Thermo-Fisher ScientificH3570Nuclei staining (1:4,000 in PBS)
OtherParaformaldehydeMilliporeSigmaP6148immunofluorescent staining (4% in PBS)
OtherTriton X-100MilliporeSigmaT-9284immunofluorescent staining (0.1%)
OtherNaK tartrateMilliporeSigmaS6170TRAP staining
OtherNaphthol AS-BI
(phosphoric acid solution)
MilliporeSigma1,802TRAP staining
Other3,3’-diaminobenzidine
(DAB) tablets
MilliporeSigmaD-5905pit staining
Other30% H2O2MilliporeSigma216,763pit staining
OtherPeroxidase-conjugated WGA
(wheat germ agglutinin) lectin
MilliporeSigmaL-7017pit staining (20 μg/ml)
OthercOmplete EDTA-free protease inhibitor cocktailMilliporeSigma4693159001protease inhibitor
OtherEnhanced chemiluminescent detection reagents (ECL)MilliporeSigmaWBKLS0100immunoblotting
OtherPolyvinylidene difluoride membrane (PVDF)MilliporeSigmaIPVH00010immunoblotting
OtherRIPA bufferMilliporeSigmaR-0278cell lysate
AntibodyMouse monoclonal anti-cathepsin K (clone 182–12 G5)MilliporeSigmaMAB33241:2000
AntibodyMouse monoclonal anti-HA.11 (clone 16B12)Biolegend901,5131:5000
AntibodyMouse monoclonal anti-Tfr1 (clone H68.4)Thermo-Fisher Scientific13–68001:500
AntibodyMouse monoclonal anti-tubulin (clone DM1A)MilliporeSigmaT90261:3000
AntibodyMouse monoclonal Total OXPHOS Antibody CocktailAbcamab1104131:1000
AntibodyGoat polyclonal HRP-anti-mouse secondary antibodyCell Signaling Technology7,0761:5000
AntibodyGoat polyclonal HRP-anti-rabbit secondary antibodyCell Signaling Technology7,0741:5000
Commercial assay or kitHigh-capacity cDNA reverse transcription kitThermo-Fisher Scientific4368813
Commercial assay or kitRNeasy mini kitQiagen74,104
Commercial assay or kitSerum TRAcP-5bImmunodiagnostic SystemsSB-TR103
Commercial assay or kitRatLaps (CTx-I) EIAImmunodiagnostic SystemsAC-06F1
Commercial assay or kitRat/mouse PINP EIAImmunodiagnostic SystemsAC-33F1
Commercial assay or kitHepcidin Murine-Complete ELISA KitIntrinsic LifesciencesHMC-001
Commercial assay or kitPointe Scientific Iron/TIBC ReagentsFisherscientific23-666-320
Commercial assay or kitIron Assay KitAbcamab83366
Commercial assay or kitActive Arf1 pull-down and detection kitThermo-Fisher Scientific16,121
Commercial assay or kitActive Rac1 pull-down and detection kitThermo-Fisher Scientific16,118
Commercial assay or kitTMTsixplex isobaric Mass Tagging kitThermo-Fisher Scientific90,064
OtherMitoTracker Green fluorescenceThermo-Fisher ScientificM7514Mitochondrial assay reagent
OtherMitoSOX RedThermo-Fisher ScientificM36008Mitochondrial assay reagent
OtherJC-1 DyeThermo-Fisher ScientificT3168Mitochondrial assay reagent
Recombinant DNA reagentMurine Nckap1lDharmacon IncMMM1013-202769283cDNA template
Commercial assay or kitTransIT-LT1Mirus Bio LLCMIR2300DNA transfection reagent
Commercial assay or kitAcp5Thermo-Fisher ScientificMm00475698_m1Quantitative PCR (qPCR) primer
Commercial assay or kitCtskThermo-Fisher ScientificMm00484039_m1qPCR primer
Commercial assay or kitmt-Cox1Thermo-Fisher ScientificMm00432648_m1qPCR primer
Commercial assay or kitDcstampThermo-Fisher ScientificMm04209236_m1qPCR primer
Commercial assay or kitNfatc1Thermo-Fisher ScientificMm00479445_m1qPCR primer
Commercial assay or kitMfsd7cThermo-Fisher ScientificMm01302920_m1qPCR primer
Commercial assay or kitMrps2Thermo-Fisher ScientificMm00475529_m1qPCR primer
Commercial assay or kitRunx2Thermo-Fisher ScientificMm00501584_m1qPCR primer
Commercial assay or kitSlc11a2Thermo-Fisher ScientificMm00435363_m1qPCR primer
Commercial assay or kitSlc39a14Thermo-Fisher ScientificMm01317439_m1qPCR primer
Commercial assay or kitSlc40a1Thermo-Fisher ScientificMm01254822_m1qPCR primer
Commercial assay or kitSlc46a1Thermo-Fisher ScientificMm00546630_m1qPCR primer
Commercial assay or kitTfrcThermo-Fisher ScientificMm00441941_m1qPCR primer
Commercial assay or kitTfr2Thermo-Fisher ScientificMm00443703_m1qPCR primer

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