mouse fzd2  (Addgene inc)

Journal: bioRxiv

Article Title: FZD2 inhibits YAP and prevents cell cycle reentry in adult murine cardiomyocytes

doi: 10.1101/2024.07.26.605158

Figure Lengend Snippet: A. Relative activity of TEAD-Gal4-UAS-Luc, a YAP reporter consisting of TEAD1 fused to the GAL4 DNA-binding domain and a UAS-driven firefly luciferase, in NVCMs transfected with empty vector or YAP1 expression plasmid and either control or FZD2 siRNA. B. Relative activity of the 8x-TEAD-Luc reporter, which uses 8 copies of the TEAD consensus binding site to express firefly luciferase, in NVCMs transfected with empty vector or YAP1-expression plasmid and control or FZD2 siRNA. C. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector or YAP expression plasmid with or without FZD2 expression vector. D. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP-expression vector, or expression plasmid for a YAP mutant with serine 127 mutated to alanine (YAP S127A) either alone or in combination with FZD2 expression vector. E. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP expression plasmid, or YAP S127A expression vector and either control or FZD2 siRNA. F. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector or YAP1 expression plasmid and FZD2 or β-catenin siRNA. G. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP1 expression vector, FZD2 expression vector, or both FZD2 and YAP1 vectors as well as control or β-catenin siRNA. Graphs represent the mean ± S.D, asterisks indicate p ≤ 0.05 by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: NVCMs were also transfected with pRK5- mFzd2 and pcDNA-Flag- Yap1 (AddGene, 42254 and 18881, respectively) and pCMV-Tag2 (Agilent Technologies, 211172) for empty vector controls.

Techniques: Activity Assay, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Control, Mutagenesis

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: HeLa wildtype (WT), stable FZD1/2/7 knockout (FZD1/2/7-KO), CSPG4-KO, and UGP2-KO cells were exposed to either recombinant TcdB1.1 ( a ), TcdB2.1 ( b ), TcdB7.2 ( g ), and TcdB12.1 ( l ), or culture supernatants from native C. difficile strains expressing TcdB3.1 ( c ), TcdB4.2 ( d ), TcdB5.1 ( e ), TcdB6.1 ( f ), TcdB8.3 ( h ), TcdB9.1 ( i ), TcdB10.1 ( j ), and TcdB11.2 ( k ) for 24 h. The percentages of round-shaped cells were plotted over toxin concentrations or supernatant dilutions. Error bars indicate mean ± s.d., N = 3 (biologically independent experiments). Strain information is listed in Supplementary Table . The activity of TcdB in each supernatant has been validated using a polyclonal TcdB antibody as shown in Supplementary Fig. . m Summary of the receptor preference of TcdB variants. The toxin concentrations inducing 50% of cell rounding (CR 50 ) were determined. The relative CR 50 values in different cell lines were normalized to WT and plotted as a bar-chart. The dependency on FZDs and CSPG4 are noted with plus and minus signs. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01; NS not significant (Student’s t -test, two-sided). n A list of residues across tested TcdB subtypes at 17 key positions mediating TcdB1.1-FZD2 interactions. These positions are based on the crystal structure of TcdB-FZD complex (PDB: 6C0B ) . o A list of residues across tested TcdB subtypes at 21 key positions mediating TcdB1.1-CSPG4 interactions. These positions are based on the cryo-EM structure of TcdB-CSPG4 complex (PDB: 7ML7 ) . Residue 1812 was highlighted with a dash box. Source data are provided as a Source Data file.

Article Snippet: 1D4-tagged full-length mouse FZD2 was obtained from Addgene (#42264).

Techniques: Knock-Out, Recombinant, Expressing, Activity Assay, Cryo-EM Sample Prep, Residue

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagram of the CRISPR-Cas9 screen process. b Genes identified by NGS were analyzed with the MAGeCK program and plotted based on the log 2 value of fold change of NGS reads and statistical significance (shown as log 10 value of RRA p -value and plotted as the y -axis). The genes involved in the GPI (glycosylphosphatidylinositol) biosynthetic pathway are colored red. c Schematic diagram of human TFPIβ (TFPI), a GPI-anchored protein with two BPTI/Kunitz protease inhibitor domains (K1 and K2). N, N-termini; C, C-termini. HeLa ( d ) or A549 ( e ) KO cells lacking TFPI, TFPI2 (a homolog of TFPI), PIGS, or PIGV were generated via the CRISPR-Cas9 approach. UGP2-KO cells were also analyzed as a control. Cells were exposed to recombinant TcdB4.2 for 24 h. Their CR 50 values are normalized to WT and plotted in a bar-chart ( f ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). g – i HeLa ( g ) or 5637 ( h ) cells overexpressing triple-HA-tagged TFPI, TFPI2, or mouse TFPI (mTFPI) via lentiviral transduction were exposed to TcdB4.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their CR 50 values are normalized to WT and plotted in a bar-chart ( i ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). j Binding of TcdB4.2 (500 nM) to Fc-tagged TFPI, TFPI2, and mTFPI (immobilized onto capture biosensors) was examined using biolayer interferometry (BLI) assays. Fc-tagged extracellular domains of FZD2 (CRD2), SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. k HeLa cells were exposed to either TcdB4.2 alone (4 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI at the indicated molar ratios (1:250 ~ 1:20,000) on ice for 1 h. The percentage of cell-rounding at 6 h incubation was plotted. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). l HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB2.1, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressing TcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations are shown in Supplementary Fig. . Their CR 50 values were normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). m – p HeLa-WT, two CSPG4 KO single clones (CSPG4#1 and CSPG4#8), and two CSPG4/TFPI double KO cells (CSPG4#1-TFPI-KO and CSPG4#8-TFPI-KO) were exposed to TcdB4.2 ( m ), TcdB2.1 ( n ), or TcdB2.2 ( o ) for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their relative CR 50 values are plotted in a bar-chart ( p ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). q HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB2.1, TcdB2.2, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressingTcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations and are shown in Supplementary Fig. . Their CR 50 values are normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: 1D4-tagged full-length mouse FZD2 was obtained from Addgene (#42264).

Techniques: CRISPR, Protease Inhibitor, Generated, Control, Recombinant, Transduction, Binding Assay, Incubation, Expressing, Clone Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagrams of TcdB4.2, TcdB4.2 1286–1805 , TcdB4.2 1835–2367 , TcdB1.1-FBD. TcdB4.1(B1.1) and TcdB1.1(B4.2) represent two mutant fragments exchanging 29 residues in the region 1432– 1600 that differ between TcdB4.1 and TcdB1.1. The numbers indicate the position of amino acid residues. GTD, glucosyltransferase domain; CPD, cysteine protease domain; DRBD, delivery/receptor-binding domain; CROPs, combined repetitive oligopeptides. Binding of 500 nM TcdB4.2 1286– 1805 ( b ) or TcdB4.2 1835– 2367 ( c ) to Fc-tagged TFPI and mTFPI was examined using BLI assays. Fc-tagged TFPI2, CRD2, SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. d HeLa cells transiently transfected with TFPI, TFPI2, mTFPI, SEMA6A, or FZD2 were exposed to FLAG-tagged TcdB4.2 1286– 1805 (5 µg/mL) on ice for 60 min, washed, fixed, permeabilized, and subjected to immunostaining analysis. Expression of exogenous proteins was confirmed by detecting fused HA or 1D4 tag. Nuclei were labeled with DAPI (blue). Scale bar, 5 µm. Representative images were from one of three independent experiments. Binding of 500 nM TcdB1.1-FBD, TcdB4.2 1286– 1805 , TcdB4.2(B1.1), and TcdB1.1(B4.2) to Fc-tagged CRD2 ( e ) and TFPI ( f ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. g Schematic diagram of TFPIβ, TFPI-K1, and TFPI-K2 fragments. Sig, signal peptide; N, N-terminal domain; K1, BPTI/Kunitz inhibitor domain 1; L1, loop 1; K2, BPTI/Kunitz inhibitor domain 2; L2, loop 2; β, GPI anchor sequence for TFPIβ. h Binding of 500 nM TcdB4.2 1286-1805 to Fc-tagged TFPI, TFPI-K1, and TFPI-K2 was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. i , j TcdB4.2 binding to TFPI-K2 prevents TFPI-K2 binding to its natural ligand coagulation factor Xa (FXa). FXa (0.5 ng/mL) cleaves its fluorescently labeled substrate and generates increasing fluorescent signal (measured as relative light unit, RLU, y -axis) over time ( x -axis). FXa’s enzymatic activity was inhibited by TFPI-K2 ( i , 7.5 ng/mL). The inhibitory effect of TFPI was blocked by adding TcdB4.2 1286– 1805 in a dose-dependent manner (1:1, 1:3, or 1:10 molar ratio). Representative curve from one of three independent experiments is shown. FXa activity was quantified by measuring the slope of RLU curves (1–10 min) and plotted as a bar-chart ( j ). The same experiments were also carried out for TFPI-Fc and mTFPI-Fc, with their curves shown in Supplementary Fig. and quantification in ( j ). Error bars indicate mean ± s.d.; N = 3; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: 1D4-tagged full-length mouse FZD2 was obtained from Addgene (#42264).

Techniques: Mutagenesis, Binding Assay, Transfection, Immunostaining, Expressing, Labeling, Sequencing, Coagulation, Activity Assay