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: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP-IC, and mainly with MP, induce the activation of autologous B cells from patients with RA. (A) From left to right: representative light microscopy pictures of MDM unstim alone; MDM unstim co-cultured with B cells; MDM differentiated in the presence of RMP or RMP-IC from patients with RA and co-cultured with B cells. (B) Representative histograms of CD80 expression on B cells from HC (top) and patients with RA (below) cultured alone (light blue) and with anti-BCR plus CD40L (yellow) or co-cultured with MDM differentiated without (Unstim, black) or with RMP (gray) and RMP-IC (green). Blue histograms represent the FMO control. (C) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with RA ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (D) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with RA ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (E) BAFF and APRIL (Top panel) levels in supernatants of MDM from patients with RA ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with RMP and RMP-IC. IgG and IgM (below panel) levels in supernatants from co-cultures of MDM differentiated with or without RMP and RMP-IC with autologous B cells from HC ( n = 5) and RA ( n = 5) patients. Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Light Microscopy, Cell Culture, Expressing, Positive Control

Journal: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP and MP-IC induce the activation and plasmablast differentiation of autologous LB from patients with SLE. (A) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with SLE ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (B) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with SLE ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (C) BAFF and APRIL levels in the supernatants of MDM from patients with SLE ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with LMP and LMP-IC. (D) Representative gating strategy to determine the frequency of plasmablasts after the co-culture of B cells with MDM differentiated without (Unstim) or with LMP and LMP-IC. (E) The frequency of plasmablasts from B cells cultured alone (Unstim, in complete medium), with anti-BCR plus CD40L (positive control), or co-cultured with autologous MDM from patients with SLE ( n = 7) and HC ( n = 6) differentiated without (Unstim) or with LMP and LMP-IC. (F) IgG and IgM levels in supernatants from the co-cultures of MDM differentiated with or without LMP and LMP-IC with autologous B cells from HC ( n = 5) and patients with SLE ( n = 5). Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Cell Culture, Positive Control, Co-Culture Assay

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Production and characterization of recombinant NSDV GP38-his/FLAG. ( A ) Alignment of partial GPC sequences from different NSDV strains (Ganjam virus IG 619 = NSDV_India; NSDV 708 = NSDV_Kenya; NSDV H. longicornis China = NSDV_China) and CCHFV (IbAr10200). Numbering corresponds to NSDV_India GPC. Amino acid sequences are displayed starting from their respective N-terminus. For CCHFV, the furin protease cleavage site (RSKR) and SKI-1/S1P cleavage site (RRLL) are underlined in blue. For NSDV, arginine-containing motifs reported to be conserved across the GPCs of different orthonairoviruses are highlighted in orange. Two potential N-glycosylation sites (Asn 220 and Asn 400 ) are highlighted with an orange star. ( B ) For protein purification, the partial NSDV_India GPC sequence (aa 138–445; NSDV GP38-his/FLAG protein) was fused to a C-terminal 6×His- and FLAG-tag both highlighted with an orange underline. ( C ) SDS-PAGE of recombinant NSDV GP38-his/FLAG his-tag purified from Sf9 cells followed by Coomassie blue staining and immunoblot analysis using anti-FLAG primary and horseradish peroxidase-conjugated secondary antibodies.

Article Snippet: NSDV Gn ( NAC-REC31904-500 ) and NSDV Gc ( NAC-REC31906-500 ) from The Native Antigen Company were diluted in 0.01 M PBS and coated overnight on Maxisorp 96-well plates at a concentration of 100 ng/well.

Techniques: Recombinant, Virus, Glycoproteomics, Protein Purification, Sequencing, FLAG-tag, SDS Page, Purification, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Elevated hepatic and serum ALCAM were observed in patients with AIH. (A) Representative immunohistochemistry images of ALCAM in liver biopsies from HC (n=3) and patients with AIH (n=6). (B) Representative immunofluorescence staining for CD4, CD6 and ALCAM in interface hepatitis lesion of liver sections from patients with AIH (n=3). (C) Concentration of serum ALCAM in HC (n=28) and AIH (n=86) was measured by ELISA assay. Individual correlation between clinical indicators and serum ALCAM was calculated in patients with AIH (n=86). (D) Correlation between the number of hepatic CD6 + cells and paired serum ALCAM concentration was calculated (n=27). ***p < 0.001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: ALCAM promoted CD6 high CD4 + T cells trans-endothelial migration in vitro . (A, B) Human CD4 + T cells were magnetically isolated from PBMC of healthy donors and stimulated with αCD3/28 for 3 days in a flat-bottom 96-well plate, the expression of CD6 and cell proliferation was detected with flow cytometry. (C) After stimulation, the expression of cytokines, surface markers and transcription factors was compared between the CD6 high and CD6 low subsets. (D) Pre-activated CD4 + T cells were placed on a transwell chamber with 5μm pore for 24 hours in the presence of rhALCAM (3ug/ml) or vehicle (PBS). The expression of CD6 and CD69 was measured by flow cytometry. Experiments were repeated at least three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration, In Vitro, Isolation, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Intrahepatic activated leukocyte cell adhesion molecule induces CD6 high CD4 + T cell infiltration in autoimmune hepatitis

doi: 10.3389/fimmu.2022.967944

Figure Lengend Snippet: Schematic diagram for this study. Upregulated ALCAM on hepatocytes promoted the trans-endothelial migration of pathogenic CD6 high CD4 + T cells, which further aggravated hepatic inflammation of patients with AIH. This study revealed a putative therapeutic approach for patients with AIH.

Article Snippet: Recombinant human ALCAM Fc chimera (R&D Systems, Minneapolis, MN, USA 7187-AL, referred as rhALCAM) was added when required.

Techniques: Migration

Journal: Nature Communications

Article Title: A bispecific CD40 agonistic antibody allowing for antibody-peptide conjugate formation to enable cancer-specific peptide delivery, resulting in improved T proliferation and anti-tumor immunity in mice

doi: 10.1038/s41467-024-53839-5

Figure Lengend Snippet: A The agonistic activity of the anti-CD40 mAbs clones incubated for 48 h with immature moDCs evaluated for upregulation of markers MHC-II, CD86, and CD83 clustered in heatmap together with IL-12 secretion. B Epitope mapping of the top agonistic clone A9 and non-agonistic clone B1 determined by HDX-MS illustrated in a CD40L-CD40 crystal structure (PDB 3QD6). C Agonistic activity of the mAbs was performed by incubation with immature moDCs for 48 h and evaluated by flow cytometry of CD86 MFI levels (illustrated as background-subtracted log-transformed values) n = 3 (independent donors) and IL-12p40 levels n = 5 (independent donors pooled from two individual experiments) in the supernatant were quantified by ELISA. P -values are shown in the graph (ns = non-significant) and calculated with Kruskal-Wallis with Dunn´s multiple (CD86) and one-way ANOVA with Dunnett´s multiple comparisons test (hIL12p40). D Agonistic activity of mAbs evaluated by upregulation of CD86 on isolated CD19 + B cells via flow cytometry 24 h post-stimulation. n = 2 (independent donors, illustrated as the mean of two technical replicates). E Immature htgCD40 BMDCs were stimulated for 48 h with the mAbs thereafter, IL-12p40 was quantified by ELISA in the supernatant. n = 1 (mean value from two technical replicates). Data is shown as mean ± SEM.

Article Snippet: Human CD40-Fc was diluted to 20 nM in HBS supplemented with 0.05% Tween-20 injected either alone or pre-incubated with 200 nM human CD40L (R&D Systems, Cat. 6420-CLB) and subsequently added.

Techniques: Activity Assay, Clone Assay, Incubation, Flow Cytometry, Transformation Assay, Enzyme-linked Immunosorbent Assay, Isolation