Journal: International journal of radiation oncology, biology, physics

Article Title: Therapeutic Intervention Using a Smad7-Based Tat Protein to Treat Radiation-Induced Oral Mucositis.

doi: 10.1016/j.ijrobp.2021.09.039

Figure Lengend Snippet: Fig. 5. Pharmacodynamics markers of Tat-PYC-Smad7 treated dog oral mucositis. (A) Biopsies of posttreatment oral mucosa were evaluated by immunohistochemistry to detect NFkB-p50, pSmad3, pH2AX, and myeloperoxidase. Representative images of vehicle treated and Tat-PYC-Smad7 treated mucosa are presented with the lower panel in each set, presenting a high-power view of the blue box identified in the panel above. Scale bar: 100 mm. (B) Quantification of positive, brown-staining in epithelium sections excluding ulcers (in vehicle group) pictured in panel A. Myeloperoxidase staining together with mor- phology evaluation were used to identify neutrophils (indicated by yellow arrows) in ulcer surface and stroma. Faint brown staining in epithelium is nonspecific. The staining quantification for each dog and the mean of each treatment § standard error of the mean is presented.

Article Snippet: We performed immunohistochemistry (IHC) staining as previously described.15 Primary antibodies used were rabbit anti-Ki67 (1:500) (Cell Signaling Technology, 12202), rabbit anti-NFkB-p105/p50 [p Ser337] (1:300) (Novus Biologicals, NB100-82074), rabbit anti-pSmad3 (1:400, Abcam, ab52903), rabbit anti-pH2AX (1:100) (Cell Signaling Technology, 9718), rabbit anti-myeloperoxidase (Ready-to-Use, Dako, GA51161-2), rabbit anti-hemagglutinin (HA) (1:700) (Cell Signaling Technology, 3724), rabbit anti-IL-1b (1:500) (Novus Biologicals, NB600-633), rabbit anti-TNF-a (1:200) (Novus Biologicals, NBP1-19532), mouse anti-CCL2 (1:50) (Novus Biologicals, MAB28171), and goat anti-IL-6 (1:40) (R&D, AF1609) antibodies.

Techniques: Drug discovery, Immunohistochemistry, Staining

Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from blood and spleen tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final dose injection. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (C) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype and function of CNS innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) CD11b + CD45 lo/neg resident microglia and (C) CNS CD11b + CD45 hi infiltrating macrophages and were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. CD11b + cells were isolated from CNS tissue of anti‐CD52‐Ab or vehicle‐treated mice at first day after the final treatment dose. (D) Respiratory burst activity of CD11b + ‐isolated cells. (E) Phagocytosis of FITC‐coated beads by CD11b + isolated cells. Unpaired two‐tailed Student's t test. (F) Cells were left unstimulated or were stimulated with LPS or Poly(I:C)(PIC) for 24 h and cytokine production determined by ELISA. Two‐way ANOVA with Sidak multiple comparisons test, * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery and CNS innate immune cells in EAE mice at three weeks post‐injection. Single‐cell suspensions were obtained from blood, spleen and CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated mice three weeks after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). (C) CNS CD11b + CD45 hi ‐infiltrating macrophages and CD11b + CD45 lo/neg resident microglia were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the function of periphery and CNS innate immune cells in SCID mice at first day post‐injection. SCID mice were treated with either vehicle or 10 mg/kg murine anti‐CD52‐Ab‐Ab for five consecutive days. Single‐cell suspensions were then obtained from blood and spleen tissues first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II, CD40, CD80 and CD86 (all n = 7). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II, CD40, CD80, CD86, and CD206 (all n = 7). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: CCL11 promotes hepatocellular carcinoma recurrence after surgery by potentiating immunosuppressive CCR5 + CD206 + M2-like macrophages and promoting tumor invasiveness

doi: 10.1038/s41419-026-08508-4

Figure Lengend Snippet: A Flow cytometry quantification of the number of monocyte-differentiated CCR5 + CD206 + M2-like macrophages after administration of recombinant human CCL11 (rhCCL11) protein. Control group: PBS treatment. B The expression Levels of M2-macrophage-related immunosuppressive markers and cytokines in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. C Cytokine array analysis of secreted cytokines in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. Red dot boxes indicate two highly upregulated cytokines: 1, MCP1; 2, IL-8. D mRNA Levels of IL-8 and MCP-1 in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. E CCL11-treated CCR5 + M2-like macrophages enhanced the induction of CD4+ Foxp3+Tregs from naive CD4 + T compared to the PBS-treated control group. F Top 10 enriched KEGG pathways from differentially expressed genes in the CCL11-treated CCR5 + M2-like macrophages. G Gene set enrichment analysis of Toll-like receptor signaling pathway. H Levels of p-IKKα/β, p-IkBα, NFkB1-P50, and PD-L1 expression via western blot analysis. I Schematic diagram of the molecular mechanism of CCL11 in regulating CCR5 + M2-like macrophages. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns not significant.

Article Snippet: Antibodies used for Western blot analysis anti-human/mouse p-IKKα/β (#2078T), anti-human/mouse p-IkBα (#2859T), anti-human/mouse NFKB1-p50 (#13586S), anti-human/mouse p-AKT (#9271S), and anti-human/mouse AKT (#9272S) from Cell Signaling Technology; anti-human/mouse PDL1 (#14-5983-82), anti-human/mouse p-PI3K (#PA5-104853), and anti-human/mouse Mafk (#PA5-115534) from Thermo Fisher Scientific; anti-human/mouse PI3K (#sc-423) and anti-human GAPDH (#sc47724) from Santa Cruz Biotechnology; and anti-human/mouse MMP13 (#18165-1-AP) and anti-mouse GAPDH (#60004-1-Ig) from Proteintech Group.

Techniques: Flow Cytometry, Recombinant, Control, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: Downregulation of transgelin 2 promotes breast cancer metastasis by activating the reactive oxygen species/nuclear factor-κB signaling pathway

doi: 10.3892/mmr.2019.10643

Figure Lengend Snippet: Loss of TAGLN2 expression induces NF-κB activation. (A) Luciferase activity of an NF-κB-luciferase reporter plasmid in TAGLN2-overexpressing 293T cells was measured 48 h after stimulation with siRNA-TAGLN2 or scramble siRNA; luciferase activity was normalized to that of Renilla . Data were representative of three independent experiments. (B) Immunoblotting of IκB, p50, p65/RelA and TAGLN2 expression in MDA-MB-231 and MCF-7 cells transfected with pLKO.1-shRNA-TAGLN2 and pLKO.1-shRNA-scramble. The results of western blotting were representative of three independent experiments. (C) Cell migration was promoted in MDA-MB-231 cells transfected with pLKO.1-shRNA-TAGLN2 compared with cells treated with scramble shRNA. Treatment with 18 µM SN50 for 24 h reversed the effects of TAGLN2 downregulation on cell invasion. Data are representative of three independent experiments. *P<0.05 vs. scramble or as indicated. DMSO, dimethyl sulfoxide; NF-κB, nuclear factor-κB; shRNA, short hairpin RNA; siRNA, small interfering RNA; TAGLN2, transgelin 2.

Article Snippet: The membranes were probed with primary antibodies against PRDX1 (1:1,000; cat. no. EPR5433; Epitomics, Abcam, Cambridge, UK), FLAG (1:500; cat. no. F7425; Sigma-Aldrich, Merck KGaA), TAGLN2 (1:1,000; cat. no. 60044-1-lg; ProteinTech Group, Inc.), IκB (1:3,000; cat. no. 66418-1-lg; ProteinTech Group, Inc.), p50 (1:200; cat. no. 15506-1-AP; ProteinTech Group, Inc.), p65 (1:1,000; cat. no 10745-1-AP; ProteinTech Group, Inc.), MMP1 (1:1,000; cat. no. 54376; Cell Signaling Technology), MMP2 (1:1,000; cat. no. 4022; Cell Signaling Technology), CXCR4 (1:1,000; cat. no. ab181020; Abcam, Cambridge, UK), Lamin B1 (1:3,000; cat. no. 12987-1-AP; ProteinTech Group, Inc.) and GAPDH (1:3,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.) on the rocking table at 4°C overnight.

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Transfection, shRNA, Migration, Small Interfering RNA