ifn β detection  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: IFN‐β was engaged in ISG56 and CXCL10 induced by poly IC in hRPTECs. Control siRNA or two different siRNAs against IFN‐β were used to transfect cultured cells, and the cells were incubated for 24 h. Poly IC (10 μg·mL −1 ) was used to stimulate the cells. (A) After 2 h (for IFN‐β analysis) and 16 h (for other analysis) incubation, RNA extraction from the cells was performed, and IFN‐β, ISG56, CXCL10 and GAPDH expression was assayed by RT‐qPCR. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. (B) After 4 h (for IFN‐β analysis) and 24 h (for CXCL10 analysis) incubation, ELISA kits were used to measure the concentration of IFN‐β and CXCL10 in the cell culture medium. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. (C) After 24 h of incubation, the cells were lysed and the lysate was used for western blotting of ISG56 and actin ( n = 1).

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques: Control, Cell Culture, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR, Transfection, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: STAT1 was scarcely involved in ISG56 and CXCL10 induced by poly IC in hRPTECs. Fludarabine, an inhibitor of STAT1 activation, was added to cultured cells at a concentration of 10 μ m , and the cells were incubated for 1 h. Poly IC (10 μg·mL −1 ) was used to stimulate the cells. (A) After 2 h (for phosphorylated STAT1 and STAT1 analysis) and 24 h (for ISG56 and actin analysis), the cells were lysed and the lysate was used for western blotting ( n = 1). (B) After 16 h of incubation, RNA extraction from the cells was performed, and ISG56, CXCL10 and GAPDH mRNA expression was evaluated by RT‐qPCR. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. (C) After 24 h of incubation, the cell culture medium was collected, and an ELISA kit was used to measure the concentration of CXCL10. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. n.s., not significant.

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques: Activation Assay, Cell Culture, Concentration Assay, Incubation, Western Blot, RNA Extraction, Expressing, Quantitative RT-PCR, Transfection, Control, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: ISG56 was engaged in CXCL10 induced by poly IC in hRPTECs. Control siRNA or two different siRNAs against ISG56 were used to transfect cultured cells, and the cells were incubated for 24 h. Poly IC (10 μg·mL −1 ) was used to stimulate the cells. (A) After 16 h of incubation, RNA extraction from the cells was performed, and ISG56, CXCL10, CXCL1 and GAPDH expression was evaluated by RT‐qPCR. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. n.s., not significant. (B) After 24 h (for ISG56 analysis) of incubation, the cells were lysed and the lysate was used for western blotting of ISG56 and actin ( n = 1). (C) After 24 h of incubation, an ELISA kit was used to measure the concentration of CXCL10 in the cell culture medium. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test.

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques: Control, Cell Culture, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR, Transfection, MANN-WHITNEY, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: ISG56 was engaged in CXCL10 induced by IFN‐β in hRPTECs. Control siRNA or two different siRNAs against ISG56 were used to transfect cultured cells, and the cells were incubated for 24 h. r(h)IFN‐β (0.1 ng·mL −1 ) was used to stimulate the cells. (A) After 16 h of incubation, RNA extraction from the cells was performed, and ISG56, CXCL10 and GAPDH expression was evaluated by RT‐qPCR. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. (B) After 24 h (for ISG56 analysis) of incubation, the cells were lysed and the lysate was used for western blotting of ISG56 and actin ( n = 1). (C) After 24 h of incubation, an ELISA kit was used to measure the concentration of CXCL10 in the cell culture medium. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test.

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques: Control, Cell Culture, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR, Transfection, MANN-WHITNEY, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: ISG56 was engaged in CXCL10 induced by JEV infection in hRPTECs. Cultured cells were transfected with control siRNA or two siRNAs against ISG56 and incubated for 24 h. JEV was added to each well at MOI = 1.0. (A) After 24 and 48 h of incubation, the medium was collected, and viral titers [focus forming unit (FFU)] were measured. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. n.s., not significant. (B) After 24 h of incubation, RNA extraction from the cells was performed, and ISG56, CXCL10 and GAPDH mRNA expression was evaluated by RT‐qPCR. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test. (C) After 24 h of incubation, the cells were lysed and the lysate was used for western blotting of ISG56 and actin ( n = 1). (D) After 24 h of incubation, the cell culture medium was harvested, and the concentration of CXCL10 was measured using an ELISA kit. The results are shown as the mean ± SD ( n = 3). * P < 0.05 relative to cells transfected with control siRNA by a Mann–Whitney U ‐test.

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques: Infection, Cell Culture, Transfection, Control, Incubation, MANN-WHITNEY, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: FEBS Open Bio

Article Title: Interferon‐stimulated gene 56 positively regulates Toll‐like receptor 3‐mediated CXCL 10 expression in human renal proximal tubular epithelial cells

doi: 10.1002/2211-5463.13851

Figure Lengend Snippet: Schematic model of ISG56 function for TLR3/IFN‐β/CXCL10 in hRPTECs.

Article Snippet: ELISA kits for detecting CXCL10 and IFN‐β were obtained from R&D Systems (Minneapolis, MN, USA) and PBL Assay Science (Piscataway, NJ, USA), respectively, and the manufacturers' recommended protocols were followed.

Techniques:

Journal: Advanced Science

Article Title: A DNA‐Modularized STING Agonist with Macrophage‐Selectivity and Programmability for Enhanced Anti‐Tumor Immunotherapy

doi: 10.1002/advs.202400149

Figure Lengend Snippet: Programmable cGAS‐STING activation process of DNDA. A) Endosomal escape of Cy5‐labeled DNDA with or without pHLIP modification in RAW264.7 cells imaged by LSCM. (Scale bar: 30 µm) B) Fluorescent native PAGE analysis of dual fluorescent‐labeled DNDA treated with indicated concentration of ATP at 37 °C for 2 h. C) Quantitative grayscale analysis of G3 dissociation rate over ATP concentration. D) The binding between G3‐biotin, DNDA‐biotin, and cGAS using co‐IP analysis after co‐incubation with RAW264.7 lysate for 6 h. E) IFN regulatory factor (IRF) response elicited by DNDA with or without pHLIP in RAW ISG cells with an IRF‐inducible luciferase reporter. Data are shown as Mean ± SD (n = 3), and statistical significance was calculated via one‐way ANOVA with Tukey's post hoc test, *** p < 0.001. F) Immunoblotting assay of phosphate STING and TBK1 level in RAW264.7 cells after incubation with DNDA with or without pHLIP. G) IFN‐β and (H) CXCL10 expression level in RAW264.7 macrophages incubated with PBS and DNDA with or without pHLIP, as measured by ELISA assay. (N/D, not detected) Data are shown as Mean ± SD (n = 3), and statistical significance was calculated via one‐way ANOVA with Tukey's post hoc test, *** p < 0.001. I) CXCL10 expression level in DNDA‐treated BMDM cells from WT and STING mut mice. (N/D, not detected) Data are shown as Mean ± SD (n = 3), and statistical significance was calculated via two‐way ANOVA with Sidak's post hoc test, *** p < 0.001.

Article Snippet: ELISA kits for IFN‐β and CXCL10 detection were purchased from Novus Biologicals (USA).

Techniques: Activation Assay, Labeling, Modification, Clear Native PAGE, Concentration Assay, Binding Assay, Co-Immunoprecipitation Assay, Incubation, Luciferase, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: A DNA‐Modularized STING Agonist with Macrophage‐Selectivity and Programmability for Enhanced Anti‐Tumor Immunotherapy

doi: 10.1002/advs.202400149

Figure Lengend Snippet: cGAS‐STING activation by DNDA in tumor‐bearing mice. A) Treatment schedule of DNDA in MC38 tumor‐bearing C57BL/6 WT and STING mut mice. B) Tumor growth curve of MC38 tumors with indicated treatment in C57BL/6 WT mice and STING mut mice. Data are shown as Mean ± SD (n = 5), statistical significance was calculated via two‐way ANOVA with Tukey's post hoc test, *** p < 0.001, ns means no significance. C) Immunoblotting assay of STING and TBK‐1 phosphorylation level and D) Real‐Time PCR analysis of mRNA level of CXCL10, IFN‐β, and Isg‐15 in tumor tissues from WT or STING mut mice with indicated treatment. Data are shown as Mean ± SD (n = 5), statistical significance was calculated via two‐way ANOVA with Sidak's post hoc test, * p < 0.05, ** p < 0.01, ns means no significance. Quantitative analysis of E) tumor‐infiltrated CD8 + T cells (gated on L/D − CD3 + CD8 + cells) and F) the ratio between tumor‐infiltrated CD8 + T cells (gated on L/D − CD3 + CD8 + cells) and regulatory T cells (gated on L/D − CD3 + CD4 + Foxp3 + cells). Data are shown as Mean ± SD (n = 5), statistical significance was calculated via two‐way ANOVA with Sidak's post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns means no significance. Representative flow cytometric plots of G) IFNγ + CD8 + T cells and I) GranB + CD8 + T cells in tumors on day 21 post‐tumor inoculation. The frequency of H) IFNγ + CD8 + T cells (gated on L/D − CD45 + CD3 + CD8 + IFNγ + cells) and J) GranB + CD8 + T cells (gated on L/D − CD45 + CD3 + CD8 + Granzyme B + cells) in tumors analyzed on day 21 post tumor inoculation. Data are shown as the Mean ± SD (n = 5), statistical significance was calculated via two‐way ANOVA with Sidak's post hoc test, ** p < 0.01, ns means no significance.

Article Snippet: ELISA kits for IFN‐β and CXCL10 detection were purchased from Novus Biologicals (USA).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: A DNA‐Modularized STING Agonist with Macrophage‐Selectivity and Programmability for Enhanced Anti‐Tumor Immunotherapy

doi: 10.1002/advs.202400149

Figure Lengend Snippet: Therapeutic effects of DNDA on inhibiting tumor growth combined with PDL1 blockade in MC38 tumor‐bearing mice. A) Treatment schedule of DNDA and anti‐PDL1 combination therapy on MC38 bearing C57BL/6 mice. B) Individual tumor growth curve and C) overall survival rate of MC38 bearing C57BL/6 mice with indicated treatment (n = 5). Statistical significance was calculated via the log‐rank Mantel‐Cox test, *** p < 0.001. D) Quantitative analysis of tumor‐infiltrated CD8 + T cells (gated on L/D − CD3 + CD8 + cells), IFNγ + CD8 + T cells (gated on L/D − CD45 + CD3 + CD8 + IFNγ + cells) and GranB + CD8 + T cells (gated on L/D − CD45 + CD3 + CD8 + Granzyme B + cells). Data are shown as Mean ± SD (n = 5), and statistical significance was calculated via one‐way ANOVA with Tukey's post hoc test, * p < 0. 05, ** p < 0.01, *** p < 0.001. E) ELISA analysis of CXCL10, IFN‐β, and TNF‐α excretion in tumor tissues. Data are shown as Mean ± SD (n = 5), statistical significance was calculated via one‐way ANOVA with Tukey's post hoc test, * p < 0. 05, ** p < 0.01, *** p < 0.001. (F) TUNEL, Ki67, and H&E staining of tumor tissue sections from mice with indicated treatment.

Article Snippet: ELISA kits for IFN‐β and CXCL10 detection were purchased from Novus Biologicals (USA).

Techniques: Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining