Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 1. Interactions between the subunits of IL-6 and IL-27 in vivo and in vitro. (AC) CoIP and immunoblotting of serum samples were performed with the indicated Abs. IL-6 interacted with IL-27A (A), IL-6 interacted with EBi3 (B), and IL-27A interacted with EBi3 (C). (DG) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with IL-27A-V5 (D), IL-6-3HA interacted with EBi3-Myc (E), IL-27A-V5 interacted with EBi3-Myc (F), and IL-6-3HA interacted with IL-27-V5 (G). (H) Colocalization of IL-6 and IL-27 was detected by confocal microscopy. A549 cells were transfected with IL-6-eGFP-KDEL and IL-27-DsRed-KDEL for 24 h, and the nuclei were stained with DAPI before observation using confocal microscopy. All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: In Vivo, In Vitro, Western Blot, Transfection, Confocal Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 2. Construction of the IL-6IL-27 complex and its antiviral activity. (A) A schematic of the IL-6IL-27 construction via flexible self-cleaved linker is shown. Immunoblotting analysis of IL-6, EBi3, IL-27A, and b-actin of HEK293T cells transfected with empty vector pcDNA3.1(1) or IL-6IL-27 complex expression plasmids for 24 h. (B) A549 cells were transfected with indicated plasmids for 24 h followed by H1N1 infection (MOI = 1) for 12 h. The NP-specific mRNA, cRNA, and vRNA levels were measured using QRT-PCR. (C) RD cells were transfected with the indicated plasmids for 24 h fol- lowed by EV71 infection (MOI = 1) for 24 h. The VP1 mRNA levels were measured using QRT-PCR. (D) Vero cells were (Figure legend continues)

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 5. The IL-6IL-27 complex interacts with MAVS. (AC) A549 cells were transfected with increasing amounts of the indicated expression plas- mids (wedge; 100, 200, and 300 ng) of the IL-6IL-27 complex expression plasmid and luciferase reporter plasmids containing the IFN-b (A), NF-kB (B), and ISRE (C) promoter for 24 h followed by VSV infection (MOI = 1) for 24 h. Reporter assays were performed. pRL-TK was used as an internal control. (DI) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with Flag-MAVS (D), IL-27A-V5 interacted with Flag-MAVS (E), EBi3-Myc interacted with Flag-MAVS (F), IL-6-HA interacted with Flag- TBK1 (G), IL-27A-V5 interacted with Flag-TBK1 (H), and EBi3-Myc interacted with Flag-TBK1 (I). All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Infection, Control, Western Blot

Journal: Nature medicine

Article Title: Interleukin-35 Induces Regulatory B Cells that Suppress CNS Autoimmune Disease

doi: 10.1038/nm.3554

Figure Lengend Snippet: (a) Schematic of the cDNA constructs used to genetically engineer IL-12p35 (p35), Ebi3 and IL-35 (p35/Ebi3) recombinant proteins. (b) Coomassie blue gels of the recombinant proteins characterized on reducing SDS or non-reducing polyacrylamide gels. (c) Detection and characterization of p35, Ebi3 or IL-35 recombinant proteins by immunoprecipitation/Western blot analysis under reducing condition. (d) Characterization of 2 independently generated batches of the purified rIL-35 preparations by Western blotting under non-reducing conditions with anti-p35 or anti Ebi3 Abs. (e, f, g) Purified B220 + CD19 + B cells from C57BL/6 mouse spleen were stimulated with LPS (5μg/ml) in medium containing 50 ng/ml pMIB, p35, Ebi3 or rIL-35 for 72 h. Lymphocyte proliferation was analyzed by [ 3 H]-thymidine incorporation assay (e) and IL-10 production was analyzed by ELISA (f) or intracellular cytokine staining assay (g). (h, i) WEHI-279 B-cells were cultured in medium containing 50 ng/ml pMIB, p35, Ebi3 or rIL-35. Lymphocyte proliferation was analyzed by [ 3 H]-thymidine incorporation assay (h) and IL-10 production was analyzed by ELISA (i). (j) CD19 + primary B cells were stimulated with rIL-35 (50ng/ml) and the purified Breg cells were co-cultured (1:5) for 3 days with freshly isolated CD19 + B cells in medium containing LPS. Proliferation was analyzed by the [ 3 H]-thymidine incorporation assay. Results represent at least 3 independent experiments (* P <0.05; ** P <0.01; *** P < 0.001; **** P <0.0001).

Article Snippet: The following antibodies were used for Western blotting and/or immunoprecipitation: anti-Flag or anti-V5 (Invitrogen Life Technologies, Grand Island, NY); antibodies specific to STAT1, pSTAT1, STAT3, pSTAT3, STAT4, pSTAT4, STAT5, pSTAT5, STAT6, pSTAT6 (Cell signaling Technology, ); antibodies specific to IL-12p35, Ebi3, IL-12Rβ1, IL-12Rβ2, IL-27Rα, gp130, and Danvers, β-actin MA(Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Construct, Recombinant, Immunoprecipitation, Western Blot, Generated, Purification, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Isolation

Journal: Nature medicine

Article Title: Interleukin-35 Induces Regulatory B Cells that Suppress CNS Autoimmune Disease

doi: 10.1038/nm.3554

Figure Lengend Snippet: (a) Purified CD19 + B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220 + B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10 − or IL-10 + B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220 lo CD19 + CD5 + or B220 lo CD19 + CD5 + IL-10 + cells in the spleen. Results are representative of at least 3 independent experiments (**** P <0.05; ** P <0.01; *** P < 0.001).

Article Snippet: The following antibodies were used for Western blotting and/or immunoprecipitation: anti-Flag or anti-V5 (Invitrogen Life Technologies, Grand Island, NY); antibodies specific to STAT1, pSTAT1, STAT3, pSTAT3, STAT4, pSTAT4, STAT5, pSTAT5, STAT6, pSTAT6 (Cell signaling Technology, ); antibodies specific to IL-12p35, Ebi3, IL-12Rβ1, IL-12Rβ2, IL-27Rα, gp130, and Danvers, β-actin MA(Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Purification, Expressing, Isolation, Staining, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Nature medicine

Article Title: Interleukin-35 Induces Regulatory B Cells that Suppress CNS Autoimmune Disease

doi: 10.1038/nm.3554

Figure Lengend Snippet: EAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19 + B-cells were isolated on a cell sorter. The purified CD19 + B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×10 7 ) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (** P < 0.01; *** P <0.001; **** P < 0.0001).

Article Snippet: The following antibodies were used for Western blotting and/or immunoprecipitation: anti-Flag or anti-V5 (Invitrogen Life Technologies, Grand Island, NY); antibodies specific to STAT1, pSTAT1, STAT3, pSTAT3, STAT4, pSTAT4, STAT5, pSTAT5, STAT6, pSTAT6 (Cell signaling Technology, ); antibodies specific to IL-12p35, Ebi3, IL-12Rβ1, IL-12Rβ2, IL-27Rα, gp130, and Danvers, β-actin MA(Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Ex Vivo, Purification, Cell Culture, Adoptive Transfer Assay

Journal: Nature medicine

Article Title: Interleukin-35 Induces Regulatory B Cells that Suppress CNS Autoimmune Disease

doi: 10.1038/nm.3554

Figure Lengend Snippet: (a) Purified rIL-35-induced Breg cells and IL-10 negative B cells were derived as described . WT mice with EAU were treated with 1×10 6 Breg or IL-10 − B cells. (b) Eyes were analyzed by fundoscopy or histology 21 days after disease induction and characteristic features of EAU are as described in . Scale bar, 500 μM. (c) EAU scores were determined as described in . (d, e) Cells from spleen or draining LN were gated on CD19 or CD4, respectively and the percentage of IL-10-, p35- or Ebi3-expressing B cells (d) or Foxp3-, IFN-γ or IL-17-expressing CD4 + T cells (e) was determined by intracellular-cytokine staining assay. (f, g) B cells from the pMIB- or rIL-35-treated mice (described in a) were co-cultured with IRBP-stimulated uveitogenic LN cells (5:1) for 3 days and the cells (1×10 7 ) were transferred into naïve CD45.1 congenic mice. Ten days after adoptive transfer, recipient (CD45.1) and donor (CD45.2) cells were analyzed for IL-10-producing B cells (gated on CD19) in spleen (f) or Foxp3-expressing T cells (gated on CD4) in LN (g). (h, i) Purified human CD19 + B cells were cultured for 3 days with PMA in medium with or without rIL-35 and analyzed by FACS or RT-PCR (h) or [ 3 H]-thymidine-incorporation assay (i). Data is representative of analysis of PBMC from 10 donors. Other results represent at least 3 independent experiments (* P <0.05; ** P <0.01; *** P < 0.001; **** P <0.0001).

Article Snippet: The following antibodies were used for Western blotting and/or immunoprecipitation: anti-Flag or anti-V5 (Invitrogen Life Technologies, Grand Island, NY); antibodies specific to STAT1, pSTAT1, STAT3, pSTAT3, STAT4, pSTAT4, STAT5, pSTAT5, STAT6, pSTAT6 (Cell signaling Technology, ); antibodies specific to IL-12p35, Ebi3, IL-12Rβ1, IL-12Rβ2, IL-27Rα, gp130, and Danvers, β-actin MA(Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Purification, Derivative Assay, Expressing, Staining, Cell Culture, Adoptive Transfer Assay, Reverse Transcription Polymerase Chain Reaction, Thymidine Incorporation Assay

Journal: Nature medicine

Article Title: Interleukin-35 Induces Regulatory B Cells that Suppress CNS Autoimmune Disease

doi: 10.1038/nm.3554

Figure Lengend Snippet: (a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [ 3 H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [ 3 H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (* P <0.05; ** P <0.01; *** P < 0.001; **** P <0.0001).

Article Snippet: The following antibodies were used for Western blotting and/or immunoprecipitation: anti-Flag or anti-V5 (Invitrogen Life Technologies, Grand Island, NY); antibodies specific to STAT1, pSTAT1, STAT3, pSTAT3, STAT4, pSTAT4, STAT5, pSTAT5, STAT6, pSTAT6 (Cell signaling Technology, ); antibodies specific to IL-12p35, Ebi3, IL-12Rβ1, IL-12Rβ2, IL-27Rα, gp130, and Danvers, β-actin MA(Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transduction, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Thymidine Incorporation Assay, Staining, Expressing, Western Blot, Immunoprecipitation, Activation Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

doi: 10.1177/1010428317692237

Figure Lengend Snippet: Figure 1. The expressions of PD-L1, PD-1, and EBI3 in rats before and after radiotherapy (×200). *p < 0.05 compared with before radiotherapy and 1 week after radiotherapy.

Article Snippet: And EBI3 small interfering RNA (siRNA; sc-39410; Santa Cruz Biotechnology Co, Ltd) was bought.

Techniques:

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

doi: 10.1177/1010428317692237

Figure Lengend Snippet: Figure 2. The expressions of EBI3, PD-1, and PD-L1 in HeLa cells detected by Western blotting: (a) the blank group, (b) the negative control group, (c) the EBI3 mimics group, and (d) the EBI3 inhibitors group. *p < 0.05 compared with the blank and negative control groups.

Article Snippet: And EBI3 small interfering RNA (siRNA; sc-39410; Santa Cruz Biotechnology Co, Ltd) was bought.

Techniques: Western Blot, Negative Control

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

doi: 10.1177/1010428317692237

Figure Lengend Snippet: Figure 5. The apoptosis rate of Treg cells after cell transfection detected by flow cytometry: (a) the blank group, (b) the negative control group, (c) the EBI3 mimics group, and (d) the EBI3 inhibitors group. *p < 0.05 compared with the blank and negative control groups.

Article Snippet: And EBI3 small interfering RNA (siRNA; sc-39410; Santa Cruz Biotechnology Co, Ltd) was bought.

Techniques: Transfection, Flow Cytometry, Negative Control

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

doi: 10.1177/1010428317692237

Figure Lengend Snippet: Figure 6. The apoptosis rates of CD4+T cells and CD8+T cells after cell transfection detected by flow cytometry: (a) the blank group, (b) the negative control group, (c) the EBI3 mimics group, and (d) the EBI3 inhibitors group. *p < 0.05 compared with the blank and negative control groups.

Article Snippet: And EBI3 small interfering RNA (siRNA; sc-39410; Santa Cruz Biotechnology Co, Ltd) was bought.

Techniques: Transfection, Flow Cytometry, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: Loss of the autophagy regulators FIP200 or Atg5 results in inhibition of directional cell motility. (A) Wild-type and FIP200 knockout (KO) MCF10A cells processed for immunofluorescence microscopy and immunostained for LC3 (green). Nuclei are labeled with Hoechst (blue). Scale bar = 10 μm. (B) Western blot analysis on lysates harvested from wild-type and FIP200 KO MCF10A cells either left untreated or treated with Bafilomycin A1 (BfnA1) for 2 and 4 hours. Immunoblotting was performed against the indicated proteins. (C) Quantitation of western blot data illustrating the fold change in p62 and LC3-II levels in untreated and Bafilomycin treated cells. Levels of p62 and LC3-II were normalized to actin and represented as fold change from untreated wild-type control cells. Results represent at least 3 independent experiments and error bars are SEM. (D) MCF10A wild-type and FIP200 KO cells were subjected to a scratch wound assay and brightfield images were taken at initial wounding (0 h) and at 6, 12, and 24 hours post-wounding. Dotted white lines highlight wound edge. Scale bar = 200 μm. (E) Quantitation of scratch wound assay was performed and represented as % of wound closure, calculated as a percentage of total area of initial wound at 0 h. Results represent at least 3 independent experiments and errors bars illustrate SEM. (F) Western blot analysis on lysates harvested from wild-type and two clones of Atg5 KO MCF10A cells either left untreated or treated with BfnA1 for 2 and 4 hours. Immunoblotting was performed against the indicated proteins. (G) MCF10A wild-type and Atg5 KO cells were subjected to a scratch wound assay and brightfield images were taken at initial wounding (0 h) and at 6, 12, and 24 hours post-wounding. Dotted white lines highlight wound edge. Scale bar = 200 μm. (H) Quantitation of scratch wound assay was performed and represented as % of wound closure, calculated as a percentage of total area of initial wound at 0 h. Results represent at least 3 independent experiments and errors bars illustrate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Inhibition, Knock-Out, Immunofluorescence, Microscopy, Labeling, Western Blot, Quantitation Assay, Control, Scratch Wound Assay Assay, Clone Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: FIP200 or Atg5 depletion increases fibronectin-induced cell adhesion and spreading. (A) Western blot analysis of cell lysates harvested along the time course of fibronectin-induced MCF10A wild-type cell spreading. Immunoblotting was performed against the indicated proteins. Quantitation of p62 expression was normalized to actin and represented as the fold change from the zero time point (cells in suspension). Results represent at least 3 independent experiments. (B) Immunofluorescence microscopy of MCF10A cells following attachment and spreading on fibronectin for indicated time points. Cells were immunostained for p62 and LC3. Scale bar = 10 μm. Quantitation was performed on multiple images from random fields of view. The number of p62 or LC3 puncta (indicated with arrowheads) were quantified from 100 cells for p62 and 13–15 cells for LC3 across 3 independent experiments. Results represent the average number of p62 or LC3 puncta per cell. Error bars represent SEM. (C) Brightfield images across the time course of adhesion to fibronectin of MCF10A wild-type, FIP200 KO, and Atg5 KO cells. Higher magnification regions illustrate phenotypic differences and dotted line represents representative cell diameter. Scale bar = 100 μm. (D) Quantitation of individual cell area represented as square pixels were plotted across the time course of fibronectin adhesion. Results represent 50–100 cells per experiment, repeated at least 3 independent times. Black bar represents the mean and error bars are the SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Western Blot, Quantitation Assay, Expressing, Suspension, Immunofluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: Differential dysregulation of FAK pY397 trafficking in FIP200 and Atg5 KO cells. (A) MCF10A wild-type and FIP200 KO cells were subjected to fibronectin induced cell adhesion and fixed at indicated time points of 120 and 240 minutes post-plating. Immunostaining was performed against FAK pY397 (green) and p62 (red). Nuclei were labeled with Hoechst (blue). Arrowheads indicate FAK pY397 spots and arrows indicate FAK pY397 and p62 colocalization. (B) Quantitation of immunofluorescence images taken from cells subjected to fibronectin induced adhesion. The number of FAK pY397 puncta per cell were quantified from between 30 and 34 cells per group across random fields of view from 2 independent experiments. Each data point indicates a cell, the red bar indicates mean value and error bars represent SEM. **** p < 0.0001. Immunofluorescence microscopy performed on wild-type (WT) and Atg5 KO MCF10A cells following attachment to fibronectin. Cells were fixed at 120 min post-plating and immunostained in (C) for FAK pY397 (red) and FIP200 (green), and in (D) for FAK pY397 (red) and LC3 (green). Nuclei are labeled with Hoechst (blue). Arrowheads indicate areas of colocalization and arrows indicate FA-like immunostaining. Scale bars = 10 μm.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Immunostaining, Labeling, Quantitation Assay, Immunofluorescence, Microscopy

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control