mouse il7  (Sino Biological)

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helices were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model was extracted and submitted to AlphaFold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as a preliminary validation of the Rosetta-remodeled protein. Iterations of the bad models (models that do not fold into the expected structure or models that did not predict to bind to the receptors) back to the design stage were performed. Models that passed the AlphaFold validation proceeded to subsequent in vitro assay using yeast display system and flow cytometry to determine their relative binding affinity to IL-7 receptors in comparison to WT-IL7.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Sequencing, Binding Assay, Biomarker Discovery, In Vitro, Flow Cytometry, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: Blueprint of the WT-IL7 was shown on the left of the figure. The connectivity of the functioning helixes was connected in a manner that requires extremely long protein loops by design (i.e. helices were not connected to the closest adjacent helixes but to the opposite helix). Loops that were not interacted with the IL-7 receptors were deleted and the helixes were reconnected in a clockwise manner via new protein linkers connecting to the adjacent helixes. The blueprint of the redesigned protein was shown at the right side of the figure. Protein structures are colored as rainbow (from N-to-C terminus with the order of Blue-Green-Yellow-Red).

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques:

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) AlphaFold validation of the first loop design version of Neo-7 (Neo-7 LDv1) using the default (left) and single sequence mode (right). ( B ) AlphaFold validation of the second loop design version of Neo-7 (left; Neo-7 LDv2) and Neo-7 LDv2 with mutations (right) favored by Rosetta fix backbone design. ( C ) Crystal structure of human IL-7 in complexation to human IL-7 receptor alpha (PDB ID = 3DI2). ( D ) Superimposition of Neo-7 structures (with or without additional disulfide bridge) predicted by AlphaFold. ( E ) Yeast display and flow cytometry validation of IL-7/Neo-7 bindings towards the IL-7 receptors. The yeast-displayed protein (different redesigned IL-7s) carries a HA-tag while the recombinant IL-7 receptors carry either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma; for detection of IL-2Rγ binding, yeast cells were first incubated with recombinant IL-7Rα, washed, and subsequently incubated with IL-2Rγ.) The signal intensity of the X-axis (conferred by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (conferred by the binding of the anti-HIS/anti-FC mAb to the recombinant receptors bound to the displayed proteins) correlates with the binding affinity of the displayed proteins towards the IL-7 receptors.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Biomarker Discovery, Sequencing, Flow Cytometry, Recombinant, Binding Assay, Incubation, Expressing

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Inspection of structural and binding interactions of residue mutations Q6P and T45I on Neo-7 towards the murine IL-7R alpha. ( B ) Yeast display and flow cytometry validation of the binding ability of IL-7/Neo-7 variants toward the IL-7 receptors.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Binding Assay, Residue, Flow Cytometry, Biomarker Discovery

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: FPLC profile of E. coli expressed ( A ) WT-IL7 ( B ) refolded WT-IL7 ( C ) Neo-7-Q6P and ( D ) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn 7 software after affinity chromatography purification. SPR (Biacore) characterization of the binding kinetics of ( E ) Neo-7-Q6P ( F ) Neo-7-Q6P-T45I and ( G ) WT-IL7 towards murine IL-7R alpha. ( H ) 2E8 proliferation assay to investigate the biological activity of the IL-7/Neo-7s expressed by E. coli . Error bars represent standard deviation (n=3).

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Software, Affinity Chromatography, Purification, Binding Assay, Proliferation Assay, Activity Assay, Standard Deviation

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In silico immunogenicity prediction of ( A ) WT-IL7 ( B ) Neo-7 ( C ) Neo-7 single mutant ( D ) Neo-7 double mutant. ( E ) Sequence comparison of Neo-7 and WT-IL7, helices of the cytokine are colored as blue, green, yellow and red; loops are colored as gray. ( F ) Alphafold predicted structural model of FC-Neo-7.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: In Silico, Immunopeptidomics, Mutagenesis, Sequencing, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: FPLC profile of CHO-S expressed ( A ) WT-IL7 ( B ) Neo-7-Q6P and ( C ) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn 7 software after affinity chromatography purification. (D–E) Murine splenocyte proliferation assays performed at day 3 and day 7 following treatment with Fc-control (gray), Fc-WT-IL7 (red), Fc-Neo-7-Q6P (blue), or Fc-Neo-7-Q6P-T45I (green). In vivo immune stimulatory ability of the Fc-fused cytokines on murine PBMCs at day 0 to day 12 post-treatment. The data are presented as a count of ( F ) total viable CD45+ cells ( G ) viable CD45+ CD3+ CD4+ T cells ( H ) viable CD45+ CD3+ CD8+ T cells ( I ) viable CD45+ CD3- NK1.1+NK cells. Treatment groups are colored as Fc-control (gray), Fc-WT-IL7 (red), Fc-Neo-7-Q6P (blue), and Fc-Neo-7-Q6P-T45I (green). All data were presented as individual data plots with error bars (SEM) (n=3). Statistical differences among groups were determined using one-way ANOVA with Turkey’s multiple comparison test. Significance levels are defined as follows *p < 0.05; **p = 0.01–0.05; ***p = 0.0001–0.001; and ****p < 0.0001.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Software, Affinity Chromatography, Purification, Control, In Vivo, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Gene ontology analysis of the gene expression data from RNA sequencing (n=3; three independent biological donors for each group). ( B ) Gene Set Enrichment Analysis (GSEA) of splenic CD8+T cells treated by Fc-Neo-7 versus Fc-WT-IL7. ( C ) Principal component analysis and ( D ) Gene expression heatmap derived from Z-scores calculated from the RNA sequencing data. The gene expression heatmap is derived from Z-scores calculated from the RNA sequencing data, with expression levels color-coded from high (red) to low (blue).

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7-dependent murine B-cell cell line, were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20% FBS.

Techniques: Gene Expression, RNA Sequencing, Derivative Assay, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: BAG2 Inhibits Cervical Cancer Progression by Modulating Type I Interferon Signaling through Stabilizing STING.

doi: 10.1002/advs.202414637

Figure Lengend Snippet: Figure 6. BAG2 positively regulates STING-mediated type I interferon responses. A) RNA seq data of SiHa cells after BAG2 overexpression were analyzed by gene set enrichment analysis (GSEA), and Hallmark gene sets were used as annotated gene sets. B) Gene heat map of Hallmark Interferon 𝛼Response gene set after BAG2 overexpression in SiHa cells. C) SiHa cells were transfected with vector or GFP-BAG2 for 48 h, and then they were treated with HT- DNA for 0, 3, 6 h or cGAMP for 0, 2, 4 h. Cells were collected for detection of STING, p-TBK1, TBK1, p-IRF3, and IRF3 protein levels by Western blot. D) Vector or BAG2 plasmid was transfected in SiHa cells for 48 h, cell supernatants were collected by HT-DNA treatment for 6 hrs, and IFN-𝛽and IL7 production was measured by ELISA. E) Vector or BAG2 plasmid was transfected in U14 cells for 48 h, cell supernatants were collected by HT-DNA treatment for 6 h, and IFN-𝛽and IL7 production was measured by ELISA. F) SiHa cells and G) U14 cells were transfected with Vector or BAG2 for 48 h, then cGAMP was added for 4 h. qRT-PCR was used to measure the mRNA expression of ISG15, IFN-𝛽, MX1, and CXCL10. Data are presented as mean values ± SD, with n = 3 (B, D-G) biological independent experiments. Statistical significance was determined by two-tailed unpaired Student’s t-test (D-G).

Article Snippet: ELISA Assay: After transfecting the cells, the supernatant was collected, centrifuged to remove the precipitate, and the samples were processed according to the instructions of IL7 kit (EK207, EK107, MultiSciences Biotech) and IFN-β kit (EK2236, EK1236,MultiSciences Biotech), and then the absorbance value was detected by a microplate reader.

Techniques: RNA Sequencing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells alleviate liver fibrosis after rat liver transplantation through JAK1/STAT5 pathway

doi: 10.1186/s13287-025-04353-y

Figure Lengend Snippet: BMSCs may alleviate liver fibrosis after liver transplantation through the JAK1/STAT5 pathway. ( A ) Box plot for FPKM values. PCA analysis. Bar plot of the statistic of differentially expressed genes. Venn graph of the differentially expressed genes. Volcano plot of the differentially expressed genes. Heat map of RNA differential expression. GO analysis reveals the potential impact of the differentially expressed RNAs on functions. KEGG analysis reveals the potential impact of the differentially expressed RNA on pathways. KEGG map of the JAK/STAT pathway. ( B ) Western blot analyses of JAK1, p-STAT5, STAT5, p-STAT1, STAT1, p-STAT3, STAT3, IL7R, and IL7 in liver tissue. TUBA1A was used as the loading control except for p-STAT5, p-STAT1, and p-STAT3. STAT5, STAT1, and STAT3 were used as the loading control for p-STAT5, p-STAT1, and p-STAT3, respectively. Jak1 , Il7r , and Il7 mRNA levels in liver tissue. ( C ) Microscopic images of each group stained with HE, Masson, Sirius Red, and ACTA2 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, and ACTA2, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. ( D ) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, ACTA2, and CASP3 in liver tissues. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Full-length blots/gels are presented in Supplementary Fig. . The samples were derived from the same experiment and processed in parallel. Col1a1 , Jak1 , Acta2 , and Casp3 mRNA levels in liver tissues. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; CASP3, caspase 3; COL1A1, collagen type I alpha 1 chain; GO, Gene Ontology; IL, interleukin; JAK, Janus-activated kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; LT, liver transplantation; STAT, signal transducer and activator of transcription; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The LT + anti-IL7 group received anti-IL7 antibody (M25; Cat# HY-P990210, MCE, Shanghai, China) (250 μg/kg of body weight, per ip, once a week) for 2 weeks after LT.

Techniques: Transplantation Assay, Quantitative Proteomics, Western Blot, Control, Staining, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells alleviate liver fibrosis after rat liver transplantation through JAK1/STAT5 pathway

doi: 10.1186/s13287-025-04353-y

Figure Lengend Snippet: BMSCs may inhibit the activation of HSC-T6 cells after hypoxia–reoxygenation and reduce their apoptosis through the JAK1/STAT5 pathway. ( A ) Western blot analyses of JAK1, p-STAT5, STAT5, p-STAT1, STAT1, p-STAT3, STAT3, IL7R, and IL7 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5, p-STAT1, and p-STAT3. STAT5, STAT1, and STAT3 were used as the loading control for p-STAT5, p-STAT1, and p-STAT3, respectively. Jak1 and Il7r mRNA levels in HSC-T6 cells. ( B ) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, and ACTA2 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1 , Jak1 and Acta2 mRNA levels in HSC- T6 cells. ( C ) Apoptosis results of flow cytometry in each group. Full-length blots/gels are presented in Supplementary Fig. . The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; IL, interleukin; JAK, Janus-activated kinase; STAT, signal transducer and activator of transcription. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The LT + anti-IL7 group received anti-IL7 antibody (M25; Cat# HY-P990210, MCE, Shanghai, China) (250 μg/kg of body weight, per ip, once a week) for 2 weeks after LT.

Techniques: Activation Assay, Western Blot, Control, Flow Cytometry, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells alleviate liver fibrosis after rat liver transplantation through JAK1/STAT5 pathway

doi: 10.1186/s13287-025-04353-y

Figure Lengend Snippet: BMSCs regulate the IL7R/JAK1/STAT5 pathway in HSCs by modulating the secretion of IL7 by hepatic cells. ( A ) Microscopic images of each group stained with HE, Masson, Sirius Red, ACTA2, p-STAT5 and STAT5 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, ACTA2, and p-STAT5/STAT5, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. ( B ) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5 and ACTA2 in liver tissue. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1 , Jak1 , and Acta2 mRNA levels in liver tissue. ( C ) Western blot analyses of IL7 in IAR20 cells. TUBA1A was used as the loading control. Il7 mRNA levels in IAR20 cells. ( D ) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5 and ACTA2 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1 , Jak1 and Acta2 mRNA levels in HSC- T6 cells. Full-length blots/gels are presented in Supplementary Fig. . The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; IL, interleukin; JAK, Janus-activated kinase; LT, liver transplantation; STAT, signal transducer and activator of transcription; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The LT + anti-IL7 group received anti-IL7 antibody (M25; Cat# HY-P990210, MCE, Shanghai, China) (250 μg/kg of body weight, per ip, once a week) for 2 weeks after LT.

Techniques: Staining, Western Blot, Control, Derivative Assay, Transplantation Assay