Journal: International Journal of Molecular Sciences

Article Title: Novel Medicinal Mushroom Blend as a Promising Supplement in Integrative Oncology: A Multi-Tiered Study using 4T1 Triple-Negative Mouse Breast Cancer Model

doi: 10.3390/ijms21103479

Figure Lengend Snippet: Primary/secondary antibodies and respective dilution used for immunocytochemical experimental procedures.

Article Snippet: Primary antibodies , Anti-Interleukin-6 (M-19) , Purified antibody raised against a peptide mapping at the C-terminus of IL-6 of mouse origin , Santa Cruz Biotechnology (Santa Cruz, CA, USA), Goat polyclonal IgG, Cat# sc-1265, RRID: AB_2127470 , 1:100.

Techniques: Purification, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: Construction, expression, and purification of rhIL-23R-CHR/Fc. ( A ) PCR products of rhIL-23R-CHR/Fc recombinant gene. Lane 1, rhIL-23R-CHR/Fc gene sequence. Lane 2, hIgG1 Fc fragment. Lane 3, rhIL-23R-CHR gene. M, DNA molecular weight markers, bp. ( B ) Identification of rhIL-23R-CHR-Fc/T vector digested by Hind III and Xho I sites. Lane 1, pcDNA3.1 (+) - IL-23R-CHR/Fc after digestion. Lane 2, pcDNA3.1 (+) - IL-23R-CHR/Fc before digestion. M, DNA marker, bp. ( C ) Plasmid map of pcDNA3.1 (+) - IL-23R-CHR/Fc. The gene sequence encoding rhIL-23R-CHR/Fc was inserted into pcDNA3.1 (+) vector at the corresponding restriction sites Hind III and Xho I. ( D ) SDS-PAGE analysis of the purified rhIL-23R-CHR/Fc fusion protein using Protein A column. Lane 1, purified rhIL-23R-CHR/Fc fusion protein at reduced state. M, protein molecular weight markers, KDa. ( E ) Western blot analysis of rhIL-23R-CHR/Fc using mAbs against human IL-23R. Lane 1, purified rhIL-23R-CHR/Fc fusion protein at non-reduced state. Lane 2, purified rhIL-23R-CHR/Fc fusion protein at reduced state. ( F ) Plasma clearance of IL23RCHR-Fc in mice.

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques: Expressing, Purification, Recombinant, Sequencing, Molecular Weight, Plasmid Preparation, Marker, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: Role of rhIL-23R-CHR/Fc in mouse Th17 cell differentiation. Mouse naive CD4 + T cells were differentiated into Th17 cells in the presence of different concentrations of rhIL23R-CHR/Fc in vitro. ( A ) Analysis of Th17 cells (CD4 + IL-17 + ) by flow cytometry. ( B ) The percentage of Th17 cells. ( C ) IL-17A level in supernatants derived from cultured spleen cells. All tests were performed in triplicate and are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Derivative Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: rhIL-23R-CHR/Fc ameliorated skin inflammation in an imiquimod (IMQ)-induced psoriasis-like model. ( A ) Schematic diagram of intravenous administration of rhIL23R-CHR/Fc on days 2, 4 and 6 during the application of IMQ. Three mice in each group were sacrificed on days 4, 7, 10 and 14 to conduct experiments. The normal group did not receive IMQ as a negative control. Cyclosporin A (CsA), an immunosuppressive agent commonly used in the clinical therapy for autoimmune diseases, was used in the treatment of psoriasis at 1.5 mg/kg as a positive control. ( B ) Phenotypic presentation of lesional skin from mice. ( C ) The size of spleens of mice. ( D ) Mice skin Psoriasis Area and Severity Index (PASI) scores. ( E ) Haematoxylin and eosin staining of lesional skin obtained from normal mice or mice treated with IMQ, CsA or rhIL-23R-CHR/Fc at day 7 (200×, 400×).

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques: Negative Control, Positive Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: rhIL-23R-CHR/Fc inhibited Th17 cell-mediated inflammatory response. ( A ) Representative flow-cytometric analysis of Th17 cells (CD4 + IL-17A + ) in spleen from normal mice or mice ( n = 3) treated with IMQ at day 4, 7, 10, and 14. ( B ) Statistical data of Th17 cell percentage. ( C , D ) Protein levels of mIL-17A(C) and mIL-23(D) in mice serum. Data are mean ± SEM of n ≥ 3 per group. * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant (Student’s t -test).

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: rhIL-23R-CHR/Fc inhibited the transcriptional levels of inflammatory-associated cytokines, transcription factors, and chemokines in mouse skin and spleen. ( A – D ) The mRNA expression of IL-17A, IL-17F, IL-22 ( A ), Rorγt ( B ), IL-23R ( C ) and CCL20 ( D ) in skin lesions from normal or mice treated with IMQ ( n > 3). ( E , F ) The mRNA expression of IL-17A, IL-17F ( E ), and IL-23R ( F ) in spleen from normal or mice treated with IMQ. All results are representative of at least 3 independent experiments with at least 3 samples in each group. Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns, not significant (Student’s t -test).

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Human IL-23R Cytokine-Binding Homology Region-Fc Fusion Protein Ameliorates Psoriasis via the Decrease of Systemic Th17 and ILC3 Cell Responses

doi: 10.3390/ijms20174170

Figure Lengend Snippet: rhIL-23R-CHR/Fc limited the production of ILC3 cells. ( A ) Percentages of ILC3s (Lin − CD45 + RORγt + ) in small intestine lamina propria from Normal, Model, rhIL-23R-CHR/Fc, and CsA group mice were analyzed by flow cytometry ( n = 3) at day 7, 10, and 14. ( B ) Statistical data of ILC3 cells percentage. ( C ) The frequency of Nkp46 − / + ILC3 cells at day 7 from Normal, Model, rhIL-23R-CHR/Fc, and CsA group mice. ( D ) Analysis of IL-22 level in small intestines by immunofluorescence staining (200×). All results are representative of at least 3 independent experiments with at least 3 samples per group in each. Data represent the mean ± SEM. * p < 0.05 (Student’s t -test).

Article Snippet: The rhIL23R-CHR/Fc fragment cleaved from rhIL-23R-CHR/Fc/T Vector pMD19 (Takara, Tokyo, Japan) with Xho I and Hind III (Takara, Tokyo, Japan).

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Single-cell Landscape Analysis of the Circulating Human B Cell Pool under Selective Pressure of Allogeneic Stem Cell Transplantation

doi: 10.1101/2022.10.13.512162

Figure Lengend Snippet: A, DEGs in the scRNA-Seq dataset (untreated B cells) reaching significance ( P adj <0.05) in four or more of the 10 B cell clusters described in , being Up in Active cGVHD (left heat map) or Down in Active cGVHD (right heat map). Colored squares in the heat maps indicate the gene reached significance in that cluster, with log2 FC values as indicated. No DEGs mapped to Cluster 6. Genes that were also depicted in are shown in bold font. B, DEG analysis between disease groups performed on total untreated B cells. Heat maps show log2 FC values for annotated genes with a statistically different ( P adj <0.05) expression between allo-HCT patient groups, representing the difference in Active cGVHD B cells compared to No cGVHD B cells (Up or Down in Active cGVHD, as indicated). C-E, Validation of CKS2 transcript overexpression in Active cGVHD B cells within the scRNA-Seq dataset and for a different cohort of allo-HCT patients. In (C), normalized expression density UMAP plots for CKS2 from the single-cell RNA-Seq dataset in the Active cGVHD (Active) and No cGVHD (No) groups are shown. Representative regions depicted by the boxes were chosen randomly and enlarged (arrows) to visualize single B cells more easily. In (D), normalized CKS2 expression values across all 10 B cell clusters for all 8 allo-HCT patients are shown, separated by disease group. In (E), qPCR analysis of CKS2 was performed on freshly isolated, untreated B cells from a different allo-HCT patient cohort having Active cGVHD ( n =10) or No cGVHD ( n =7). Results indicate the fold change in CKS2 expression based on the mean value in the No cGVHD group normalized to 1. ACTB (β-ACTIN) was the housekeeping gene in the qPCR analysis. Statistical comparison was performed using a two-tailed Mann-Whitney test (GraphPad Prism 9 software; **, p <0.01). ( F ) Representative phosphoprotein capture arrays for detection of various intracellular signaling molecules phosphorylated on key sites involved in their regulatory activity, performed on whole cell lysates of purified, untreated B cells isolated from Active cGVHD ( n =3) and No cGVHD ( n =3) patient blood samples (see also fig. S6 ). Dashed boxes and protein IDs indicate the location and assay results for duplicate spots of capture antibodies against P27 KIP1 (phospho-T198), AMPKα2 (phospho-T172), and RSK1/2/3 (phospho-S380/S386/S377, respectively). Reference control spots on the arrays are indicated (ref). ( G ) Combined density results from the 3 independent phosphoprotein array assays shown in (F) and fig. S6 . Each bar indicates the results from one experiment and represents the ratio of the average dual spot intensity for Active cGVHD B cells over No cGVHD B cells for the protein indicated (dashed line represents a ratio of 1 as a guide). ( H ) Western blot analysis of total P27 KIP1 protein levels relative to β-ACTIN in whole cell lysates of B cells isolated from Active cGVHD ( n =4) and No cGVHD ( n =4) patient blood samples. Statistical comparisons were performed using a two-tailed, unpaired t-test (GraphPad Prism 9 software; **, p <0.01). ( I ) Model for P27 KIP1 dysregulation in Active cGVHD B cells.

Article Snippet: Membranes were blocked in 2% fish gelatin buffer for 75 min and then incubated with rabbit anti-human P27 KIP1 polyclonal antibody (C-19, Santa Cruz Biotechnology, cat#sc-528, lot#KO413) overnight at 4°C.

Techniques: Expressing, Over Expression, RNA Sequencing Assay, Isolation, Two Tailed Test, MANN-WHITNEY, Software, Activity Assay, Purification, Western Blot

Journal: Frontiers in Immunology

Article Title: An Integrated Platform for Serological Detection and Vaccination of COVID-19

doi: 10.3389/fimmu.2021.771011

Figure Lengend Snippet: Detection of SARS-CoV-2 infection using our N-cell-based ELISA and comparison to protein-based ELISA kits. Serum samples (1:200 dilution) of 10 healthy controls (HC) and 21 patients (PT) were subjected to N-cell-based ELISA (A) , N-protein-based ELISA kit-1 (B) , and N-protein-based ELISA kit-2 (C) to determine levels of anti-N antibodies. Pink bars represent the three samples from patients infected with the Alpha variant. Cell-based ELISA reads have been normalized to the read derived from individual serum interacting with wt-cells. Dotted line: cutoff value using the mean + 3SD of HC samples.

Article Snippet: Four different commercialized protein-based ELISA kits were used to compare detection sensitivity with our cell-based ELISAs, namely N-protein-based ELISA kit-1 (Abclonal, Catalog No. RK04139, Woburn, MA, USA), N-protein-based ELISA kit-2 (Proteintech, Catalog No. KE30001, Rosemont, IL, USA), S1-protein-based ELISA kit (Abclonal, Catalog No. RK04138, Woburn, MA, USA), and RBD-protein-based ELISA kit (Proteintech, Catalog No. KE30003, Rosemont, IL, USA).

Techniques: Infection, In-Cell ELISA, Enzyme-linked Immunosorbent Assay, Variant Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: An Integrated Platform for Serological Detection and Vaccination of COVID-19

doi: 10.3389/fimmu.2021.771011

Figure Lengend Snippet: P/N ratios calculated from three N-based ELISAs applied to 21 patient samples.

Article Snippet: Four different commercialized protein-based ELISA kits were used to compare detection sensitivity with our cell-based ELISAs, namely N-protein-based ELISA kit-1 (Abclonal, Catalog No. RK04139, Woburn, MA, USA), N-protein-based ELISA kit-2 (Proteintech, Catalog No. KE30001, Rosemont, IL, USA), S1-protein-based ELISA kit (Abclonal, Catalog No. RK04138, Woburn, MA, USA), and RBD-protein-based ELISA kit (Proteintech, Catalog No. KE30003, Rosemont, IL, USA).

Techniques: Enzyme-linked Immunosorbent Assay