Journal: Journal of Virology

Article Title: H5N1 Virus-Like Particle Vaccine Elicits Cross-Reactive Neutralizing Antibodies That Preferentially Bind to the Oligomeric Form of Influenza Virus Hemagglutinin in Humans

doi: 10.1128/jvi.05406-11

Figure Lengend Snippet: FIG. 2. Characterization of bacterially expressed and purified H5N1 HA proteins. (A and B) Characterization of purified H5N1 HA proteins from E. coli by Superdex S-200 gel filtration chromatography. Purified H5N1 HA1 proteins with an intact N terminus (positions 1 to 320) (A) and HA1 with an N-terminal deletion (28 to 320) (B) were subjected to gel filtration. The panels show superimposed elution profiles of purified HA1 proteins (red lines) overlaid with calibration standards (gray lines). The elution volumes of protein species are shown in parentheses. (C and D) Steady-state binding equilibrium analysis of conformation-dependent human H5N1 neutralizing MAbs FLD21.40 (C) and FLA3.14 (D) at 10 g/ml to purified bacterially expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group and on a blank flow cell, free of peptide. Binding was recorded using a ProteOn system surface plasmon resonance biosensor instrument (Bio-Rad Labs, Hercules, CA). (E) Agglutination of human RBCs by properly folded oligomeric H5N1 rHA1(1-320) protein and its monomeric H5N1 rHA1(28-320) counterpart along with rgH5N1 virus. Serial dilutions of purified HA1 proteins were mixed with washed RBCs, and hemagglutination was read after 30 min at room temperature. Reassorted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1) was used as a positive control.

Article Snippet: Binding kinetics for the selected human vaccine sera and data analyses were calculated using Bio-Rad ProteOn manager software (version 2.0.1).

Techniques: Chromatography, Binding Assay, SPR Assay, Agglutination, Virus, Positive Control

Journal: Journal of Translational Medicine

Article Title: DB-1314, a novel DLL3-targeting ADC with DNA topoisomerase I inhibitor, exhibits promising safety profile and therapeutic efficacy in preclinical small cell lung cancer models

doi: 10.1186/s12967-024-05568-y

Figure Lengend Snippet: In vitro property characterization of anti-DLL3 ADC DB-1314. (A) Conjugated drug distribution was measured by hydrophobic interaction chromatography. (B) Epitope binning of DB131401 and Rovalpituzumab by BLI. His-tagged human DLL3 (60 nM) was loaded onto biosensor, followed by binding of the first antibody Rovalpituzumab at saturating concentration of 100 nM. Subsequently, either the saturation antibody Rovalpituzumab or the completion antibody DB131401 was applied. CH5 represented 100 nM DB131401. CH8 represented 100 nM Rovalpituzumab. Increment of layer thickness in the last step suggested that the epitopes of the two antibodies did not overlap. (C) Affinity of DB131401 and Rovalpituzumab binding to human DLL3 was characterized using SPR. Internalization of DB131401 and Rovalpituzumab tagged with Incucyte ® Fabfluor-pH sensitive dye in SHP-77 cancer cells (D) and highly expressing human HER3 HEK293 (H_DLL3 HEK293) cells (E) was analyzed by Incucyte ® Live-Cell Analysis System and quantified using the IncuCyte ® software. (F) In vitro bystander killing effect of DB-1314. DLL3-positove NCI-H82 cells and DLL3-negative Raji-Luc cells were cocultured for 5 days and incubated with DB-1314, followed by the measurement of viable Raji-Luc cell numbers by detecting the luciferase activity using the luminometer. (G) ADCC activity of DB-1314 was measured using isolated PBMCs cocultured with NCI-H82 cells Abbreviations: BLI, biolayer interferometry; PBMC, peripheral blood mononuclear cell; ADCC, antibody-dependent cell-mediated cytotoxicity; LDH, lactate dehydrogenase; ADC, antibody-drug conjugate; DLL3, Delta-like ligand 3; SPR, surface plasmon resonance; iso-IgG1, isotype IgG1; conc., concentration

Article Snippet: Antibodies including DB131401, Rovalpituzumab, and human IgG1 isotype control were labeled with Incucyte ® Human Fabfluor-pH Red Antibody Labeling Reagent (Sartorius, Germany) at 1:3 molar ratio for 15 min and subsequently added to cells.

Techniques: In Vitro, Hydrophobic Interaction Chromatography, Binding Assay, Concentration Assay, Expressing, Cell Analysis, Software, Incubation, Luciferase, Activity Assay, Isolation, SPR Assay

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B supports SARS-CoV-2 infection, and TMEM106B-specific monoclonal antibodies are internalized into cells, related to Figures 1 and (A) Confirmation of TMEM106B and ACE2 knockout in monoclonal NCI-H1975 cell lines generated by CRISPR-Cas9. For each sgRNA, the target sequence is shown. The cut site is indicated by an arrowhead, and the protospacer adjacent motif (PAM) is underlined. Wild-type (WT) sequences of the corresponding exons were determined by Sanger sequencing and are presented with chromatograms. Sequences of knockout cells were determined by next-generation sequencing. For each detected sequence variant, the detection frequency and the type of mutation are shown. (B) NCI-H1975 cells expressing sgRNAs targeting ACE2 (monoclonal) or both ACE2 and TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Viral RNA in cells was measured by qPCR at the indicated time points (n = 8 wells examined over two independent experiments.). p values for differences between ACE2 KO and ACE2 KO / TMEM106B KO on day 1 were calculated using Mann-Whitney test with Holm-Šídák correction for multiple comparisons. ∗∗∗ 0.0001 < p < 0.001. (C) Huh7 cells transduced with sgRNAs targeting ACE2 and with cDNA encoding luciferase ( Luc ) or TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cells were stained for nucleocapsid after 48 h. Infected cells were quantified by high-content imaging analysis (n = 8 wells examined over two independent experiments). Data were analyzed using two-sided unpaired t test with Welch’s correction. Data are mean ± SEM. ∗∗∗∗ p < 0.0001. (D) Binding of TMEM106B-specific monoclonal antibodies to A549 cells stably overexpressing TMEM106B . Cells were incubated with antibody at the indicated concentrations and stained with Alexa Fluor 647-labeled anti-human IgG. The geometric mean fluorescence intensity (GMFI) compared with an hIgG1 isotype control is shown. (E) Binding of Ab09 to recombinant AviHis-TMEM106B-luminal domain (LD) was confirmed by surface plasmon resonance. Ab09 bound to recombinant AviHis-TMEM-LD that was captured by immobilized rabbit anti-Avi antibody. This confirmed that the epitope bound by Ab09 was intact on the recombinant protein. (F) WT and TMEM106B KO NCI-H1975 cells incubated with Ab09 for 2 h at ambient temperature to assess antibody internalization. Extracellular Ab09 was stained on live cells (green), followed by fixation, permeabilization, and staining of both extracellular and intracellular Ab09 (red) and nuclei (blue). The presence of red foci in WT cells indicates the endocytic uptake of anti-TMEM106B. Scale bars, 20 μm. (G) NCI-H1975 cells incubated with Ab09 for 50 min at ambient temperature and stained for Ab09 (green), LAMP-1 (red), and nuclei (blue). Four representative images are shown. Scale bars, 10 μm.

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Infection, Bioprocessing, Knock-Out, Generated, CRISPR, Sequencing, Next-Generation Sequencing, Variant Assay, Mutagenesis, Expressing, MANN-WHITNEY, Transduction, Luciferase, Staining, Imaging, Binding Assay, Stable Transfection, Incubation, Labeling, Fluorescence, Control, Recombinant, SPR Assay

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: Different SARS-CoV-2 isolates can employ TMEM106B for infection, and spike substitution E484D enhances TMEM106B usage (A) NCI-H1975 cells expressing sgRNAs targeting ACE2 (monoclonal) or TMEM106B (polyclonal) infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cell viability was determined by MTS assay after 4 days (n = 6 wells from two experiments). Fit curves were calculated by least squares regression. (B) Huh7 cells transduced with luciferase ( Luc ) or TMEM106B cDNA and infected with SARS-CoV-2 isolates or HCoV-229E. Cells were stained for nucleocapsid (SARS-CoV-2) after 24 h or double-stranded RNA (dsRNA) (HCoV-229E) after 48 h (n = 9 wells from three experiments). Data were analyzed using two-sided unpaired t test with Welch’s correction. (C) NCI-H1975 wild-type (WT) or monoclonal TMEM106B KO cells infected with SARS-CoV-2 isolates at multiplicity of infection (MOI) 10 or HCoV-229E at MOI 2. Viral RNA in cells was measured by qPCR (SARS-CoV-2: n = 8 wells from three experiments; HCoV-229E: n = 4 wells from one experiment). p values for differences between WT and TMEM106B KO on day 1 were calculated using Mann-Whitney test with Holm-Šídák correction for multiple comparisons. (D) Left: alignment of spike protein sequences of SARS-CoV-2 stocks used in this study. Right: NCI-H1975 cells infected with different stocks of SARS-CoV-2 Belgium/GHB-03021/2020 at MOI 1. Viral RNA in cells was measured by qPCR (n = 4 wells from two experiments). (E) Close-up view of the interactions between ACE2 (cyan) and the SARS-CoV-2-receptor-binding domain (RBD; orange), with Glu484 in red. Side chains are shown for important residues at the ACE2-spike interface. (F) HCT-116 or Huh7 cells transduced with luciferase ( luc ), TMEM106B , or ACE2 cDNA and infected with pseudoparticles harboring SARS-CoV-2 spike (VSV-spike; sequence of isolate Belgium/GHB-03021 passage 6) containing the indicated substitutions. GFP expression was quantified 24 h post-infection (n = 6 wells [HCT-116] from two experiments or n = 3 wells [Huh7] from one of two experiments with similar results). Data were log-transformed and analyzed using two-way ANOVA with Tukey’s multiple comparison test. (B and F) Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ∗∗∗ 0.0001 < p < 0.001; ∗ 0.01 < p < 0.05; ns, not significant. See also Figure S1 .

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Infection, Expressing, MTS Assay, Transduction, Luciferase, Staining, MANN-WHITNEY, Binding Assay, Sequencing, Transformation Assay, Comparison

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B-specific monoclonal antibodies block SARS-CoV-2 entry (A) NCI-H1975 cells pretreated with TMEM106B-specific antibodies at 20 μg/mL and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Positive controls: remdesivir (Rem) and hamster anti-SARS-CoV-2 serum (αSARS2). Cell viability was determined by MTS assay after 3 days (n = 6 wells (infected; blue) or 4 wells (uninfected; red) from two experiments). (B) Heatmap representation of the viability of NCI-H1975 cells pretreated with different concentrations of TMEM106B-specific antibodies and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cell viability was determined by MTS assay after 3 days. (C) Correlation plot showing the ability of TMEM106B-specific antibodies to neutralize SARS-CoV-2 infection of NCI-H1975 cells at 2 μg/mL (y axis) and their ability to bind A549 cells overexpressing TMEM106B. Binding is expressed as the geometric mean fluorescence intensity (GMFI) relative to a hIgG1 isotype control. r, Spearman correlation. Fit curve was calculated by linear regression. (D) NCI-H1975 cells pretreated with TMEM106B-specific antibodies or remdesivir and infected with SARS-CoV-2 Belgium/GHB-03021/2020 at MOI 10. Viral RNA in cells was measured by qPCR (n = 3 wells from one of two experiments with similar results). (E) NCI-H1975 cells pretreated with TMEM106B-specific antibodies or remdesivir and infected with SARS-CoV-2 Belgium/GHB-03021/2020, HCoV-229E, or RSV. Cells were stained with crystal violet after 4 days. (F) NCI-H1975 cells pretreated with anti-TMEM106B (Ab09) or remdesivir and infected with SARS-CoV-2 Belgium/GHB-03021/2020, HCoV-229E, or RSV. After 24 h, cells were stained for nucleocapsid (SARS-CoV-2), dsRNA (HCoV-229E), or F (RSV). Left: representative confocal images. Scale bars, 100 μm. Right: quantification (SARS-CoV-2: n = 8–12 wells from three experiments; HCoV-229E and RSV: n = 6–9 wells from two experiments). Dotted line: lower detection limit. (G) Monoclonal TMEM106B KO or wild-type (WT) NCI-H1975 cells transduced with luciferase ( Luc ) or TMEM106B cDNA and stained with Ab09. Intensities were quantified using ImageJ (n = 4 wells), and representative images are shown. Scale bars, 50 μm. (H) NCI-H1975 cells treated at different time points with Ab09, anti-SARS-CoV-2, E64d, or remdesivir. At 11 h post-infection, viral RNA in cells was measured by qPCR (n = 4 wells from two experiments). Data are normalized to infected untreated cells. Fit curves were calculated by robust regression. (A, D, F, and G) Data are represented as mean ± SEM. See also Figure S1 .

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Bioprocessing, Blocking Assay, Infection, MTS Assay, Binding Assay, Fluorescence, Control, Staining, Transduction, Luciferase

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B directly interacts with the RBD of SARS-CoV-2 spike, related to Figure 3 (A) TMEM106B crystal structure is shown as sticks with 2Fo-Fc electron density in blue mesh (contoured at 1 RMSD) and positive and negative Fo-Fc density in green and red mesh (contoured at 3 RMSD), respectively. The regions shown correspond to α1 helix (residues 208–216), left, and glycosylated Asn151, right. Carbon atoms of TMEM106B amino acid residues are colored magenta (chain A) or pink (molecules related by crystal symmetry). Carbon atoms of NAG residues are shown in gray. Other atoms are colored according to the standard format: oxygen, red; nitrogen, blue; and sulfur, yellow. (B) Examples of 2D class averages of the trimeric spike ectodomain. TMEM106B LD is visible in some 2D class average (purple arrowheads). (C) Result of the classification of the spike particles into four 3D classes. The class containing 201,270 particles selected for further processing is boxed. (D) Unmasked 3D reconstruction using particles images selected after initial 3D classification (B). The cryo-EM map is shown as a semi-transparent surface with the feature corresponding to associated TMEM106B indicated with dotted purple circle. Fitted is an atomistic model of the spike trimer in 1RBD-up conformation (PDB: 7NTA ); RBD and NTD domains are indicated. (E) Results of focused 3D classification after signal subtraction, as detailed in the section. The displayed cryo-EM reconstructions were obtained after reversion to the original (non-subtracted) particles. One class selected for the final reconstruction is boxed. (F) Resolution and particle orientation metrics for final cryo-EM reconstructions. Half-map Fourier shell correlations (FSCs) and distribution of the refined particle orientations for the result of the final global non-uniform refinement, as implemented in cryoSPARC. (G) The final 3D reconstruction, colored by local resolution. (H) Half-map FSCs and particle orientation for local refinement using a soft mask covering TMEM106B LD and the associated RBD.

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B directly interacts with the RBD of SARS-CoV-2 spike (A) The crystal structure spanning residues 118–261 of human TMEM106B, shown as cartoons, colored by the rainbow gradient from N (blue) to C (red) terminus. The remainder of the protein, comprising the transmembrane region (TM, residues 97–117) and cytoplasmic tail (residues 1–96), is schematically represented as thick gray lines. Secondary structure elements (α1, β1–β7), N-acetylglucosamine (NAG) residues, Cys214–Cys253 disulfide, the TM, and the cytoplasmic tail are indicated. NAG residues are shown as sticks with carbon atoms in gray. (B) Cryo-EM map of the spike trimer in complex with TMEM106B LD . Protein chains are colored by protomer: subunits of the spike trimer in green, yellow, and blue and TMEM106B in magenta. The cryo-EM map features corresponding to glycans are light gray. (C) Local reconstruction of TMEM106B LD bound to the erect RBD within the spike trimer. The cryo-EM map is shown as a semi-transparent surface, colored as in (B). The atomistic models are placed by rigid body docking and shown as cartoons. The RBD regions showing protection from HDX in the presence of excess TMEM106B LD are colored dark green. (D) Biolayer interferometry results of S1 binding to immobilized TMEM106B. Data are represented as plots of variation of fractional saturation with S1 concentration for the S1 E484 (Wuhan-Hu-1; red) versus S1 D484 (Belgium/GHB-03021; blue) spike subunits. Symbols are measured values, and solid lines are computed best fits. (E) NCI-H1975 monoclonal TMEM106B KO cells transduced with wild-type (WT) or mutant TMEM106B cDNA, infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E. Cells were stained for dsRNA after 24 h (n = 12 wells from three experiments). Data were log-transformed and analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with WT TMEM106B. (F) NCI-H1975 cells or monoclonal TMEM106B KO cells transduced with WT or mutant TMEM106B cDNA, infected with SARS-CoV-2 VOC omicron. Viral RNA in cells was measured by qPCR at 0 and 24 h post-infection (n = 8 wells from two experiments). Data were log-transformed and analyzed using one-way ANOVA with Tukey’s multiple comparison test. (G) Close-up view of the spike-TMEM106B interface shown in (C). TMEM106B and the RBD are shown as purple and green cartoons with side chains of TMEM106B Met210 and Phe213 and spike Asp484 as sticks. Consistent with binding data (D and Figure S3 F), the model predicts that the three residues project into the protein-protein interface. (E and F) Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ns, not significant. See also Figures S2 , , and .

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Cryo-EM Sample Prep, Binding Assay, Concentration Assay, Transduction, Mutagenesis, Infection, Staining, Transformation Assay, Comparison

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: Spike residue D484 and TMEM106B residues M210 and F213 enhance spike-TMEM106B binding, related to Figure 3 (A) Differences in hydrogen-deuterium exchange (ΔHDX) between the spike S1 subunit of the Belgium/GHB-03021 isolate alone and when in the presence of excess TMEM106B. Negative values indicate protection and positive values deprotection from exchange in the presence of TMEM106B. The threshold of significance calculated with 98% CI at ±0.42 Da is indicated with a dashed gray line. Peptides are arranged from the N to C terminus according to their peptide center residue. See Table S3 and for HDX data tables and deuterium uptake plots. (B) Biolayer interferometry results of S1 binding to immobilized TMEM106B. Data are represented as the dependence of the observed rate constant on S1 concentration for S1 E484 (Wuhan-Hu-1) and S1 D484 (Belgium/GHB-03021). (C) Thermodynamic parameters for S1 E484 and S1 D484 subunit binding to immobilized TMEM106B. For each variant, k on was determined from the slope of the plot of k obs against (S1) for the association phase, k off was obtained from the intercept of the plot of k obs against (S1) for the association phase, K D kinetic was calculated as k off /k on , and K D equilibrium was calculated from the plot of the amplitude versus (S1). (D) Biolayer interferometry traces of 6 μM S1 D484 (blue) and 6 μM S1 D484 premixed with 8 μM ACE2 (green) binding to immobilized TMEM106B (luminal domain). (E) NCI-H1975 monoclonal TMEM106B knockout cells transduced with cDNA encoding wild-type (WT) TMEM106B or TMEM106B containing single amino acid changes, infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cells were stained for dsRNA after 24 h. Infected cells were quantified by high-content imaging analysis (n = 8 wells examined over two independent experiments). Data were log-transformed and analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with WT TMEM106B. (F) Biolayer interferometry traces of 13.5 μM S1 D484 binding to immobilized wild-type TMEM106B (blue) or mutant TMEM106B M210A/F213A (red). (G) NCI-H1975 monoclonal TMEM106B knockout cells transduced with cDNA encoding WT or mutant TMEM106B and stained with anti-TMEM106B (Ab09) and DAPI. Representative images are shown, scale bars, 10 μm. (H) WT or TMEM106B KO NCI-H1975 cells transduced with cDNA encoding human, mouse ( mus musculus ), hamster ( Mesocricetus auratus ), or African green monkey ( Chlorocebus sabaeus ) TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cell viability was determined by MTS assay after 3 days (n = 6 wells examined over two independent experiments). Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with TMEM106B KO cells. (E and G) Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ∗∗ 0.001 < p < 0.01; ∗ 0.01 < p < 0.05; ns, not significant.

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Residue, Binding Assay, Concentration Assay, Variant Assay, Knock-Out, Transduction, Infection, Staining, Imaging, Transformation Assay, Comparison, Mutagenesis, MTS Assay

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B is required for a post-endocytic stage of virus entry (A) Wild-type (WT) or monoclonal TMEM106B KO NCI-H1975 cells, untransduced (control) or transduced with ACE2 cDNA and infected with SARS-CoV-2 Belgium/GHB-03021/2020 at MOI 0.03. Viral RNA in cells was measured by qPCR (n = 8 wells from two experiments). (B) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E in the presence of anti-TMEM106B (Ab09). After 6 h (SARS-CoV-2) or 24 h (HCoV-229E), cells were stained for dsRNA (n = 8 wells from three experiments; untreated, n = 36). (C) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E pretreated with different concentrations of heparin or heparan sulfate. After 6 h (SARS-CoV-2) or 24 h (HCoV-229E), cells were stained for dsRNA (n = 6 wells from two experiments; untreated, n = 28). (D) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , with or without an sgRNA targeting EXT1 ( EXT1 KO ), infected with SARS-CoV-2 Belgium/GHB-03021/2020. After 6 h, cells were stained for dsRNA (n = 8 wells from two experiments). (E) WT NCI-H1975 cells, ACE2 KO (monoclonal), TMEM106B KO (monoclonal), ACE2/EXT1 KO (monoclonal), or ACE2/EXT1/TMEM106B KO (polyclonal) cells, incubated with SARS-CoV-2 Belgium/GHB-03021/2020 on ice. Viral RNA bound on cells was measured by qPCR (n = 12 wells from two experiments). Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with WT cells. (F) NCI-H1975 cells, untransduced (control) or transduced with ACE2 cDNA, treated with E64d or camostat, infected with SARS-CoV-2 Belgium/GHB-03021/2020, and stained for nucleocapsid after 6 h (n = 6 wells from two experiments). (G) NCI-H1975 WT or monoclonal TMEM106B KO cells, incubated with SARS-CoV-2 at MOI 8 on ice, followed by virus internalization at 35°C for 0 or 2 h in the presence of 20 μg/mL cycloheximide to block translation. Cells were stained for nucleocapsid before permeabilization (green) and after permeabilization (red) and nuclei (blue). Shown are representative images, scale bars, 10 μm. A magnification of the area in the square is shown in each upper right corner. (H) Quantified results from (G) (n = 12 wells from two experiments.). (B–D, F, and H) Upper dotted line: untreated level. Lower dotted line: detection limit. Data were log-transformed (B, C, and F) and analyzed using two-way ANOVA with Tukey’s (H), Šidák’s (B and D), or Dunnett’s (C and F) multiple comparison test, comparing each condition with the untreated control. (I) HEK293T cells co-transfected with three plasmids, encoding (1) SARS-CoV-2 Belgium/GHB-03021/2020 spike and mNeonGreen, (2) TMPRSS2, and (3) a receptor (ACE2 or TMEM106B) or control protein (Luc). Left: representative images, scale bars, 100 μm. Right: quantified syncytium area (n = 2 wells from one of two experiments with similar results). The area under the curve was calculated, followed by one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with Luc. (C, F, and I) Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ∗∗∗ 0.0001 < p < 0.001; ∗∗ 0.001 < p < 0.01; ∗ 0.01 < p < 0.05; ns, not significant. See also Figure S5 .

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Virus, Control, Transduction, Infection, Staining, Incubation, Comparison, Blocking Assay, Transformation Assay, Transfection

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: Analysis of TMEM106B cell surface expression, SARS-CoV-2 uptake into TMEM106B KO cells, and ACE2 expression in various cell lines, related to Figures 4 and (A) Confirmation of EXT1 knockout in monoclonal NCI-H1975 cell lines generated by CRISPR-Cas9. The cut site within the sgRNA is indicated by an arrowhead, and the protospacer adjacent motif (PAM) is underlined. Sequences of wild-type and EXT1 knockout cells were determined by Sanger sequencing and are shown as chromatograms. Inserted nucleotides are shown in red. (B and C) Wild-type (WT) or TMEM106B KO NCI-H1975 cells stained for TMEM106B (Ab09; green), membranes (CellBrite Fix 640; red), and nuclei (blue). Shown are representative images from one out of two independent experiments with similar results. Cells were either permeabilized before staining (B) or not permeabilized (C) to visualize only TMEM106B expressed on the cell surface. Scale bars, 10 μm. (D) WT or TMEM106B KO NCI-H1975 cells incubated with SARS-CoV-2 at MOI 1 for 24 h and stained for SARS-CoV-2 N (red), LAMP-1 (green), and nuclei (blue). Shown are representative images from one out of three independent experiments with similar results. Scale bars, 10 μm. Note that WT cells show more widespread N staining due to the translation of new N protein during productive infection. (E) Analysis of ACE2 expression levels in different cell lines. Lysates of the indicated wild-type cell lines or HEK293T cells transduced with an ACE2 overexpression construct were analyzed using a ProteinSimple Wes system, with antibodies specific for ACE2 and the endogenous controls vinculin and GAPDH.

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Expressing, Knock-Out, Generated, CRISPR, Sequencing, Staining, Incubation, Infection, Transduction, Over Expression, Construct

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: Cells from various organs support SARS-CoV-2 infection via TMEM106B (A) U-87 MG cells transduced with sgRNAs targeting TMEM106B or safe harbor locus AAVS1 , infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E, fixed after 24 (SARS-CoV-2) or 96 h (HCoV-229E). (B) Patient-derived glioblastoma cells infected with SARS-CoV-2 Belgium/GHB-03021/2020 in the presence of anti-TMEM106B (Ab09), fixed after 24 h. (C) iPSC-derived astrocytes infected with SARS-CoV-2 Belgium/GHB-03021/2020 in the presence of Ab09, fixed after 48 h. (D) HIEC-6 cells infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E in the presence of Ab09, fixed after 96 (SARS-CoV-2) or 72 h (HCoV-229E). (A–D) Cells were stained for nucleocapsid (SARS-CoV-2) or dsRNA (HCoV-229E). Data were analyzed using two-sided unpaired t test with Welch’s correction (n = 6 wells—A, B, and D—or n = 8 wells—C —from two experiments). Upper dotted line: untreated level. Lower dotted line: detection limit. Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ∗∗∗ 0.0001 < p < 0.001; ∗∗ 0.001 < p < 0.01; ∗ 0.01 < p < 0.05; ns, not significant. (E) Hypothetical model summarizing the two SARS-CoV-2 infection mechanisms characterized here. See also Figure S5 .

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Infection, Transduction, Derivative Assay, Staining

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet:

Article Snippet: Mouse: C57BL/6N TMEM106B knockout (female) , Taconic, Rensselaer, NY , N/A.

Techniques: Virus, Recombinant, Transfection, Staining, Lysis, Proliferation Assay, Gene Expression, Expressing, Knock-Out, Plasmid Preparation, Construct, Software, Cell Analysis, Simple Western

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Recombinant monomeric tau binds to SORL1 and the VPS10 domain of SORL1. A , equilibrium analysis of the binding of increasing concentrations of recombinant 2N4R tau to full-length SORL1 ( blue circles ) and the SORL1 VPS10 domain ( orange triangles ) coupled to a Biacore CM5 sensor chip. b and c , recombinant SORL1 ( B ) or VPS10 ( C ) was immobilized on the chip and binding of 25 nM tau ± 500 nM VPS10 was assessed. Shown are representative images. D , binding of tau isoforms 2N4R, 0N3R, 2N3R, and tau MBD to SORL1 VPS10 domain assessed by SPR equilibrium analysis. E and F , the binding of tau produced by Sf9 cells along with two mutant forms of tau to SORL1 VPS10 domain ( E ) or SORL1 ( F ) was measured by SPR; 6A (T181, S199, S202, S396, S400, and S404 are all converted to alanine) and 6E, in which all these residues are converted to glutamic acid. G and H , binding of mutant forms of tau to SORL1 VPS10 domain ( G ) or SORL1 ( H ): 3XKQ in which lysine residues 311, 317, and 321 were converted to glutamine residues and 9XKQ tau in which lysine residues 311, 217, 321, 340, 343, 347, 353, 369, and 375 are all converted to glutamine residues. The data were normalized to Rmax to correct for slight differences in coupling to the SPR surfaces. Each experiment was repeated at least three times. Shown are mean ± SEM. MBD, microtubule-binding domain; SORL1, sortilin-related receptor 1; SPR, surface plasmon resonance; VPS10, vacuolar protein sorting 10.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Recombinant, Binding Assay, Produced, Mutagenesis, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Kinetic and equilibrium binding constants for the interaction of various forms of tau with the VPS10 domain of SORL1 and full length SORL1 a

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Binding Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 mediates the internalization of tau and colocalizes with internalized tau. WT CHO and LRP1-deficient CHO 13-5-1 cells were transfected with SORL1 plasmid or empty vector (Mock). A , transfection efficiency was validated via immunoblot analysis. B , transfected cells were incubated with 20 nM 125 I-labeled tau ± 1 μM RAP for 2 h, and the amount of tau internalized by the cells was quantified. (Mean ± SEM, two-way ANOVA followed by Tukey multiple comparisons test). C , H4 cells were transfected with SORL1-GFP plasmid, then incubated with 40 nM tau labeled with Alexa Flour 594 for 2 h. Cells were fixed and imaged on a Nikon spinning disk confocal microscope at 60×. Cells expressing SORL1 ( teal ) internalize more tau ( magenta ) than neighboring cells not expressing SORL1-GFP (nuclei marked by DAPI staining, blue ). Tau and SORL1 colocalization is represented in white in the merged image. The scale bar represents 10 μm. D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software. Shown is mean ± SD, n = 4. CHO, Chinese hamster ovary; DAPI, 4′,6-diamidino-2-phenylindole; LDL receptor-related protein 1; RAP, receptor associated protein; LRP1; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Transfection, Plasmid Preparation, Western Blot, Incubation, Labeling, Microscopy, Expressing, Staining, Single-cell Analysis, Software

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 knockdown does not impact tau uptake in H4 cells. A , time course for 125 I-tau internalization in H4 cells. Cells were incubated with 20 nM 125 I-labeled tau ± 1 μM RAP for designated times and internalized tau measured. B and C , H4 cells were treated with vehicle control, nonsense siRNA, or siRNA targeting SORL1, then incubated with 20 nM 125 I-labeled tau ± 1 μM RAP or 300 μg/ml R2629 for 2 h. B , SORL1 knockdown confirmed by immunoblot analysis. C , internalized tau was quantified. Shown are mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test, n = 3. RAP, receptor associated protein; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Knockdown, Incubation, Labeling, Control, Western Blot, Comparison

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Tau colocalizes with endogenous SORL1 in H4 cells. A , H4 cells were incubated with 40 nM tau labeled with Alexa Flour 594 for 2 h, then fixed and immunostained with anti-SORL1 antibody to label endogenous SORL1. The scale bar represents 10 μm. ( B ) Inset from panel ( A ) zoomed in to 200%, scale bar is 2 μm. All cells were fixed and imaged on Nikon spinning disk confocal microscope at 60×. Imaris Bitplane software was used to identify colocalization ( white ) between SORL1 ( teal ) and tau (magenta). Nuclei marked by DAPI staining, blue . C , single-cell analysis of the percent of tau that colocalizes with SORL1. Shown is mean ± SD, n = 7. DAPI, 4′,6-diamidino-2-phenylindole; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Incubation, Labeling, Microscopy, Software, Staining, Single-cell Analysis

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Tau binds SORL1 at pH 7.4 as well as pH 5.5. SPR analysis of tau binding LRP1 or SORL1 at pH 7.4 or pH 5.5. Increasing concentrations of tau (11, 33, 100, 300, and 900 nM were titrated on CM5 sensor chips coated with LRP1 ( A and B ) or SORL1 ( C and D ). The experiment was performed at pH 7.4 ( blue lines ) or pH 5.5 ( orange lines ). B and C , equilibrium fits of the data in panels a and c. Shown are mean ± SEM, n = 3. LRP1, LDL receptor-related protein 1; SORL1, sortilin-related receptor 1; SPR, surface plasmon resonance.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Binding Assay, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 transfection reconstitutes pathogenic internalization and seeding in HEK293T reporter cells. HEK293T Tau RD P301S FRET Biosensor cells were transfected with SORL1 , then incubated with ( A ) human brain homogenate from an Alzheimer’s patient (AD) or age-match healthy control (Ctrl) fraction or ( B ) HMW SEC fractions from AD patient brain (AD) or healthy control (Ctrl) and tau seeding was quantified. C , siRNA was used to knockdown SORL1 in H4 cells and was confirmed by immunoblot analysis, SORL1 band marked. Lane 1, nontransfected, lane 2, transfected with pcDNA SORL1, lane 3, control siRNA, and lane 4, SORL1 siRNA. D , H4 cells stably expressing the FRET reporter system with SORL1 knockdown were incubated with 300 ng/well AD brain derived HMW tau seeding material and tau seeding was quantified. (Mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test). SEC, size exclusion chromatography; HMW, high molecular weight; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Transfection, Incubation, Control, Knockdown, Western Blot, Stable Transfection, Expressing, Derivative Assay, Comparison, Size-exclusion Chromatography, High Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 harboring the N1358S mutation exhibits increased seeding capacity in HEK293T cells. HEK293T Tau RD P301S FRET Biosensor cells were transfected with plasmid containing WT SORL1 or SORL1 harboring the G511R or N1358S mutations. A , expression level of different variants was measured by Western blot (GAPDH was used as a loading control). These cells were incubated with ( B ) HMW or LMW SEC fractions from human AD patient brain or vehicle control and tau seeding was quantified. In a separate experiment, transfected cells expressing equal levels of the SORL1 variants ( C ) were incubated with ( D ) 20 nM 125 I-labeled tau in the presence or absence of 1 μM RAP for 2 h and then internalized tau was quantified. (Mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test). AD, Alzheimer’s disease; HMW, high molecular weight; LMW, low molecular weight; RAP, receptor associated protein; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Incubation, Labeling, Comparison, High Molecular Weight, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Model of LRP1 and SORL1-mediated tau trafficking. LRP1 mediates the internalization of monomeric forms of tau, resulting in rapid and efficient tau degradation. Internalized tau colocalizes with SORL1, in compartments that remain to be identified, and likely impacts tau trafficking through the endosomal pathways. Tau associates with SORL1 at endosomal pH of 5.5, but not LRP1, indicating a possible hand-off of tau from LRP1 to SORL1 as it traffics through endocytic pathways. LRP1 and SORL1 both mediate tau seeding in response to tau from human AD patient brains, and which could be through direct or indirect mechanisms. We postulate that AD pathogenic tau escapes the major degradative pathway to reach the extracellular space where it can induce cytoplasmic seeding. Furthermore, the AD-associated N1358S mutation in SORL1 increases the propensity of tau to seed aggregation, likely through impacting tau intracellular trafficking. Created with BioRender.com . AD, Alzheimer’s disease; LRP1, LDL receptor-related protein 1; SORL1, sortilin-related receptor 1.

Article Snippet: Slides were washed with DPBS, and in some cases anti-SORL1 antibody (Cell Signaling SORL1 (D8D4G) Rabbit mAb 79322) was used to label endogenously expressed SORL1.

Techniques: Mutagenesis

Journal: Journal of Translational Medicine

Article Title: DB-1314, a novel DLL3-targeting ADC with DNA topoisomerase I inhibitor, exhibits promising safety profile and therapeutic efficacy in preclinical small cell lung cancer models

doi: 10.1186/s12967-024-05568-y

Figure Lengend Snippet: In vitro property characterization of anti-DLL3 ADC DB-1314. (A) Conjugated drug distribution was measured by hydrophobic interaction chromatography. (B) Epitope binning of DB131401 and Rovalpituzumab by BLI. His-tagged human DLL3 (60 nM) was loaded onto biosensor, followed by binding of the first antibody Rovalpituzumab at saturating concentration of 100 nM. Subsequently, either the saturation antibody Rovalpituzumab or the completion antibody DB131401 was applied. CH5 represented 100 nM DB131401. CH8 represented 100 nM Rovalpituzumab. Increment of layer thickness in the last step suggested that the epitopes of the two antibodies did not overlap. (C) Affinity of DB131401 and Rovalpituzumab binding to human DLL3 was characterized using SPR. Internalization of DB131401 and Rovalpituzumab tagged with Incucyte ® Fabfluor-pH sensitive dye in SHP-77 cancer cells (D) and highly expressing human HER3 HEK293 (H_DLL3 HEK293) cells (E) was analyzed by Incucyte ® Live-Cell Analysis System and quantified using the IncuCyte ® software. (F) In vitro bystander killing effect of DB-1314. DLL3-positove NCI-H82 cells and DLL3-negative Raji-Luc cells were cocultured for 5 days and incubated with DB-1314, followed by the measurement of viable Raji-Luc cell numbers by detecting the luciferase activity using the luminometer. (G) ADCC activity of DB-1314 was measured using isolated PBMCs cocultured with NCI-H82 cells Abbreviations: BLI, biolayer interferometry; PBMC, peripheral blood mononuclear cell; ADCC, antibody-dependent cell-mediated cytotoxicity; LDH, lactate dehydrogenase; ADC, antibody-drug conjugate; DLL3, Delta-like ligand 3; SPR, surface plasmon resonance; iso-IgG1, isotype IgG1; conc., concentration

Article Snippet: Imaging was captured at the indicated time points by the Incucyte ® Live-Cell Analysis System (Sartorius, Germany) and internalization intensity was assessed using the IncuCyte ® software (Sartorius, Germany).

Techniques: In Vitro, Hydrophobic Interaction Chromatography, Binding Assay, Concentration Assay, Expressing, Cell Analysis, Software, Incubation, Luciferase, Activity Assay, Isolation, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Recombinant monomeric tau binds to SORL1 and the VPS10 domain of SORL1. A , equilibrium analysis of the binding of increasing concentrations of recombinant 2N4R tau to full-length SORL1 ( blue circles ) and the SORL1 VPS10 domain ( orange triangles ) coupled to a Biacore CM5 sensor chip. b and c , recombinant SORL1 ( B ) or VPS10 ( C ) was immobilized on the chip and binding of 25 nM tau ± 500 nM VPS10 was assessed. Shown are representative images. D , binding of tau isoforms 2N4R, 0N3R, 2N3R, and tau MBD to SORL1 VPS10 domain assessed by SPR equilibrium analysis. E and F , the binding of tau produced by Sf9 cells along with two mutant forms of tau to SORL1 VPS10 domain ( E ) or SORL1 ( F ) was measured by SPR; 6A (T181, S199, S202, S396, S400, and S404 are all converted to alanine) and 6E, in which all these residues are converted to glutamic acid. G and H , binding of mutant forms of tau to SORL1 VPS10 domain ( G ) or SORL1 ( H ): 3XKQ in which lysine residues 311, 317, and 321 were converted to glutamine residues and 9XKQ tau in which lysine residues 311, 217, 321, 340, 343, 347, 353, 369, and 375 are all converted to glutamine residues. The data were normalized to Rmax to correct for slight differences in coupling to the SPR surfaces. Each experiment was repeated at least three times. Shown are mean ± SEM. MBD, microtubule-binding domain; SORL1, sortilin-related receptor 1; SPR, surface plasmon resonance; VPS10, vacuolar protein sorting 10.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Recombinant, Binding Assay, Produced, Mutagenesis, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Kinetic and equilibrium binding constants for the interaction of various forms of tau with the VPS10 domain of SORL1 and full length SORL1 a

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Binding Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 mediates the internalization of tau and colocalizes with internalized tau. WT CHO and LRP1-deficient CHO 13-5-1 cells were transfected with SORL1 plasmid or empty vector (Mock). A , transfection efficiency was validated via immunoblot analysis. B , transfected cells were incubated with 20 nM 125 I-labeled tau ± 1 μM RAP for 2 h, and the amount of tau internalized by the cells was quantified. (Mean ± SEM, two-way ANOVA followed by Tukey multiple comparisons test). C , H4 cells were transfected with SORL1-GFP plasmid, then incubated with 40 nM tau labeled with Alexa Flour 594 for 2 h. Cells were fixed and imaged on a Nikon spinning disk confocal microscope at 60×. Cells expressing SORL1 ( teal ) internalize more tau ( magenta ) than neighboring cells not expressing SORL1-GFP (nuclei marked by DAPI staining, blue ). Tau and SORL1 colocalization is represented in white in the merged image. The scale bar represents 10 μm. D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software. Shown is mean ± SD, n = 4. CHO, Chinese hamster ovary; DAPI, 4′,6-diamidino-2-phenylindole; LDL receptor-related protein 1; RAP, receptor associated protein; LRP1; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Transfection, Plasmid Preparation, Western Blot, Incubation, Labeling, Microscopy, Expressing, Staining, Single-cell Analysis, Software

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 knockdown does not impact tau uptake in H4 cells. A , time course for 125 I-tau internalization in H4 cells. Cells were incubated with 20 nM 125 I-labeled tau ± 1 μM RAP for designated times and internalized tau measured. B and C , H4 cells were treated with vehicle control, nonsense siRNA, or siRNA targeting SORL1, then incubated with 20 nM 125 I-labeled tau ± 1 μM RAP or 300 μg/ml R2629 for 2 h. B , SORL1 knockdown confirmed by immunoblot analysis. C , internalized tau was quantified. Shown are mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test, n = 3. RAP, receptor associated protein; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Knockdown, Incubation, Labeling, Control, Western Blot, Comparison

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Tau colocalizes with endogenous SORL1 in H4 cells. A , H4 cells were incubated with 40 nM tau labeled with Alexa Flour 594 for 2 h, then fixed and immunostained with anti-SORL1 antibody to label endogenous SORL1. The scale bar represents 10 μm. ( B ) Inset from panel ( A ) zoomed in to 200%, scale bar is 2 μm. All cells were fixed and imaged on Nikon spinning disk confocal microscope at 60×. Imaris Bitplane software was used to identify colocalization ( white ) between SORL1 ( teal ) and tau (magenta). Nuclei marked by DAPI staining, blue . C , single-cell analysis of the percent of tau that colocalizes with SORL1. Shown is mean ± SD, n = 7. DAPI, 4′,6-diamidino-2-phenylindole; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Incubation, Labeling, Microscopy, Software, Staining, Single-cell Analysis

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Tau binds SORL1 at pH 7.4 as well as pH 5.5. SPR analysis of tau binding LRP1 or SORL1 at pH 7.4 or pH 5.5. Increasing concentrations of tau (11, 33, 100, 300, and 900 nM were titrated on CM5 sensor chips coated with LRP1 ( A and B ) or SORL1 ( C and D ). The experiment was performed at pH 7.4 ( blue lines ) or pH 5.5 ( orange lines ). B and C , equilibrium fits of the data in panels a and c. Shown are mean ± SEM, n = 3. LRP1, LDL receptor-related protein 1; SORL1, sortilin-related receptor 1; SPR, surface plasmon resonance.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Binding Assay, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 transfection reconstitutes pathogenic internalization and seeding in HEK293T reporter cells. HEK293T Tau RD P301S FRET Biosensor cells were transfected with SORL1 , then incubated with ( A ) human brain homogenate from an Alzheimer’s patient (AD) or age-match healthy control (Ctrl) fraction or ( B ) HMW SEC fractions from AD patient brain (AD) or healthy control (Ctrl) and tau seeding was quantified. C , siRNA was used to knockdown SORL1 in H4 cells and was confirmed by immunoblot analysis, SORL1 band marked. Lane 1, nontransfected, lane 2, transfected with pcDNA SORL1, lane 3, control siRNA, and lane 4, SORL1 siRNA. D , H4 cells stably expressing the FRET reporter system with SORL1 knockdown were incubated with 300 ng/well AD brain derived HMW tau seeding material and tau seeding was quantified. (Mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test). SEC, size exclusion chromatography; HMW, high molecular weight; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Transfection, Incubation, Control, Knockdown, Western Blot, Stable Transfection, Expressing, Derivative Assay, Comparison, Size-exclusion Chromatography, High Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: SORL1 harboring the N1358S mutation exhibits increased seeding capacity in HEK293T cells. HEK293T Tau RD P301S FRET Biosensor cells were transfected with plasmid containing WT SORL1 or SORL1 harboring the G511R or N1358S mutations. A , expression level of different variants was measured by Western blot (GAPDH was used as a loading control). These cells were incubated with ( B ) HMW or LMW SEC fractions from human AD patient brain or vehicle control and tau seeding was quantified. In a separate experiment, transfected cells expressing equal levels of the SORL1 variants ( C ) were incubated with ( D ) 20 nM 125 I-labeled tau in the presence or absence of 1 μM RAP for 2 h and then internalized tau was quantified. (Mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test). AD, Alzheimer’s disease; HMW, high molecular weight; LMW, low molecular weight; RAP, receptor associated protein; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Incubation, Labeling, Comparison, High Molecular Weight, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: SORL1 is a receptor for tau that promotes tau seeding

doi: 10.1016/j.jbc.2024.107313

Figure Lengend Snippet: Model of LRP1 and SORL1-mediated tau trafficking. LRP1 mediates the internalization of monomeric forms of tau, resulting in rapid and efficient tau degradation. Internalized tau colocalizes with SORL1, in compartments that remain to be identified, and likely impacts tau trafficking through the endosomal pathways. Tau associates with SORL1 at endosomal pH of 5.5, but not LRP1, indicating a possible hand-off of tau from LRP1 to SORL1 as it traffics through endocytic pathways. LRP1 and SORL1 both mediate tau seeding in response to tau from human AD patient brains, and which could be through direct or indirect mechanisms. We postulate that AD pathogenic tau escapes the major degradative pathway to reach the extracellular space where it can induce cytoplasmic seeding. Furthermore, the AD-associated N1358S mutation in SORL1 increases the propensity of tau to seed aggregation, likely through impacting tau intracellular trafficking. Created with BioRender.com . AD, Alzheimer’s disease; LRP1, LDL receptor-related protein 1; SORL1, sortilin-related receptor 1.

Article Snippet: D , single cell analysis of the percent of tau that colocalizes with SORL1-GFP using Imaris Bitplane Software.

Techniques: Mutagenesis

Journal: Journal of Molecular Biology

Article Title: Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation

doi: 10.1016/j.jmb.2020.166795

Figure Lengend Snippet: Defining the SKP2 binding site on cyclin A. (a) Surface plasmon resonance to measure the interaction between p27 and CDK2-cyclin A and CDK2-cyclin Amut5. GST-p27M (residues 1–106) was bound to a CM5 sensor-chip functionalized with anti-GST antibody and the CDK2-cyclin A complexes were flowed over at increasing concentrations. K d values were derived using Biacore S200 evaluation software. SPR sensorgrams are representative of two repeats using independently prepared protein samples. (b) Isothermal titration calorimetry (ITC) to measure the interaction between p27MS (residues 23–51) and CDK2-cyclin A complexes. K d values and reaction conditions are tabulated in . (c) Cyclin A mutants distinguish SKP2 and p27 binding sites on cyclin A and reveal their shared and divergent binding elements. ITC thermograms for the binding of p27MS (residues 23–51) and SKP1-SKP2N to cyclin A_W217K, cyclin A_I213D (within the cyclin A α1 MRAIL sequence), cyclin A_R293E (on cyclin α5). ITC thermograms for other mutants discussed in the text are included in Figure S3(A). ITC thermograms shown are of one of two replicates carried out using independently prepared proteins. Data analysis is described in .

Article Snippet: SPR analysis was carried out using a Biacore S200 (GE Healthcare).

Techniques: Binding Assay, SPR Assay, Derivative Assay, Software, Isothermal Titration Calorimetry, Sequencing

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: ( A ) Steps 2 and 3 of the CPD heme biosynthesis pathway. ( B ) The fold changes of proteins with significant changes in thermal stability after treatment with SC-43 (0.5 µg/mL) under different temperatures. ( C ) Melting curves for CpfC comparing DMSO (use M to represent) and SC-43 (0.5 µg/mL) (use S to represent) treatment. ( D ) Surface plasmon resonance (SPR) binding kinetic traces of the interactions of SC-43/vancomycin and CpfC. ( E ) Kinetic analysis of the inhibitory effect of SC-43 on CpfC ferrochelatase activity and the ferrochelatase activity of CpfC with Y12A, H181A, or E263A point mutations. 4 µM CpfC was assayed in the presence of varying concentrations of coproporphyrin III (CPIII) and a fixed concentration of 0.7 nM of iron. Michaelis–Menten and Lineweaver–Burk plots (insets), curves were generated using the kinetic constants obtained from nonlinear regression. 0 vs 200 nM, * P = 0.0412. Error bars indicate mean ± standard deviation from biological replicates ( n = 3). Two-way ANOVA, * P < 0.05. .

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Stability, SPR Assay, Binding Assay, Analysis, Activity Assay, Concentration Assay, Generated, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: The antibacterial activity of SC-43.

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Activity Assay

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: ( A ) Fe 2+ -bound porphyrins in S. aureus ATCC 29213 (SA 29213 ) after treatment with SC-43 (0.8 µg/mL), paired t -test. Control vs SC-43, ** P = 0.0022. ( B ) The intracellular ATP levels of SA 29213 after treatment with varying concentrations of SC-43. Control vs 0.4 µg/mL, **** P < 0.0001; 0.8 µg/mL, **** P < 0.0001; 1.6 µg/mL, **** P < 0.0001; 3.2 µg/mL, **** P < 0.0001. ( C ) The growth curves of SA 29213 in the medium in the presence or absence of varying concentrations of heme. Optical density at 600 nm was monitored at the indicated time points. Control vs 0.2 µg/mL SC-43, *** P = 0.0001; 0.2 µg/mL SC-43 vs 0.2 µg/mL SC-43 + 0.2 µg/mL heme, *** P = 0.0009; ( D ) The change in the membrane potential of SA 29213 incubated with varying concentrations of SC-43, indicated by fluorescence of the dye DiSC3(5) (0.5 μM). a.u. arbitrary units. Control vs 0.25 × MIC, * P = 0.0112; 0.5 × MIC, ** P = 0.0012; 1 × MIC , **** P < 0.0001; 2 × MIC, **** P < 0.0001. Error bars indicate mean ± standard deviation from biological replicates ( n = 3 for ( A – C ), n = 8 for ( D )). One-way ANOVA, **** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05. .

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Control, Membrane, Incubation, Fluorescence, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: ( A ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis illustrating the top pathways that have been enriched by differentially expressed metabolites after SC-43 treatment. The number of differentially expressed metabolites enriched in the corresponding pathways are displayed on the right side of the box. ( B ) The comparison of four differentially altered metabolites in the porphyrin metabolism pathway. Control vs 0.2 µg/mL SC-43, biliverdin, * P = 0.0331; coproporphyrinogen III, * P = 0.0408; coproporphyrin III, ** P = 0.0015; D-1-aminopropan-2-ol O-phosphate, **** P < 0.0001. Error bars indicate mean ± standard deviation from biological replicates ( n = 3). Hypergeometric test for ( A ), unpaired t -test for ( B ), **** P < 0.0001, ** P < 0.01, and * P < 0.05. .

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Analysis, Comparison, Control, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: ( A ) RMSD plots for the protein SA CpfC and the ligand SC-43 over the simulation period; ( B ) Time-course analysis of protein-ligand contacts; ( C ) Detailed schematic representation of atomic interactions between the ligand and specific protein residues; ( D ) Three-dimensional visualization of the ligand (carbon atoms in wheat) interacting with the protein (carbon atoms in greencyan); hydrogen bonds are shown as red dashed lines, and π–π interactions are indicated by magenta dashed lines. .

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Analysis, Docking Assay

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: Surface plasmon resonance (SPR) analysis of the interaction between SC-43 and SA CpfC wild-type and mutants.

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: SPR Assay, Analysis

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: ( A ) The dosing schedule and route of drug administration for the mouse model. ( B ) The size of infected wounds in mice treated with DMSO, SC-43 (0.1 mg/kg), and mupirocin (0.1 mg/kg) ( n = 20 per group). ( C ) The comparison of the size of the original wound with that of the wound on day 7. No infection vs Control, * P = 0.0190; No infection vs SC-43, **** P < 0.0001; Control vs SC-43, * P = 0.0111 ; ( D ) Bacterial loading of the wound site in mice treated with DMSO, SC-43 (0.1 mg/kg), and mupirocin (0.1 mg/kg) were recorded continuously. At day 3, No infection vs SC-43, ** P = 0.0077, No infection vs mupirocin, * P = 0.0189 ; At day 5, No infection vs SC-43, **** P < 0.0001; No infection vs mupirocin, ** P = 0.0086; At day 7, No infection vs SC-43, **** P < 0.0001; Error bars indicate mean ± standard deviation ( n = 5 per group); One-way ANOVA, **** P < 0.0001, ** P < 0.01, and * P < 0.05. .

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Infection, Comparison, Control, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: CpfC is a key enzyme in the process by which S. aureus synthesizes heme using the coproporphyrin-dependent (CPD) pathway, and it is responsible for catalyzing the insertion of iron ions into coproporphyrin III (CPIII). SC-43 can inhibit the activity of CpfC and affect the heme synthesis of S. aureus . Since heme is a cofactor of various complexes in the bacterial electron transport chain, SC-43 may potentially inhibit ATP synthesis by affecting the electron transport chain. The Y12, H181, and E263 of CpfC are the key sites that determine its interaction with SC-43.

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Activity Assay

Journal: EMBO Molecular Medicine

Article Title: SHP-1 agonist SC-43 limits methicillin-resistant Staphylococcus aureus infection through inhibition of heme biosynthesis

doi: 10.1038/s44321-026-00418-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: SC-43 , TargetMol , T8478.

Techniques: Recombinant, Sequencing, Saline, Software, Bicinchoninic Acid Protein Assay, ATP Bioluminescent Assay