recombinant human light tnfsf14 protein  (R&D Systems)

Journal: PLOS One

Article Title: Genetic ablation of interleukin-17A augments fibrosis in a mouse model of cholestatic liver injury

doi: 10.1371/journal.pone.0342251

Figure Lengend Snippet: A. The abundance of total IHL isolated from WT and Il-17a -/- mice is quantified (N = 4-6 per group). B. Total number of IHL isolated from WT and Il-17a -/- mice reared on control or DDC diet is normalized to liver weight (N = 4-6 per group). C. The proportion of CD45 + cells in the IHL as determined by flow cytometry (N = 4-6 per group). D. (Left panel) CD4 + T cells abundance in gated CD45 + immune cells, and (Right panel ) the proportion of LIGHT + cells in the CD4 + T cell fraction (N = 3 per group). E. (Left panel) NK T cell abundance in gated CD45 + immune cells, and (Right panel ) proportion of LIGHT + cells in the NK T cell fraction (N = 4-6 per group). F. LIGHT positivity in CD4 + T cell subsets, Th1, Th2, Th17, and T-reg, was determined by flow cytometry (N = 3 per group). G. WT primary mouse splenocytes when stimulated with PMA/ionomycin or vehicle display (left panel) a similar proportion of CD3 + CD4 + T cells, (middle panel) an increased abundance of IFNγ + CD 4 + T cells and (right panel) an increased proportion of LIGHT + CD4 + T cells (N = 3 per group). H. The expression of pro-fibrogenic genes ACTA2, COL1A1, PDGFRA and CTGF is elevated in primary human hepatic stellate cells when stimulated with recombinant LIGHT (N = 3 per group). * - p < 0.05, ** - p < 0.01, *** - p < 0.005, **** - p < 0.001.

Article Snippet: Cells were rested for 4 hours and stimulated with medium containing vehicle or recombinant human LIGHT (664-LI, R&D systems, Minneapolis, MN) for 72 hours refreshing the medium at 48 hours.

Techniques: Isolation, Control, Flow Cytometry, Expressing, Recombinant

Journal: The EMBO Journal

Article Title: TNFSF14 + natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence

doi: 10.1038/s44318-024-00220-3

Figure Lengend Snippet: ( A – C ) C57BL/6 pregnant mice were fed with control (1.2% leucine) ( n = 3 biological replicates) or high leucine (6%) ( n = 4 biological replicates) fodder, the SAβG activity of DSCs were measured by flow cytometry, and expression of CDKN2A, CDKN1A, and TP53 of DSCs were detected by immunofluorescence. ( D ) The embryo resorption rate, the weight of placenta and embryo of pregnant mice were quantified at the gestation of day 13.5 (control fodder: n = 3 biological replicates, high leucine fodder: n = 4 biological replicates). ( E ) Decidualized hESCs were co-cultured with dNK cells for another 48 h, and CDKN2A, CDKN1A, and TP53 expression were detected by western blotting ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference GAPDH. ( F ) Compared the expression of TNFSF14 on dNK cells of NP ( n = 6 biological replicates) and SA ( n = 6 biological replicates) by flow cytometry. ( G ) Compared the expression of TNFRSF14 on DSCs of NP ( n = 6 biological replicates) and SA ( n = 6 biological replicates) by Immunohistochemistry. Scale bar, 100 μm. ( H , I ) Decidualized si TNFRSF14 -hESCs were co-cultured with dNK cells for another 48 h, western blotting indicated CDKN2A, CDKN1A, and TP53 expression ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference GAPDH, and immunofluorescence showed SAβG + cells ( n = 4 biological replicates per group). NP: normal pregnancy; RSA: recurrent spontaneous abortion. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significance, using a one-tailed ( A , B , D ), two-tailed, unpaired Student’s t test ( E , F , H , I ). .

Article Snippet: To investigate the effects of LIGHT, decidualized hESCs or DSCs were treated with vehicle or human recombinant TNFSF14 (250 ng/mL; DQU0119011, R&D Systems, Minneapolis, MN, USA) for 48 h. Decidualized hESCs were co-cultured with dNK cells for 48 h to explore the role of NK cells.

Techniques: Control, Activity Assay, Flow Cytometry, Expressing, Immunofluorescence, Cell Culture, Western Blot, Immunohistochemistry, One-tailed Test, Two Tailed Test

Journal: The EMBO Journal

Article Title: TNFSF14 + natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence

doi: 10.1038/s44318-024-00220-3

Figure Lengend Snippet: ( A , B ) At the gestation of day 13.5, embryo resorption, weight of placenta and embryo in Tnfrsf14 knockout pregnant mice ( Tnfrsf14 −/− ♀×WT♂, n = 5 biological replicates) and WT pregnant mice (WT♀× Tnfrsf14 −/− ♂, n = 5 biological replicates) were recorded. ( C , D ) The SAβG activity of DSCs were measured by flow cytometry, and expression of CDKN2A, CDKN1A and TP53 of DSCs were detected by immunofluorescence ( n = 5 biological replicates per group). ( E ) Schematic diagram of decidualized hESCs treated with recombinant TNFSF14 protein. ( F , G ) After decidualized hESCs were treated with TNFSF14 (250 ng/mL, 48 h), SAβG + cells were detected by immunofluorescence, and CDKN2A, CDKN1A, and TP53 expression were measured by western blotting ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference GAPDH. Scale bar, 100 μm. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significance, using a one-tailed ( B – D ), two-tailed, unpaired Student’s t test ( F , G ). .

Article Snippet: To investigate the effects of LIGHT, decidualized hESCs or DSCs were treated with vehicle or human recombinant TNFSF14 (250 ng/mL; DQU0119011, R&D Systems, Minneapolis, MN, USA) for 48 h. Decidualized hESCs were co-cultured with dNK cells for 48 h to explore the role of NK cells.

Techniques: Knock-Out, Activity Assay, Flow Cytometry, Expressing, Immunofluorescence, Recombinant, Western Blot, One-tailed Test, Two Tailed Test

Journal: The EMBO Journal

Article Title: TNFSF14 + natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence

doi: 10.1038/s44318-024-00220-3

Figure Lengend Snippet: ( A ) Bubble diagram showed the enrichment of pathway for DSCs treated with TNFSF14. ( B ) Heat map showed the relative expression of differential gene for DSCs treated with TNFSF14. ( C , D ) Decidualized hESCs were treated with TNFSF14 (250 ng/mL), or co-cultured with dNK cells (5 × 10 5 ) for 48 h, SLC3A2 expression levels were detected with western blotting ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference Tubulin. ( E , F ) Decidualized si TNFRSF14 hESCs were co-cultured with dNK cells for 48 h, SLC3A2 expression were detected by western blotting ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference Tubulin. ( G ) Decidualized hESCs were treated with TNFSF14 (250 ng/mL), or decidualized si TNFRSF14 hESCs were co-cultured with dNK cells for 48 h, and then the concentration of BCAA was measured ( n = 6 biological replicates per group). ( H ) After decidualized hESCs were treated with TNFSF14 (250 ng/mL, 48 h), the transcriptional levels of SLC3A2 and HSF1 were detected by RT-PCR ( n = 6 biological replicates per group). ( I ) Dual luciferase reporter assays were conducted in hESCs to verify the combination of HSF1 and WT or mutated SLC3A2 promoter region ( n = 4 biological replicates per group). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, using a two-tailed, unpaired Student’s t test. .

Article Snippet: To investigate the effects of LIGHT, decidualized hESCs or DSCs were treated with vehicle or human recombinant TNFSF14 (250 ng/mL; DQU0119011, R&D Systems, Minneapolis, MN, USA) for 48 h. Decidualized hESCs were co-cultured with dNK cells for 48 h to explore the role of NK cells.

Techniques: Expressing, Cell Culture, Western Blot, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Two Tailed Test

Journal: The EMBO Journal

Article Title: TNFSF14 + natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence

doi: 10.1038/s44318-024-00220-3

Figure Lengend Snippet: After C57BL/6 pregnant mice were treated with NK1.1 neutralizing antibody ( n = 5 biological replicates) or isotype control IgG antibody ( n = 5 biological replicates) by intraperitoneal injection, and at the same time, mice were injected with recombinant TNFSF14 protein intraperitoneally ( n = 5 biological replicates). ( A , B ) SAβG activity of DSCs were measured by flow cytometry, and CDKN2A, CDKN1A, and TP53 expression of DSCs were verified by immunofluorescence. ( C , D ) The pregnancy outcome, embryo resorption, weight of placenta, and embryo were counted at the gestation of day 13.5. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.01, using a two-tailed, unpaired Student’s t test. .

Article Snippet: To investigate the effects of LIGHT, decidualized hESCs or DSCs were treated with vehicle or human recombinant TNFSF14 (250 ng/mL; DQU0119011, R&D Systems, Minneapolis, MN, USA) for 48 h. Decidualized hESCs were co-cultured with dNK cells for 48 h to explore the role of NK cells.

Techniques: Control, Injection, Recombinant, Activity Assay, Flow Cytometry, Expressing, Immunofluorescence, Two Tailed Test

Journal: The EMBO Journal

Article Title: TNFSF14 + natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence

doi: 10.1038/s44318-024-00220-3

Figure Lengend Snippet: ( A ) DSCs of spontaneous abortion were treated with metformin (2.5 mM) for 48 h, CDKN2A, CDKN1A, and TP53 expression were detected by western blotting ( n = 3 biological replicates per group), relative expression levels of proteins were standardized using internal reference GAPDH. ( B , C ) After treatment with metformin (200 mg/kg, every other day) in aging pregnant female mice, number of blastocyst implantation, embryo absorption, weight of placenta and embryo were counted at the gestation of day 13.5 (D-galactose group: n = 3 biological replicates, metformin group: n = 3 biological replicates). ( D , E ) Tnfrsf14 -/- pregnant mice were treated with metformin by gavage ( n = 3, biological replicates per group, 200 mg/kg, every other day), embryo resorption, weight of placenta and embryo were calculated at the gestation of day 13.5. ( F , G ) SAβG activity of DSCs were measured by flow cytometry, and CDKN2A, CDKN1A, and TP53 expression of DSCs were verified by immunofluorescence ( n = 3 biological replicates per group). Scale bar, 100 μm. ( H ) Schematic roles of decidual NK cells in preventing spontaneous abortion by maintaining homeostasis of DSC senescence during early pregnancy. During decidualization, increased BCAA transport mediated by SLC3A2 induces senescent DSCs, whereas TNFSF14 + dNK cells control DSC senescence by the TNFSF14/TNFRSF14-SLC3A2/leucine regulatory axis. Excessive senescence of DSC induced by the imbalance of TNFSF14/TNFRSF14-SLC3A2/leucine regulatory axis increases the risk of adverse pregnancy outcomes. The image was drew by the Figdraw. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 using a one-tailed ( C , E – G ), two-tailed, unpaired Student’s t test ( A ). .

Article Snippet: To investigate the effects of LIGHT, decidualized hESCs or DSCs were treated with vehicle or human recombinant TNFSF14 (250 ng/mL; DQU0119011, R&D Systems, Minneapolis, MN, USA) for 48 h. Decidualized hESCs were co-cultured with dNK cells for 48 h to explore the role of NK cells.

Techniques: Expressing, Western Blot, Activity Assay, Flow Cytometry, Immunofluorescence, Control, One-tailed Test, Two Tailed Test