Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: F/G‐actin binds nuclear proteins. A) Upper, HEK 293T cells were cultured in 0.5 µ m Jasp, 1 µ m LatB, or 10 µ m CytD for 1 h, lysed with lysis and F‐actin stabilization buffer 2 (LAS2), and subjected to F/G‐actin fractionation. Western blot showed that Jasp induced actin polymerization, while LatB and CytD induced actin depolymerization. Middle, The above cells were also resuspended in the fractionation buffer, and subjected to nuclear fractionation and F/G‐actin fractionation. Western blot showed that Jasp induced nuclear actin polymerization, while LatB and CytD treatment induced nuclear actin depolymerization. Lower, After cultured in 0.25 µ m Jasp, 0.5 µ m LatB, or 5 µ m CytD for 4 h, HEK 293T cells were subjected to nuclear fractionation. Nuclear extracts were lysed with LAS2, and subjected to immunoprecipitation with antibody against actin. The pulldown products were subjected to mass spectrometry analysis, showing that precipitation of nuclear actin pulled down α‐catenin, β‐catenin, and filamin A (FLNA) in Jasp treated cells, and MYBBP1A, NKRF, and profilin 1 (PFN1) in LatB and CytD treated cells. B) The cells were transfected with α‐catenin siRNAs. Western blot showed that silencing α‐catenin decreased α‐catenin expression, but did not affect β‐catenin expression. Silencing α‐catenin decreased both α‐catenin and β‐catenin in the nuclei. The nuclear extracts were incubated with 25 µL biotin‐X Phalloidin, and subjected to biotin‐labeled Phalloidin pulldown assay. Precipitation of F‐actin pulled down α‐catenin and β‐catenin, and pulled down less α‐catenin (23.4%) and β‐catenin (39.8%) by silencing α‐catenin. C) Western blot showed that silencing FLNA decreased its expression, but did not change SMAD2/SMAD3 levels. Silencing FLNA decreased FLNA, SMAD2, and SMAD3 in the nuclei. Precipitation of nuclear F‐actin pulled down FLNA, SMAD2, and SMAD3, but less FLNA (25.7%), SMAD2 (27.8%) and SMAD3 (10.5%) by silencing FLNA. D) Silencing PFN1 decreased PFN1 and MYPOP levels in cell lysate and nuclei. Precipitation of G‐actin pulled down PFN1 and MYPOP, but less of them by silencing PFN1. E) The nuclear fractions of the above cells were lysed with LAS2 and subjected to F/G‐actin fractionation and immunoprecipitation with antibody against actin. Precipitation of nuclear F‐actin pulled down β‐catenin, SMAD2, SMAD3, and precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP. F, HEK 293T cells were cultured in 0.1 µ m Jasp, 0.2 µ m LatB, and 1 µ m CytD for 16 h, lysed with lysis buffer, and subjected to Western blot. Jasp treatment repressed E‐cadherin, enhanced N‐cadherin and vimentin proteins. LatB and CytD treatment increased E‐cadherin, decreased N‐cadherin and vimentin levels.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Cell Culture, Lysis, Fractionation, Western Blot, Immunoprecipitation, Mass Spectrometry, Transfection, Expressing, Incubation, Labeling

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: F/G‐actin binds EMT‐related functional transcription factors in the nuclei. A) HEK 293T cells were transfected with MYBBP1A, NKRF, or MYPOP, and processed to ELISA. Overexpression of MYBBP1A, NKRF, or MYPOP increased E‐cadherin, and decreased N‐cadherin and vimentin levels. ** p < 0.01 versus vector ( n = 6). B) Left, HEK 293T, MDA‐MB231, and MDA‐MB468 cells were transfected with MYBBP1A, NKRF, or MYPOP, and processed to chamber migration assays for indicated time points, showing that expression of MYBBP1A, NKRF, or MYPOP suppressed cell migration. ** p < 0.01 versus vector ( n = 6). Right, The transfected cells were cultured in basal medium with 700 µ m H 2 O 2 for 24 h. Expression of MYBBP1A, NKRF, or MYPOP suppressed cell survival. ** p < 0.01 versus vector ( n = 6). C) Left, HEK 293T, HaCaT, and MCF‐7 cells were transfected with MYBBP1A, NKRF, or MYPOP siRNAs, and processed to chamber migration assays for indicated time points, showing that silencing MYBBP1A, NKRF, or MYPOP enhanced cell migration. ** p < 0.01 versus oligo ( n = 6). Right, The cells were cultured in basal medium with 650 µ m H 2 O 2 for 24 h, showing that silencing MYBBP1A, NKRF, or MYPOP enhanced cell survival. ** p < 0.01 versus oligo ( n = 6). D) The transfected cell lysates were subjected to ELISA analysis, showing that silencing MYBBP1A, NKRF or MYPOP repressed E‐cadherin, and increased N‐cadherin and vimentin expression. ** p < 0.01 versus oligo ( n = 6). E) The nuclear extracts of the above transfected cells were lysed with LAS2, and subjected to F/G‐actin fractionation and immunoprecipitation with antibody against actin. Western blot showed that precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP in the nuclei, but precipitation of F‐actin did not pull down these proteins. Western blot showed that precipitation of MYBBP1A, NKRF, or MYPOP pulled down actin in the nuclei. F) Western blot showed that precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP, which pulled down less MYBBP1A (25.5%), NKRF (12.8%), and MYPOP (12.1%) in the siRNA knock‐down samples, but precipitation of F‐actin did not pull down MYBBP1A, NKRF, and MYPOP in the nuclear fraction. Precipitation of nuclear MYBBP1A, NKRF, and MYPOP also pulled down actin.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Functional Assay, Transfection, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Migration, Expressing, Cell Culture, Fractionation, Immunoprecipitation, Western Blot

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Silencing XPO6/IPO9 alters both F‐actin and G‐actin levels in the nuclei. A) HEK 293T cells were transfected with Exportin 6 (XPO6) or Importin 9 (IPO9) siRNAs, and subjected to nuclear fractionation. Western blot showed that silencing XPO6 with siRNAs (si‐XPO6) increased actin, β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP in the nuclei, while silencing IPO9 (si‐IPO9) decreased actin, β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP in the nuclei. B) Western blot showed that silencing XPO6/ IPO9 did not affect total actin, E‐cadherin, N‐cadherin, and vimentin expression levels in the cells.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Transfection, Fractionation, Western Blot, Expressing

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Effect of silencing XPO6/IPO9 on levels of nuclear F/G‐actin in the actin polymerization/depolymerization models. A) ELISA showed that medium/si‐XPO6 increased both F‐actin and G‐actin in the nuclei compared to the medium/oligo cells. Jasp/si‐XPO6 cells showed same cell actin dynamics and nuclear G‐actin levels as Jasp/oligo, and increased F‐actin in the nuclei. LatB/si‐XPO6 cells showed the same cell actin dynamics and nuclear F‐actin levels as LatB/oligo, and increased G‐actin in the nuclei. ** p < 0.01 versus oligo ( n = 6). B) The cells were lysed and subjected to Western blotting. Jasp/si‐XPO6 cells showed decreased E‐cadherin, but increased N‐cadherin and vimentin compared to Jasp/oligo, while LatB/si‐XPO6 cells presented increased E‐cadherin, but decreased N‐cadherin and vimentin compared to the LatB/oligo cells. Jasp/si‐XPO6 cells showed enhanced β‐catenin, SMAD2, and SMAD3 expression in the nuclei, while LatB/si‐XPO6 showed increased MYBBP1A, NKRF, and MYPOP expression in the nuclei compared to LatB/oligo treated cells. C) Nuclear F‐actin/G‐actin fraction from the above cells was subjected to immunoprecipitation with antibody against actin. Precipitation of F‐actin pulled down β‐catenin, SMAD2, and SMAD3, which was more evident in Jasp/si‐XPO6 cells. Precipitation of G‐actin pulled down MYBBP1A, NKRF, and MYPOP, which was more evident in LatB/si‐XPO6 cells. D) ELISA confirmed a decrease in both F‐actin and G‐actin in the nuclei of the medium/si‐IPO9 cells compared to the medium/oligo cells. Jasp/si‐IPO9 cells showed similar actin dynamics and nuclear G‐actin levels as Jasp/oligo, but decrease in F‐actin in the nuclei. LatB/si‐IPO9 cells showed similar actin dynamics and nuclear F‐actin levels as LatB/oligo, but decrease in G‐actin in the nuclei. ** p < 0.01 versus oligo ( n = 6). E) Nuclear F‐actin/G‐actin fraction was subjected to immunoprecipitation with antibody against actin. Precipitation of F‐actin pulled down β‐catenin, SMAD2 and SMAD3, while precipitation of G‐actin pulled down MYBBP1A, NKRF, and MYPOP.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Nuclear F/G‐actin regulates EMT via binding and modulating β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP expression in the nuclei. A) Upper, HEK293T cells were transfected with mDia2 with or without XPO6 siRNAs. The cells and nuclear extracts were subjected to F/G‐actin fractionation. Overexpression of mDia2 enhanced actin polymerization, and silencing XPO6 did not affect F/G‐actin in the cells. mDia2+/si‐XPO6 cells showed increased nuclear F‐actin. Lower, ELISA confirmed that mDia2+/si‐XPO6 cells expressed increased nuclear F‐actin, decreased E‐cadherin, but increased N‐cadherin and vimentin compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6). B) mDia2+/si‐XPO6 cells expressed increased β‐catenin, SMAD2, and SMAD3 in the nuclei compared to mDia2+/oligo cells. C) Left, HEK293T, HaCaT, and MCF‐7 cells were co‐transfected with mDia2 and XPO6 siRNAs, and processed to chamber migration assays for indicated time points. mDia2+/si‐XPO6 cells showed enhanced cell migration compared to mDia2+/oligo cells. Right, The transfected cells were cultured in basal medium with 650 µ m H 2 O 2 for 24 h. mDia2+/si‐XPO6 cells showed enhanced cell survival compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6 ). D) HEK293T cells were transfected with the control vector or mDia2 with or without IPO9 siRNAs. ELISA showed that mDia2+/si‐IPO9 cells expressed decreased nuclear F‐actin, increased E‐cadherin, but decreased N‐cadherin and vimentin compared to the mDia2+/oligo treated cells. ** p < 0.01 versus oligo ( n = 6). E) mDia2+/si‐IPO9 cells displayed cuboidal epithelial shape compared to mDia2+/oligo cells. The ratios of cell length/width were quantified, which showed that silencing IPO9 decreased cell elongation ** p < 0.01 versus oligo ( n = 25). F) HEK293T cells were transfected with mDia2 siRNAs with or without XPO6 siRNAs and processed for nuclear extraction. The cells and nuclear extracts were subjected to F/G‐actin fractionation. ELISA showed that si‐mDia2/si‐XPO6 cells expressed increased nuclear G‐actin, increased E‐cadherin, but decreased N‐cadherin and vimentin compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6). G) ELISA showed that si‐mDia2/si‐IPO9 cells expressed decreased nuclear G‐actin, decreased E‐cadherin, but increased N‐cadherin and vimentin expression compared to mDia2+/oligo treated cells. ** p < 0.01 versus oligo ( n = 6).

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Binding Assay, Expressing, Transfection, Fractionation, Over Expression, Enzyme-linked Immunosorbent Assay, Migration, Cell Culture, Plasmid Preparation, Extraction

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Overexpression of the nuclear F/G‐actin with constructs modulates EMT. A) Upper, HEK293T cells were transfected with YFP‐NLS‐β‐actin (NLS‐β‐actin), YFP‐NLS‐β‐actin S14C (S14C), YFP‐NLS‐β‐actin G13R (G13R), pmCherry‐NLS‐β‐actin R62D (R62D), and the vector. Transfection of NLS‐β‐actin expressed both F‐actin and G‐actin (β‐actin) in the nuclei, transfection of S14C expressed F‐actin (β‐actin), while transfection of G13R or R62D expressed G‐actin (β‐actin). Lower, The nuclear extracts were subjected to Western blotting. Transfection with S14C enhanced β‐catenin, SMAD2, and SMAD3, while transfection with G13R or R62D enhanced MYBBP1A, NKRF, and MYPOP levels in the nuclei. B) Transfection with S14C repressed E‐cadherin, and enhanced N‐cadherin and vimentin expression, while expression of G13R or R62D enhanced E‐cadherin, and repressed N‐cadherin and vimentin. C) ELISA showed that transfection with S14C repressed E‐cadherin, and enhanced N‐cadherin and vimentin expression, while expression of G13R or R62D enhanced E‐cadherin, and repressed N‐cadherin and vimentin. ** p < 0.01 versus vector ( n = 6). D) HEK293T cells were transfected with NLS‐β‐actin, S14C, G13R, or R62D. Immunofluorescence showed that transfection with S14C enhanced nuclear F‐actin and cell N‐cadherin levels, but repressed E‐cadherin levels. Transfection with G13R or R62D enhanced nuclear G‐actin and cellular E‐cadherin levels, but repressed N‐cadherin levels. E) HEK293T cells were transfected with S14C and G13R. Immunofluorescence showed the co‐localization of MYBBP1A, NKRF, and MYPOP with the nuclear G‐actin in the G13R‐transfected cells. F) Upper, HEK293T, MDA‐MB‐231, MDA‐MB‐468, HaCaT, and MCF‐7 cells were transfected with the above constructs, and processed to chamber migration assays for indicated time points. Transfection with actin S14C enhanced cell migration, while transfection with G13R or R62D repressed cell migration. Lower, The cells were cultured in basal medium with 700 µ m H 2 O 2 for 24 h. Transfection with S14C enhanced cell survival, while transfection with G13R or R62D repressed cell survival. ** p < 0.01 versus vector ( n = 6).

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Over Expression, Construct, Transfection, Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Migration, Cell Culture

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Association of EMT with wound repair. A) Absolute values of E‐cadherin, N‐cadherin, vimentin, and nuclear F‐actin/G‐actin protein levels were quantified by ELISA in 133 cell lines including, MDA‐231‐BoM‐1833, 786‐O, 4T1, 4T07, 66C14, 67NR, A431, A549, A2058, A2780, AC16, ACHN, ARH77, AU565, B16, BC3, BEAS‐2B, BTH‐1, BT‐20, BT‐474, BT‐549, BxPC‐3, C2C12, Caco‐2, Caki‐1, Caki‐2, CI3K, CO‐115, COLO‐201, COLO‐205, COLO‐775, CW‐9019, CRL‐1476, Cos‐1, Cos‐7, CV‐1, DLD‐1, DU145, EMT6, ES2, FHC, H460, HaCaT, HCC1393, HCT6, HCT8, HCT15, HCT116, HDL100, HEK293T, Hela, Hep3B, HepG2, HEY, HGF, HL‐1, HL‐60, HLE, HT‐1080, HT‐29, HTB‐123, HTB‐126, Ho, Hs5787, Huh6, Huh7, ICE6, ICE18, JHH‐1, Jurkat, JR75‐1, JR‐75‐30, K652, KTC‐1, LAPC‐4, LAPC‐9, Li7, LNCaP, MC3T3, MCF, MCF‐7, MCF‐10A, MDA‐MB‐157, MDA‐MB‐175, MDA‐MB‐231, MDA‐MB‐436, MDA‐MB‐468, MDA‐MB‐453, NIH3T3, NMuMG, PAN3, PANC‐1, PC3, PC12, PLC/PRF/5, OV‐2008, OVCAR‐3, Raji, Rat2, RD, RFL‐6, RH1, RH2, RH3, RH4, RH6, RH14, RH18, RH28, RH30, RIE‐1, SK‐NEP‐1, SNU‐16, SNU‐378, SNU‐449, Saos‐2, SW48, SW480, SW620, SW837, SW1116, SW1353, T3M‐4, T47V, T860, TOV‐112, SK‐BR‐3, UO‐31, U87, U118, U343, U937, and YPEN‐1. Pearson correlation analysis showed that E‐cadherin levels were negatively correlated with N‐cadherin in the cell lines. p < 0.0001, n = 133, R 2 = 0.6741. B) Pearson correlation analysis showed a positive correlation between the ratio of nuclear F‐actin/G‐actin proteins and N‐cadherin/E‐cadherin. p < 0.0001, n = 133, R 2 = 0.6818. C) A total of 52 wound healing samples were selected, which were collected from 6 day‐wounded mice. All samples contained normal skin and wound healing skin in the same section, which was confirmed by H&E staining. A typical image of wound healing sample is shown. D) Typical z‐stack images (xy, xz, and yz projection and orthogonal view) showing nuclear F‐actin (Phalloidin staining, red) and G‐actin (Deoxyribonuclease I staining, green) in wound healing and normal skin cells (epidermis). The images for wound healing skins were randomly selected from central wound healing areas, while the normal skin images were randomly selected from the normal epidermis areas far from the wound region. E) ImageJ analysis showed that the wound healing cells expressed higher levels of nuclear F‐actin and lower levels of nuclear G‐actin than the normal skin cells. The intensity of Phalloidin (F‐actin)/Deoxyribonuclease I staining (G‐actin) within the cell nucleus (DAPI staining) was analyzed by ImageJ. Phalloidin/Deoxyribonuclease I staining regions that overlapped with DAPI were defined as nuclear F/G‐actin stained. The average intensity value of five cells from each image represented F/G‐actin intensity of the sample image. Phalloidin/Deoxyribonuclease I stained areas around the edge of the nucleus were excluded, and only the regions stained away from the nuclear edge were counted as stained positive for nuclear F/G‐actin. ** p < 0.01 versus normal ( n = 6). F) A diagram showing that nuclear actin polymerization regulates cell epithelial‐mesenchymal transition process.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: HA-49K orthologs from the A549-based expression system function comparably to natural E3/49K and exhibit a consistent binding activity to CD45 expressing target cells. Cell supernatants containing individual E3/49K variants were incubated together with target cells and bound E3/49K was measured via flow cytometry. Binding activity of HA-49K of HAdV-D64 from the A549-based producer cell line was compared to E3/49K obtained from cells infected with HAdV-D64, HAdV-D64ΔE3 and HAdV-D64ΔE3 + 49K viruses . Supernatants were incubated with wild-type Jurkat (red), Ramos (blue) and the CD45-deficient Jurkat (orange) and Ramos (light blue) cells, respectively. The grey dashed vertical line separates results obtained from transfected cells (left) from those of infected cell lines (right). Binding of HA-49K and E3/49K was detected with 4D1 mAb (A) . Binding activity to Jurkat and Ramos cell lines of recombinant HAdV-D64 HA-49K of was compared with HA-tagged orthologs of HAdV-D8, -D19 and -D36, respectively. Binding was determined with the target cell system as applied in the previous experiment using α-HA Ab (B) . Contrasting the binding of HA-49K and untagged E3/49K from the A549-based expression system to Jurkat cells reveals no negative influence to the binding activity by the HA-tag. Bound E3/49K versions were detected using HA-specific (blue) or E3/49K-specific (4D1, red) mAbs (C) . The binding specificity of HA-49K orthologs was further characterized by competition with untagged E3/49K of HAdV-D64. Residual binding activity of HA-49K orthologs was monitored by flow cytometry using α-HA Abs and a two-step sequential incubation of Jurkat cells with supernatants, containing E3/49K variants. The order of the individual incubations for competition is indicated within the figure. Significant differences to single incubations were analyzed (D) . Cell supernatants from normal A549 cells were utilized as negative controls. The columns represent the mean-MFIs obtained in independent experiments, each depicted as dots, and error-bars represent the standard deviations. Statistical significance (****P<0.0001) was determined via the two-way ANOVA test and is indicated within the panel (D) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Expressing, Binding Assay, Activity Assay, Incubation, Flow Cytometry, Infection, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Activation of Jurkat T cells is inhibited by HA-49K orthologs to an equal extent. Jurkat cells were previously incubated with cell supernatants (red) containing HA-49K orthologs. Cells were washed and stimulation was conducted via receptor cross-linking using immobilized α-CD3 and soluble α-CD28 Abs for 6h. After cell fixation the activation level was determined by flow cytometry-based monitoring of the cell surface expression of the early activation marker CD69. Relative numbers of CD69 positive cells were normalized to CD3/CD28 stimulation control (not shown). Untreated (unstim.) and isotype control (ISO) treated samples were used as negative controls (grey). To show the efficiency of the CD3/CD28 stimulation we treated the cells also with 50 ng/ml Phorbol-12-myristate-13-acetate and 1 µg/ml ionomycin (PMA/Iono, blue). Administration using CD3/CD28 stimulation and unreactive A549 supernatant was utilized to control the effect of the supernatant on CD3/CD28 stimulation (A549, blue) (A) . pErk1/2 levels were identified by immunoblot analysis upon CD3 stimulation of Jurkat cells with 1 µg/ml for 2 min. Sample loading was controlled by detection of β-actin. One representative blot for Jurkat and CD45-/- Jurkat is shown (B) and the relative expression levels of pErk1/2 to β-actin ratios were normalized to the pos. ctrl. (C) . The columns represent the mean of 3 individual experiments (dots), the error-bars represents the standard deviation for A and (C) Statistical differences compared toPMA/ionomycin treatment in (A) and CD3-stimulation in the presence of A549 supernatants in (C) positive controls were analyzed using the two-way ANOVA test. Only significant results were indicated in the figure.

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Activation Assay, Incubation, Flow Cytometry, Expressing, Marker, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Ramos B cell signaling is inhibited by HA-49K orthologs to comparable levels. Ramos B cell signaling was determined to assess the inhibitory potential of HA-49K orthologs in B cells. Previous incubation of Ramos cells with cell supernatants (red) containing various HA-49K orthologs was performed. Unstimulated cells (unstim.) were used as a negative control (grey), co-incubation with unreactive A549 supernatant (A549) served as a positive control indicated in blue. Cells were washed and stimulated via receptor cross-linking using α-λ Abs. The cellular calcium-response was detected by flow cytometry and the mean peak Ca2+-levels (columns) of 3 individual experiments (dots) including standard deviation are shown. Statistical differences compared to A549 were analyzed using two-way ANOVA test (A) . Immunoblot analysis of pErk1/2 levels upon BCR stimulation of Ramos B cell lines with 1 µg/ml α-λ Abs for 2 min. Unstimulated cells (unstim.) served as negative control while α-λ treated cells (pos. crtl.) and co-incubation with A549 supernatant (A549) served as positive controls. Detection of β-actin levels was used as loading control. One representative blot for Ramos and CD45-/- Ramos cells is shown (B) . The relative detection levels of pErk1/2 to β-actin ratios in Ramos cells were normalized to the positive ctrl. The mean (columns) of 3 individual experiments (dots) including standard deviation is shown. Statistical differences to the positive ctrl. were analyzed using the two-way ANOVA test. Only significant results were indicated in the panel (C) . A two-fold dilution series of cell supernatants containing HA-49K-D64 (D) and purified HA-49K-D64 proteins starting at 8 µg per sample (E) was performed. Samples were either supplemented with 0.5 µg per sample hCD45-Fc (+hCD45-Fc, red) or without (hCD45-Fc, blue). After a 1 h incubation period, the binding of HA-49Ks to Ramos cells was detected using α-HA-based flow cytometry. Undiluted supernatant from untransfected A549 cells (A549) or the 8 µg MT protein were utilized as negative controls and shown as single values at the end of the x-axis separated by the grey dashed line. The mean MFI of 3 individual experiments is displayed for each, including standard deviation in the form of error bars. Erk1/2 phosphorylation was analyzed to investigate the prevention of the inhibitory effect HA 49K-D64 by hCD45-Fc receptors via immunoblotting. The supernatant containing HA-49K-D64 proteins was diluted 1:10 and 4 µg purified HA-49K-D64 proteins were incubated for 1 h with 500 ng hCD45-Fc decoy receptors as indicated in the figure. Subsequently, reagents were incubated with Ramos cells for 1 h Cells were lysed after BCR stimulation with 2 µg/ml α-λ Abs for 2 min. Sample loading was controlled by the detection of β-actin levels. One representative blot is presented (F) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Incubation, Negative Control, Positive Control, Flow Cytometry, Standard Deviation, Western Blot, Control, Purification, Binding Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Only HAdV-D infected A549 cells bind soluble hCD45-Fc. A549 cells were infected with HAdV-A12, -B7, -B35, -C5, -D8, -D19, -D36, -D64 and -D64ΔE3 and -E4, viruses with an MOI of 5 for 24 h Efficient infection was confirmed by internal hexon protein expression (red) using 2Hx-2 mAbs in comparison to isotype control staining (grey) of infected A549 cells in flow cytometry. Displayed are representative histograms of productive infections (A) . Infected cells were treated with +/- (red/blue) 0.5 µg hCD45-Fc per sample. Bound CD45 molecules were detected by CD45-ECD staining using α-human pan-CD45 MEM-28 in flow cytometry. Mock infected cells as well as cells infected with HAdV-D64ΔE3 virus served as negative controls. As positive control the cell clone stably expressing HA-49K of HAdV-D64 was applied. The mean of 3 individual experiments (columns) from (dots) including standard deviation, presented as error bars, is shown. Significant differences between +/- hCD45-Fc treatment were determined using the two-way ANOVA test and are indicated in the panel (B) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Infection, Expressing, Comparison, Control, Staining, Flow Cytometry, Virus, Positive Control, Stable Transfection, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical abstract of the putative functional mechanism of E3/49K action in B cells. During BCR antigen ligation, the catalytic activity of CD45 shifts the equilibrium of functional Lyn toward activated Lyn (Lyn Y396). The removal of the inhibitory phosphate group from Y507 sites primes the auto-phosphorylation of Lyn at Y396 sites to induce Lyn kinase activity. Active Lyn promotes phosphorylation of ITAMs and pITAM-attached Syk to facilitate BCR signal transduction. pSyk initiates several signaling pathways, including the MAPK pathway. pErk1/2 and the calcium flux results in transcriptional and cellular activation (A) . E3/49K-mediated dimerization of CD45 molecules prevents the catalytic activity of CD45. As a result, the equilibrium of functional Lyn is shifted toward inactive Lyn (Lyn Y507), increasing the activation threshold during BCR stimulation. As a result of reduced Lyn kinase activity, less pSyk, pErk1/2, and calcium flux are generated, resulting in decreased transcriptional and cellular activation (B) . Dimerization of CD45 by E3/49K may disrupts CD22-CD45 interaction. Active Lyn induces CD22 which enhances its inhibitory effect in reducing BCR signals by affecting pErk1/2 and calcium flux (C) . The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Functional Assay, Ligation, Activity Assay, Transduction, Activation Assay, Generated

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical representation about E3/49K functions. CD45 modulation via binding of E3/49K proteins to its ECD is a common feature of HAdVs of species D. E3/49K ECDs are shed from infected cells and bind to and inhibit CD45 positive target cells. Based on the current hypothesis, inhibition is mediated by enforced dimerization of CD45 , which inhibits leukocyte receptor signaling. B cells are here identified as a new target for E3/49K-mediated immune evasion. Since there are more CD45 expressing leukocytes existing, it is hypothesized that they serve as targets for E3/49K as well. The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Binding Assay, Infection, Inhibition, Expressing

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a KRas expression and activity in Caco2, HKE3, and HCT116 cells. Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological). Test t ** p < 0.01, ( n = 4); e–g ROS production after 72 h of PDIA1 silencing, measured by HPLC analysis of DHE oxidation products. DHE oxidation produces, among many others, two major products: 2-hidroxyethidium (EOH), which is representative of superoxide species and ethidium, representative of other oxidant species. Ctrl negative, si-RNA control; si-PDI, si-RNA against PDIA1 protein. Representative immunoblots of PDI silencing for each cell type Test t * p < 0.05; ** p < 0.01. For Caco2 cells, n = 3; HKE3 cells, n = 4; HCT116 cells, n = 5

Article Snippet: Following cell lysis, soluble PDIA1 antigen was measured using the Human P4HB Pair Set enzyme-linked immunosorbent assay (ELISA) (SinoBiological Inc) according to the protocol described by the supplier.

Techniques: Expressing, Activity Assay, Western Blot, Software, Titration, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a Interaction map fashioned with String 10.5 program ( http://string-db.org ), Network nodes represent proteins of Nox1, PDIA1 (P4HB), RhoGDIα (ARHGDIA), RAC1, BRAF, KRAS, GSK3β (GSK3B), STAT3, and E-cadherin (CDH1). Different colored lines displays predicted functional links. Interactions experimentally determined appear in pink, interactions curated from databases in blue, co-expression was represented in black and text data mining in green. The analysis showed: nine nodes with an average node degree of 3.56; number of edges: 16; expected number of edges: 4; average local clustering coefficient: 0.333; PPI enrichment p -value: 2.02e −05 . b Top 10 ranked enriched proteins KEGG pathway analysis. Bar graphs show the –log of FDR (False discovery rate). High values correlated to higher probabilities. c Cellular component Gene ontology (GO) analysis

Article Snippet: Following cell lysis, soluble PDIA1 antigen was measured using the Human P4HB Pair Set enzyme-linked immunosorbent assay (ELISA) (SinoBiological Inc) according to the protocol described by the supplier.

Techniques: Functional Assay, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: The specificity of EGFR recognition by EGFR scFv and the construction of EGFR-CXCR5 chimeric antigen receptor (CAR)-Ts (A) Graphical representation of the CAR designed using the anti-EGFR scFv, CD8a hinge, and transmembrane domain, 4-1BB and CD3zeta endodomain. EGFR-CXCR5 was constructed with an additional CXCR5 sequence after the CD3zeta endodomain. (B) ELISA of anti-EGFR scFv with recombinant human immunoglobulin G1 (IgG1) Fc-conjugated EGFR (ErbB1), HER2 (ErbB2), HER3 (ErbB3), MUC1, Flk1 (VEGFR2), and FLT4 (VEGFR3). Recombinant proteins were coated in the plate wells at 5 μg/mL. Anti-EGFR scFv concentration started from 5,000 pg/mL and was diluted 5-fold repeatedly until 8 pg/mL. (C) FACs analysis of A549 and PC9 (LUAD cell lines), H929 (myeloma cell line), Raji (human Burkitt’s lymphoma cell line), and K562 (human myelogenous leukemia cell line) stained with anti-EGFR scFv. Concentration started from 20,000 ng/mL and was diluted 10-fold repeatedly until 0.2 ng/mL. (D) The expression of transgenes in lentivirus-transduced T cells was analyzed by flow cytometry using protein L and anti-CXCR5 antibody. Single dot represents individual sample. Error bars represent mean ± SD for each T cell population (n = 4).

Article Snippet: In addition, rabbit anti-human MUC1, HER2, HER3, VEGFR2, and VEGFR3 antibodies (Sino Biological) were used as positive controls for detecting recombinant human MUC1, HER2, HER3, VEGFR2, and VEGFR3 proteins.

Techniques: Construct, Sequencing, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Staining, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting the model system employed to visualise TIGIT on the surface of T cells when interacting with Raji B cells expressing different nectin ligands. b Flow cytometry analysis showing the expression of TIGIT in Jurkat cells (above) and CD111 and CD155 in Raji cells (below), in both the parental and expression lines together with isotype-matched controls. c Confocal microscopy images showing TIGIT-GFP (green) on the surface of Jurkat cells (T) conjugated for 20 mins with different Raji cell (B) populations, as indicated to the left of the panel. CD19 (yellow) is used to mark Raji cells and a V5 stain labels expressed nectins (magenta). Respective brightfield images (BF) are also provided. The bottom two rows show Jurkat T cells that have been preincubated with either an antagonistic TIGIT antibody or an isotype-matched control. d Mean log 2 fold change in synaptic TIGIT enrichment in Jurkat cells, from the conjugates shown in c (±S.D.; n = 3 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; ns = not significant). e Representative confocal microscopy images showing TIGIT (green) on the surface of primary T cells conjugated with different Raji B cell populations, as indicated to the left. CD4 and CD8 (yellow) were stained to mark T cell subsets, and BF provided. f Mean log 2 fold change (±S.D., n = 3 independent donors matched by colour) in synaptic TIGIT enrichment in primary T cells, from the conjugates shown in e . Adjusted P values from a paired T-test are given (Holm-Šídák method). g Schematic depicting the model system employed to test the inhibitory effect of TIGIT on the surface of Jurkat T cells when interacting with cells expressing different nectin ligands. Staphylococcal Enterotoxin E (SEE) was used to stimulate Jurkat cells. h Relative amounts of IL-2 released from either parental or TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells for 6 h. Data is shown as the mean log 2 fold changes between Raji-CD155 conjugates compared to Raji-CD111 conjugates (±S.D., n = 5 independent experiments with adjusted P values from a one-way ANOVA with Holm-Šídák’s multiple comparisons displayed). Cells pre-incubated with an antagonistic TIGIT antibody (αT) or an isotype-matched control (iso) are shown, as indicated. All scale bars = 5 µm. Source data are provided as a Source Data file.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Staining, Control, Incubation

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting the model system employed to visualise TIGIT at the IS of T cells upon ligation. TIGIT expressing T cells interact with Planar Lipid Bilayers (PLB) containing laterally mobile ligands and imaged with Total Internal Reflection Fluorescence (TIRF) microscopy. b TIRF microscopy images showing TIGIT-GFP at the IS of Jurkat cells that have interacted with PLBs loaded with ICAM-1 (100 molecules/μm 2 ), and either CD111 or CD155 (400 molecules/μm 2 ) for 20 mins. Cells preincubated with an antagonistic TIGIT antibody or an isotype-matched control are shown, as indicated. c Mean degree of TIGIT clustering measured from the images shown in b (±S.D.; n = 3 independent experiments with adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons shown; ns = not significant). d Representative TIRF microscopy images showing the spatial distribution of TIGIT at the IS of primary CD4+ and CD8 + T cells that have interacted with PLBs loaded with ICAM-1, and the ligands CD111 or CD155 for 20 mins, as in b . In both b and d the fluorescent intensities have been scaled equally, and the colour scales provided. e Mean degree of TIGIT clustering measured from the images shown in d (±S.D., n = 3 individual donors). Adjusted P values from a paired T-test with Holm-Šídák’s multiple corrections are displayed. f Video stills from live TIRF microscopy imaging of Jurkat T cells expressing TIGIT-SNAP interacting with PLBs containing ICAM-1 and either CD111 or CD155 (as in b ). Acquisition times are indicated at the top left (mins). g Kymographs showing a single spatial position, as indicated by the dashed yellow line in f , over time. h Zoomed video stills from Jurkat TIGIT-SNAP on PLBs containing ICAM-1 and CD155, from f , displaying occurrences where TIGIT clusters appear to split (top row, yellow arrow) or fuse (bottom row, magenta arrow). Arrows mark specific xy locations, and time intervals are displayed above. i Confocal microscopy images of a FRAP experiment showing the recovery of both CD155-AF647 within the PLB and TIGIT-GFP on the surface of Jurkat cells within clusters. PLBs contain both ICAM-1 and CD155-AF647. Images were taken before photobleaching (Pre-bleach), and at the indicated times (in seconds) following photobleaching (Post-bleach). j FRAP profiles of both CD155-AF647 and TIGIT-GFP from cells measured as shown in i . Data is presented as the mean ±S.D. ( n = 11 cells from 2 independent experiments). Scale bars = 5 μm ( b , d , f ) and 1 µm ( h, i ). Source data are provided as a Source Data file.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Ligation, Expressing, Fluorescence, Microscopy, Control, Imaging, Confocal Microscopy

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting the model system employed to visualise TIGIT and the TCR at the Immune Synapse (IS) of T cells upon co-ligation. Both Jurkat T cells expressing TIGIT-SNAP, and peripheral blood-isolated primary T cells that express TIGIT endogenously interact with PLBs containing nectin ligands (CD111 or CD155), ICAM-1 and the directly labelled, mono-biotinylated stimulatory TCR antibody OKT3 and imaged with TIRF microscopy. b Video stills of Jurkat T cells expressing TIGIT-SNAP and labelled with dye (magenta) interacting with PLBs containing ICAM-1 (100 molecules/μm 2 ), CD111 or CD155 (400 molecules/μm 2 ) and fluorescently labelled OKT3 (100 molecules/μm 2 ; green), using live TIRF microscopy. Acquisition times are indicated at the top right of each column of images (mins:secs). Brightfield images are shown above. The data are representative of 3 independent experiments. c Kymographs showing a single spatial position, as indicated by the dashed yellow line in b , over time. d Representative TIRF microscopy images showing the relative localisation of TIGIT (antibody labelled; magenta) and the TCR (green) upon interaction with PLBs, as in b , in fixed primary CD4+ and CD8 + T cells at the indicated times. Throughout, scale bars = 5 μm. The data are representative of 3 independent donors. Pearson correlation coefficients ( r ) are displayed on merged images.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Ligation, Expressing, Isolation, Microscopy

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting individual point mutations introduced into TIGIT-SNAP. b Representative confocal microscopy images showing WT and mutant forms of TIGIT-SNAP (green) on the surface of Jurkat T cells conjugated for 20 mins with different Raji B cell populations (either CD111- or CD155-expressing; stained via V5 and shown in magenta). A merged fluorescence-BF image is also provided. c Mean log 2 fold change (±S.D., n = 3 independent experiments) in synaptic TIGIT enrichment in Jurkat T cells, from the conjugates shown in b . Adjusted P values from a one-way ANOVA with Šídák’s multiple comparisons are displayed, with differences from the WT-111 condition displayed in black and the WT-155 condition displayed in grey. d Representative TIRF microscopy images of WT and mutant forms of TIGIT-SNAP at the IS of Jurkat cells that have interacted with PLBs loaded with ICAM-1, and CD111 or CD155 for 20 mins, as in Fig. . Intensities have been scaled equally, and colour scales provided. e Mean degree of TIGIT clustering measured from the images shown in d (±S.D., n = 3–4 independent experiments, as indicated). Adjusted P values from a one-way ANOVA with Dunnett’s multiple comparisons are displayed, and coloured as in c . f ELISA data showing the relative amount of IL-2 released from either parental or different forms of TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells. Data is shown as the mean log 2 fold changes between Raji-CD155 conjugates compared to Raji-CD111 conjugates, ± S.D. ( n = ≧5 independent experiments with adjusted P values from a one-way mixed-effects analysis with a Dunnett’s multiple comparison test displayed). Differences from the parental condition are displayed above in black and from the WT condition displayed below in grey. g Western blot analysis of TIGIT using either Phos-tag SDS-PAGE (left) or standard SDS-PAGE to examine TIGIT phosphorylation in Raji-Jurkat conjugates, as labelled above. Data are representative of 3 independent experiments. h Representative TIRF microscopy images of different forms of TIGIT (SNAP labelled; magenta) and the TCR (OKT3 in PLB; green) in Jurkat cells upon interaction with PLBs containing ICAM-1, either CD111 or CD155, and fluorescently labelled OKT3 (100 molecules/μm 2 ), for 10 mins. i Mean Pearson’s correlation coefficient (±S.D; n = ≧50 cells from 2 independent experiments) between TIGIT and OKT3 from the images shown in h . Adjusted P values from a Kruskal-Wallis test with Dunn’s multiple comparisons are shown, and coloured as in c . All scale bars = 5 μm. Source data are provided as a Source Data file.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Confocal Microscopy, Mutagenesis, Expressing, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Western Blot, SDS Page