Journal: Cell Stem Cell

Article Title: Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging

doi: 10.1016/j.stem.2024.05.010

Figure Lengend Snippet:

Article Snippet: APC anti-human CD271 (clone ME20.4-1.H4) , Miltenyi , Cat# 130-113-418; RRID: AB_2733363.

Techniques: Recombinant, Reverse Transcription, DNA Library Preparation, DNA Sequencing, RNA Sequencing, Purification, Sequencing, Software, Sterility

Journal: Journal of Neuroscience

Article Title: Human Immunodeficiency Virus Type 1 Alters Brain-Derived Neurotrophic Factor Processing in Neurons

doi: 10.1523/jneurosci.0865-12.2012

Figure Lengend Snippet: Figure 5. p75NTR-dependent reduction of neuronal survival. A, B, CGCs were exposed to gp120(A)orproBDNF(50ng/ml;B)aloneorinthepresenceofK252a(100nM),TAT-Pep5(100 nM),orp75ba(10g/ml).InA,BDNF(50ng/ml)wasadded3hbeforegp120.Neuronalsurvival wasdetermined18hlaterasdescribedinFigure4.DataareexpressedasthemeanSEM.n 18. pˆ 0.05 vs proBDNF or gp120; *p 0.001 vs control (ANOVA and Sheffe’s test).

Article Snippet: TAT-Pep5 was purchased from EMD4 Biosciences. p75NTR antibody (clone 192-IgG) was from Santa Cruz Biotechnology.

Techniques: Control

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [35S]met–cys or [35S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [35S]met–cys or [35S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Pulse-chase analysis and purification on sucrose gradients of transmembrane forms of PLAP and p75NTR in transfected FRT cells. (A) Schemes of p75NTR and PLAP-PS321 (a fusion protein containing the ectodomain of PLAP and the transmembrane and the cytoplasmic domain of the PS321 NTR mutant). (B) Pulse-chase and TX-100 extraction of p75NTR and PLAP-PS321. FRT cells expressing p75NTR and PLAP-PS321 were labeled with [35S]cys or [35S]met–cys, respectively, and subsequently treated as described in Figure ​Figure2A.2A. (C) Purification of p75NTR- and PLAP-PS321–enriched fractions on sucrose density gradients. Labeled proteins of FRT cells expressing p75NTR and PLAP-PS321 were solubilized in TX-100 and purified as described in Figure ​Figure2B.2B. Both transmembrane proteins are soluble in TX-100 and do not float on sucrose density gradients.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Pulse Chase, Purification, Transfection, Mutagenesis, Expressing, Labeling

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [35S]met–cys or [35S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [35S]met–cys or [35S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Stable Transfection, Labeling, Immunoprecipitation

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored and transmembrane proteins after cholesterol depletion. (A) FRT cells expressing PLAP, NTR-PLAP, gD1-DAF, p75NTR, or PLAP-PS321 were grown on filters and treated with mevinolin (Mev.; 10 μM) for 48 h and M-β-CD (10 mM) for 60 min. Cells were then pulse labeled for 30 min, chased for 2 h, and biotinylated as usual. (B) Amounts of labeled proteins were quantified in three independent experiments and are shown as means ± SD. Hatched bars represent apical (Ap) proteins, and black bars represent basolateral (BL) proteins. Removal of cholesterol does not alter the surface expression of apical and basolateral GPI-anchored and transmembrane proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Labeling

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure 1. The most abundant p75 NTR oligomer is a trimer. Expression of p75 NTR in transfected HEK293 cells detected by nonreducing(non-red.)orreducingSDS-PAGEusingantibodiesspecificfortheICD(A–D)orECD(F)ofp75 NTR.Iodoacetamidewas used in the lysis buffer to inhibit disulfide bond formation during cell lysis, and its inclusion enhanced p75 NTR detection. A, In nonreducing conditions, p75 NTR migrated as a 72 kDa monomer and a 200 kDa oligomer, suggesting trimerization of the receptor. B, In nonreducing conditions, chemical crosslinking allowed the detection of p75 NTR species consistent with dimers resolvedat140kDa(2timesthemolecularmassofthe72kDamonomerinthiscondition).C,Reducingconditionsshifted the molecular weight of the monomer to 80–85 kDa, and a second band of 70–75 kDa was detected. The 250 mM DTT and 5 min at 95°C reduced the oligomers to monomers. D, After the addition of ME and 5 min at 95°C, p75 NTR oligomers were detectedat240kDaand175kDa,stronglysuggestingatrimeranddimer,respectively(2or3timesthemolecularmassof the80–85kDamonomerinthiscondition).E,Identificationofthe80–85kDaandthe70–75kDap75 NTR-positivebands found in reducing conditions. p75 NTR has one asparagine-linked glycosylation in the first cysteine-rich domain and several O-linked glycosylations in the ECD juxtamembrane region. Therefore, we tested whether the bands correspond to differentially glycosylated isoforms. Lysates from p75 NTR-transfected HEK293 cells were deglycosylated with N-glycanase, sialidase A, O-glycanase,orwiththethreeenzymestogether.N-glycanasereducedthemolecularmassofbothbands,suggestingthatbothare N-linkedglycosylated.NeithersialidaseAnorO-glycanaseaffectedp75 NTRmolecularmass.However,sequentialdeglycosylation using the three enzymes together shifted the molecular weights as follows: (1) the higher band shifted more than with N-glycanase alone and started to collapse on top of the lower band, confirming that this isoform is N- and O-linked glycosylated; and (2) the lower band was resolved at the same molecular mass as after the N-glycanase, suggesting that this isoform is only N-linked glycosylated but is missing O-linked oligosaccharides. Overall, the molecular mass shifts were partial after deglycosyla- tion. This may be due to iodoacetamide affecting the enzymatic activity and/or on the partial reducing efficacy of ME in the experimentalconditionsused,whichmayonlyincompletelyunfoldtheECDandconsequentlypartiallyexposetheoligosaccharides for enzymatic removal. F, Antibodies specific for the p75 NTR ECD displayed the same pattern of bands as the ICD antibody in nonreducing and DTT reducing conditions.

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques: Expressing, Transfection, Lysis, Molecular Weight, Glycoproteomics, Activity Assay

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure 2. p75 NTR oligomers are formed by full-length monomers, and p75 NTR monomers and trimers coexist at the plasma membrane. A, Electroelution of the 200 kDa complex found in nonreducing conditions, followed by DTT reduction of the resolubilizedprotein,showedthattheoligomersareformedbymonomersoffull-lengthp75 NTR(80–85kDa).B,Electroelution of the 240 kDa p75 NTR oligomer observed in the 3% ME 5 min at 95°C condition. The resolubilized protein was further reduced with DTT and showed that the oligomer is formed by monomers of full-length p75 NTR (80–85 kDa). C, Electroelution ofthe175kDaformobservedinthe3%ME5minat95°C.TheresolubilizedproteinwasfurtherreducedwithDTT,andthis showedthattheoligomerisformedbymonomersoffull-lengthp75 NTR(80–85kDa).D,p75 NTRoligomerizationattheplasma membranewasstudiedintransfectedandsurface-biotinylatedHEK293cells(Biotin).Monomersandtrimerscoexistatthecell surface.Biotin,Transfectedbutnotbiotinylatedcells.E,Administrationof50ng/mlofproNGFfor30or60mindidnotaffectthe p75 NTR monomer/trimer ratio at the cell surface in transfected HEK293 cells.

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques: Clinical Proteomics, Membrane, Transfection

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure 3. p75 NTR monomers and trimers are present in the mouse brain, DRGs in culture, and in PC12 cells. A–C, Endogenous p75 NTR oligomerization was studied in postnatal day 0 (P0) mouse cortical lysates. A, Nonreducing SDS-PAGE of C57BL/6 cortical lysatesshowedthatp75 NTRmonomersareresolvedat70kDa,whereasoligomersappearedat230kDa,stronglysupporting trimerizationofthereceptor.B,C,p75NTRflx/flxconditionalmicewereusedtostudythespecificityofthebands.B,Recombination of the conditional alleles with Cre under the control of EIIa promoter results in loss of 70 kDa and 230 kDa p75 NTR bands observedinnonreducingconditions.C,MEreducedthehighmolecularmassoligomersandshiftedthemolecularweightofthe monomers to 75 kDa. Ponceau red staining was used as the loading control. D, E, Endogenous p75 NTR oligomerization in the adultbrainwasstudiedin12-week-old(12W)mousehippocampallysates,afterloading150g/laneintheSDS-PAGE,andusing pilocarpine-inducedseizures(Pilo)toincreasep75 NTRendogenouslevels.D,Representativeblotdetectingp75 NTRbyregularECL. E, The same blot shown in D developed using enhanced ECL signal was necessary to detect the 230 kDa oligomers. F, p75 NTR

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques: SDS Page, Control, Staining

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure4. Thecysteine256inthetransmembranedomainisrequiredfortrimerization.Representativeimagesofnonreducing andreducing(3%MEand5minat95°C)SDS-PAGEdisplayingHEK293cellstransfectedwithp75 NTRpointmutations.Agraphic representation of p75 NTR mutations and the oligomers found for each one are displayed on top of each image. Wild-type p75 NTR

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques:

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure 5. The cysteine-rich domains are required for trimerization. Representative images of nonreducing and reducing (3% MEand5minat95°C)SDS-PAGEdisplayingHEK293cellstransfectedwithp75 NTRdeletionmutants.Agraphicrepresentationof p75 NTRdeletionsandtheoligomersfoundforeachonearedisplayedontopofeachimage.Wild-typep75 NTRisshownas“WT”(in nonreducing conditions, monomers run at 72 kDa and trimers at 200 kDa; in reducing conditions, monomers run at 80–85 kDa, dimers at 175 kDa, and trimers at 240 kDa). A, Deletion of the intracellular domain (ICD) did not affect trimerization(ICDmonomer:50kDa;ICDtrimer:150kDa).B,C,Deletionoftheextracellulardomain(ECD)orthefour cysteine-rich domains (CRDs) prevented trimerization, but dimers were detected. B, In nonreducing conditions, ECD mono- mers were detected at 25 kDa, ECD dimers at 50 kDa, CRDs monomers at 28 kDa, and CRDs dimers at 55 kDa. C, In reducing conditions, ECD monomers were detected at 27 kDa and CRDs monomers at 30 kDa. D, E, Deletion of the cysteine-richdomain1(CRD1)partiallypreventedtrimerizationandincreasedtheproportionofdimers.D,Innonreducingconditions, CRD1 monomers were detected at 60 kDa, dimers at 125 kDa, and trimers at 180 kDa. E, In reducing conditions, CRD1 monomers were detected at 75 kDa, dimers at 150 kDa, and trimers at 225 kDa. F, Deletion of the cysteine-rich domains 2–4 (CRD2–4)preventedtrimerization,butdimerswerestilldetected(CRD2–4monomer:40kDa,CRD2–4dimer:80kDa).

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques:

Journal: Journal of Neuroscience

Article Title: Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation

doi: 10.1523/jneurosci.0591-15.2015

Figure Lengend Snippet: Figure 6. p75 NTR trimerization is not required for proNGF-induced growth cone retraction. A, E17-dissociated hippocampal neurons were treated with proNGF (20 ng/ml) for 30 min, fixed, and stained for p75 NTR and actin. ProNGF induced growth cone retraction in endogenously p75 NTR-expressing hippocampal neurons. Quantification of growth cone area (m 2) was performed todeterminegrowthconeretraction.ErrorbarsindicatemeanSEM.B,E17-dissociatedhippocampalneuronsweretransfected withratwild-typep75 NTR-HA,C257A-HA,orCRD1-HAconstructs.AtDIV3,culturesweretreatedwithproNGFfor30min,fixed, andstainedforHAandactin.Quantificationofgrowthconearea(m 2)toassessgrowthconeretractionintransfectedp75 NTR-HA, C257A-HA, or CRD1-HA-positive neurons, and HA-negative (HA) neurons of the same cultures. Error bars indicate mean SEM. Statistical comparisons were made by Kruskal–Wallis nonparametric analysis of the variance test. n 3 independent experiments; at least 2 coverslips per experiment per condition were analyzed. *p 0.05. ns, Nonsignificant. C, Representative image of a p75 NTR-HA-positive and a p75 NTR-HA-negative neuron after proNGF treatment. Arrows indicate expanded growth cones. * indicates retracted growth cones. Scale bar, 20 m.

Article Snippet: Membranes were incubated with the following combination of antibodies: (1) antibody specific for the ICD of p75 NTR (1:10,000, 1 h at 20°C–25°C, Covance, catalog #PRB-602C), followed by anti-rabbit HRP secondary antibody (1:10,000, 1 h at 20°C–25°C, Calbiochem-Millipore); and (2) antibody specific for the ECD of p75 NTR (1:1000, 16 h at 4°C, R&D Systems, catalog #AF367), followed by anti-goat HRP secondary antibody (1:7000, 1 h at 20°C–25°C, Calbiochem-Millipore).

Techniques: Staining, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in p75NTR mRNA expression level at different steps relative to STEP-0: * P<0.05. (F) Cell morphology on 35th day of differentiation in STEP-3 medium. (G) Immunostaining for αA-crystallin in the same area as indicated in (F). (H) Immunostaining for SOX2 in the same area as in (G). (I) Merged image of αA-crystallin and SOX2 immunostaining. Scale bar in (F-I), 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Derivative Assay, Expressing, Immunostaining

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Changes in gene expression levels at each step of differentiation induction using Ex.4 the method. (A) SOX2 , (B) PAX6 , (C) p75NTR , (D) αA-crystallin , (E) type-IV collagen ; fold-changes are relative to gene expression level in STEP-0: * P<0.05, ** P<0.01, *** P<0.005. Ex, experiment.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.4 method. (A) Gross observation of cell aggregate. Reduced opacity is observed in certain areas. Scale bar, 2 mm. (B) H&E-stained specimen of cell aggregate. Scale bar, 200 µm. (C) Enlarged view of H&E staining in Area 1 of the specimen in (B). Cells displaying epithelial-like morphology have infiltrated inside the cell aggregate (yellow arrowhead). (D-G and I-L) Immunostaining of cell aggregates for (D and I) SOX2, (E and J) p75NTR, (F and K) αA-crystallin, and (G and L) type IV collagen. Blue in immunostaining images: DAPI staining of nuclei. (H) Enlarged view of H&E staining of Area 2 in the specimen in (B), where the cells on the surface of the aggregate have transitioned from a multilayer to a monolayer (white arrowhead). Scale bar in (C-L), 50 µm. Ex, experiment; H&E, hematoxylin and eosin.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Staining, Immunostaining

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) Stacked violin plots of stromal, osteochondrogenic, and fibroblastic gene expression in different stromal clusters. Corresponding stromal cell groups are indicated on the x-axis legend. The color bar indicates gene expression in each cluster. ( B ) mRNA fold change of CD81 in FACS soreted A1-A4 subsets (n=3). Results are shown as mRNA fold change after standardization with GAPDH levels. For comparison, expression level of CD81 in A4 is set as one fold. *: p<0.05 (Kruskal-Wallis test). ( C ) The relative contribution of each sorted stromal cell populations in ( B ) to total CFU-F. Data are given as a normalized mean percentage as indicated in the figure (n=3). The sum of CFU-F of all populations is defined as 100%. ( D ) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for CD271 (pink), CD81 (green), NCAM1 (cyan) and CD45 (yellow) and scanned with the OlympusVS120 slide scanner. Single staining for each marker is shown as indicated under each picture. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone ( b ), adipocytes ( a ), and capillaries (*) are indicated. ( E ) FFPE human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Scale bars represent 20 µm.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Gene Expression, Comparison, Expressing, Formalin-fixed Paraffin-Embedded, Staining, Marker

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) Dot plot of surface marker gene expression in different stromal cell clusters. Cluster numbers and corresponding stromal cell groups are indicated on the y-axis legend. Dot sizes represent the percentage of cells expressing a certain gene in each cluster and dot colors represent the scaled average expression of that gene. ( B ) FACS plots illustrating the gating strategy for the isolation of different stromal subsets. The displayed cell populations are indicated on top of the plot. Following exclusion of doublets, dead cells, CD45- and CD235a-expressing cells, CD45 low/- CD235a - CD71 - CD271 + cells (left panel) were gated based on CD52 and NCAM1 expression (middle panel). The resulting three populations were labelled as A-C (middle panel), corresponding to the stromal cell groups in ( A ) (A, CD52 - NCAM1 - ; B, CD52 - NCAM1 + ; C, CD52 + NCAM1 - ). CD45 - CD235a - CD71 - CD271 + CD52 - NCAM1 - (group A) cells were further divided based on CD81 expression and four populations were identified ( A1–A4 ) (right panel). A1, CD81 ++ ; A2, CD81 + ; A3; CD81 +/- ; A4, CD81 - . ( C ) CFU-F frequencies of sorted stromal cell populations as shown in ( B ). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=3–6). Symbol colors and x-axis labels correspond to the cell population colors in ( B ). *: p<0.05; ****: p<0.0001 (Kruskal-Wallis test). ( D ) In vitro differentiation capacity of sorted stromal cell populations (as indicated in B) towards the adipogenic, osteoblastic, and chondrogenic lineage. Non-induction controls are shown in the left panel. Scale bars represent 200 µm. A representative set of pictures from a total of three independent experiments is shown. ( F ) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Different staining combinations are shown as indicated under each picture to provide better visualization of individual staining obtained from the same FFPE slide. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone ( b ), adipocytes ( a ), and capillaries (*) are indicated.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Marker, Gene Expression, Expressing, Isolation, In Vitro, Formalin-fixed Paraffin-Embedded, Staining

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) CFU-F frequency of single-cell and bulk sorted MSSCs, i.e. sorted A1 cells (CD45 low/- CD235a - CD71 - CD271 + NCAM1 - CD52 - CD81 ++ ). Data are shown as individual data points and medians (horizontal lines), n=3. **: p<0.01 (Mann-Whitney test). ( B ) A dot plot overview of top-ranked ligand-receptor pairs between different bone marrow stromal clusters. P values are indicated by circle color. The means of the average expression levels are indicated by circle size. Scale bars are provided on the right side of the plot. Stromal cell cluster pairs are indicated by ‘cluster number_cluster number’ (y-axis labels). Dashed lines separate the different stromal clusters. HAGEP, highly adipocytic gene-expressing progenitors; Bal., balanced progenitors; Pre-ob, pre-osteoblasts; Pre-fb, pre-fibroblasts. Interacting molecule pairs are indicated in the x-axis labels. ( C ) UMAP (as in ) illustration of the normalized expression of genes involved in alpha 1 beta 1 integrin complex (a1b1 complex). ( D ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor genes. ( E ) Fold change of total number of CD34 + cells (left) and CD34 + CD90 + cells (right) produced after seven days in culture. Results are shown as fold change relative to the cell number of standard CD34 + culture without stroma support (No stroma). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=2).

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: MANN-WHITNEY, Expressing, Produced

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A–D, F–G ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in ( G ) is to better visualize PDGFA- and PDGFB-expressing cells. ( E ) Left panel: Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Left upper image: all markers are shown; left lower image: just NCAM1 channel is shown. Middle panel: confocal laser scanning analysis of BM biopsies co-stained with CD271 (red), NCAM1 (green), SPP1 (white), and DAPI (blue) presented as 3D orthographic cross-section view. Right panel: Single staining channel data for CD271 (red), NCAM1 (green), SPP1 (white), and corresponding blow-ups for indicated areas. NCAM1, SPP1 and CD271(NGFR). White scale bars represent 50 μm and yellow scale bars represent 25 μm. White dashed lines indicate the trabecular bone surface lining regions.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A–E, G–H, J ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. ( F ) Flow cytometric analysis of SPP1 expression of primary CD45 low/- CD235a - CD71 - CD271 + CD56 + cells (red histogram), CD45 low/- CD235a - CD71 - CD271 + CD56 - CD81 ++ cells (blue histogram) and corresponding isotype control (orange histogram). ( I ) CFU-F assay of sorted CD45 low/- CD235a - CD71 - CD271 + cells (100 cells/well) in the presence (upper panel) or absence (lower panel) of sorted bone marrow CD45 + cells (3x10 5 cells/well), respectively.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Expressing, Control

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 1. p75NTR is a marker for human muscle satellite cells and regenerating fibers. (A) p75NTR immunoreactivity in a satellite cell of adult skeletal muscle. (B) Confocal imaging for p75NTR and CD56/neural cell adhesion molecule (NCAM) on satellite cells in vivo. Laminin staining was used to define myofibers. In (A) and (B), arrows indicate positively stained satellite cells. (C) Percen- tages of CD56/NCAM-positive satellite cells expressing p75NTR in human adult skeletal muscle. Circles represent distinct tissue samples; horizontal bar represents mean. (D) Quantification of CD56/NCAM-positive regenerating myofibers. Black bars indicate average values for each group, dots represent distinct tissue samples. (E, F) p75NTR expression (F) on CD56/NCAM-positive (E) regenerating fibers. Stainings were performed on serial sections from a dermatomyositis (DM) muscle. Arrows indicate positively stained fibers. Scale bars = (A, B) 5 Km; (E, F) 10 Km. ***p G 0.001 versus control. CTRL, control; PM, polymyositis; IBM, inclusion body myositis; BMD, Becker muscular dystrophy.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Marker, Imaging, In Vivo, Staining, Expressing, Control

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 2. p75NTR is expressed in cultured muscle precursor cells and transiently upregulated upon differentiation. (A) Double immunofluorescence for p75NTR and CD56/NCAM in cultured myoblasts. (B) Upregulation of p75NTR mRNA levels upon dif- ferentiation. Quantitative real-time PCR for p75NTR was performed for each time point. For statistics, p75NTR transcript levels at distinct time points were compared with baseline (Day 0). (C) Flow cytometry experiments showing percentages of p75NTR- positive mononucleated myoblasts after exposure to differentiation medium. (D) Immunofluorescence for p75NTR and dystrophin in myotubes (Day 11). (E) Kinetics of p75NTR expression on myotubes and correlation with dystrophin induction. Costaining for p75NTR and dystrophin was performed on differentiating cultures. The percentages of p75NTR (square) or dystrophin (circle) or p75NTR-positive dystrophin-positive (triangle) myotubes were measured at different time points. Experiments were performed in triplicate, and at least 100 myotubes per replicate were counted. Similar observations were obtained in at least 3 independent experiments in at least 2 primary cell lines. Error bars represent SD. Scale bars = (A, D) 30 Km. *p G 0.05, ***p G 0.001.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Expressing

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 4. p75NTR regulates myogenesis and dystrophin expression in myotubes. (A) Fusion index in a blocking experi- ment with anti-p75NTR antibody or isotype control adminis- tered during cell differentiation. (B) Extent of myogenesis in p75NTRlow, p75NTRhigh, and p75NTRhigh myoblasts treated with anti-p75NTR blocking antibody. In (A) and (B), fusion was evaluated at Day 6 after differentiation induction. (C) p75NTR silencing in myoblasts and effects on myogenesis. Silencing efficiency and relative fusion index were evaluated at Day 8 after differentiation induction. p75NTR mRNA (first panel) and protein (second panel) levels in p75NTR-silenced myotubes were significantly lower than in control myotubes. p75NTR RNA interference decreased cell differentiation (third panel). (D) p75NTR silencing did not alter myoblast proliferation. Small- interfering RNA administration was performed at Day 0; cells were maintained in growth medium for a few days and counted. (E) Cell cycle analysis was detected by propidium iodide incor- poration in silenced myoblasts. Evaluation was performed at Day 3 after transfection with p75NTR or control siRNA. Cell cycle entry in p75NTR-silenced cells was unchanged. (F) Quantifica- tion of dystrophin, A-dystroglycan, and dysferlin-positive myo- tubes in differentiated cultures at Day 8 after differentiation induction. Shown experiments were performed in triplicate, and at least 100 myotubes per replicate were counted. Similar observations were obtained in at least 3 independent experi- ments. Error bars represent SD. Results in (A, C, D, and E) were confirmed in 2 primary cell lines. **p G 0.01, ***p G 0.001; ns, not significant.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Expressing, Blocking Assay, Control, Cell Differentiation, Small Interfering RNA, Cell Cycle Assay, Transfection