cd4 t lymphocytes  (Bio-Rad)

Journal: Research

Article Title: NUFIP1-Mediated Ribophagy Alleviates PANoptosis of CD4 + T Lymphocytes in Sepsis via the cGAS-STING Pathway

doi: 10.34133/research.0895

Figure Lengend Snippet: Sepsis induces activation of ribophagy in CD4 + T lymphocytes. (A) The transfection efficiency of lentivirus in different groups was measured by flow cytometry. n = 3 technical repetitions. (B) The expression of NUFIP1 protein in different groups of Jurkat T cells was detected by WB. n = 3 technical repetitions. (C) The expression and colocalization of NUFIP1, LAMP2, and LC3B in Jurkat T cells stimulated with LPS were detected by LSCM. The scale bar represents 25 μm. (D) The expression and colocalization of NUFIP1, LAMP2, and LC3B in splenic CD4 + T cells stimulated with LPS were detected by LSCM. The scale bar represents 25 μm. (E) The expression and colocalization of NUFIP1, LAMP2, and LC3B in splenic CD4 + T cells by LSCM after CLP operation. The scale bar represents 25 μm. (F) The expression of ribophagy-related proteins in Jurkat T cells stimulated with LPS was detected by WB. (G) The expression of ribophagy-related proteins in splenic CD4 + T cells stimulated with LPS was detected by WB. (H) The expression of ribophagy-related proteins in splenic CD4 + T cells was detected by WB after CLP operation. (I) The morphological changes of CD4 + T lymphocyte organelles and ribophagy in sepsis were determined by TEM. The red stars indicate the ribosomes, and the blue arrows represent autophagosomes. The scale bars represent 500 nm (top) and 100 nm (bottom). Data were expressed as means ± SEM. An unpaired 2-sided Student’s t test was applied to test the statistical significance. * P < 0.05, *** P < 0.001. ### P < 0.001 compared with the empty-LPS group.

Article Snippet: CD4 + T lymphocyte-specific NUFIP1 cKO mice ( Cd4 cre Nufip1 fl/fl ) and their corresponding control mice ( Nufip1 fl/fl ) were generated and raised to 6 to 8 weeks of age by Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Activation Assay, Transfection, Flow Cytometry, Expressing

Journal: Research

Article Title: NUFIP1-Mediated Ribophagy Alleviates PANoptosis of CD4 + T Lymphocytes in Sepsis via the cGAS-STING Pathway

doi: 10.34133/research.0895

Figure Lengend Snippet: Effect of NUFIP1-mediated ribophagy on ZBP1-PANoptosis in sepsis. (A) The expression and colocalization of ASC/caspase-8/RIPK3 in Jurkat-empty cells stimulated with LPS were determined by LSCM. The yellow arrows represent PANoptosomes, and the scale bar represents 25 μm. (B) The expression and colocalization of ASC/caspase-8/RIPK3 in Jurkat-KD cells stimulated with LPS were determined by LSCM. The yellow arrows represent PANoptosomes, and the scale bar represents 25 μm. (C) The expression of PANoptosis-related proteins in Jurkat T cells after transfection with lentivirus was detected by WB. (D) The morphological characteristics of PANoptosis in Jurkat T cells after transfection with lentivirus were observed by TEM. The red arrow denotes the intracellular vesicles associated with pyroptosis; the green arrow signifies the nuclear condensation and chromatin shrinkage characteristic of cell apoptosis; the blue arrow indicates the loss of cell membrane integrity in necroptosis; and the scale bar represents 500 nm. (E and F) Expression of 3 key proteins mediating PANoptosome formation in Jurkat T cells of empty and KD groups under LPS stimulation by WB. n = 3 technical repetitions. (G and H) Expression of key proteins mediating PANoptosome formation in splenic CD4 + T cells of the Flox and cKO groups under LPS stimulation by WB. n = 3 technical repetitions. (I and J) The expression of key proteins mediating PANoptosome formation in splenic CD4 + T cells of the Flox and cKO groups was examined by WB after CLP operation. n = 3 technical repetitions. Data were expressed as means ± SEM. A 2-way ANOVA test was applied to test the statistical significance. *** P < 0.001 compared with the empty/Flox group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the KD-PBS/cKO-PBS/cKO-sham group.

Article Snippet: CD4 + T lymphocyte-specific NUFIP1 cKO mice ( Cd4 cre Nufip1 fl/fl ) and their corresponding control mice ( Nufip1 fl/fl ) were generated and raised to 6 to 8 weeks of age by Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Expressing, Transfection, Membrane

Journal: Research

Article Title: NUFIP1-Mediated Ribophagy Alleviates PANoptosis of CD4 + T Lymphocytes in Sepsis via the cGAS-STING Pathway

doi: 10.34133/research.0895

Figure Lengend Snippet: Impact of conditional deletion of NUFIP1 on PANoptosis of splenic CD4 + T lymphocytes in septic mice. (A and B) The necrosis of splenic CD4 + T lymphocytes in mice stimulated with LPS was detected by SYTOX-Green. The scale bar represents 100 μm. n = 3 technical repetitions. (C) The expression and colocalization of ASC/caspase-8/RIPK3 in splenic CD4 + T lymphocytes of Flox mice stimulated with LPS were detected by LSCM. The yellow arrows represent PANoptosomes, and the scale bar represents 25 μm. (D) The expression and colocalization of ASC/caspase-8/RIPK3 in splenic CD4 + T lymphocytes of cKO mice stimulated with LPS were detected by LSCM. The yellow arrows represent PANoptosomes, and the scale bar represents 25 μm. (E) Expression of PANoptosis-related proteins in splenic CD4 + T lymphocytes of cKO mice stimulated with LPS under WB. (F) Morphological characteristics of PANoptosis in splenic CD4 + T lymphocytes of Flox and cKO mice stimulated with LPS under TEM. The red arrow denotes the intracellular vesicles associated with pyroptosis; the green arrow signifies the nuclear condensation and chromatin shrinkage characteristic of cell apoptosis; the blue arrow indicates the loss of cell membrane integrity in necroptosis; and the scale bar represents 500 nm. (G) Cytokine levels in the culture supernatant of splenic CD4 + T cells were measured by ELISA in Flox and cKO groups. n = 6 technical repetitions. Data were expressed as means ± SEM. A 2-way ANOVA test was applied to test the statistical significance. * P < 0.05, *** P < 0.001 compared with the Flox group. ## P < 0.01, ### P < 0.001 compared with the cKO-PBS group.

Article Snippet: CD4 + T lymphocyte-specific NUFIP1 cKO mice ( Cd4 cre Nufip1 fl/fl ) and their corresponding control mice ( Nufip1 fl/fl ) were generated and raised to 6 to 8 weeks of age by Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Expressing, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: NUFIP1-Mediated Ribophagy Alleviates PANoptosis of CD4 + T Lymphocytes in Sepsis via the cGAS-STING Pathway

doi: 10.34133/research.0895

Figure Lengend Snippet: Impact of conditional deletion of NUFIP1 on immune response of CD4 + T lymphocytes, organ injury, and the 1-week survival rate of mice in sepsis. (A) The proliferative activity of splenic CD4 + T cells was measured by CCK-8 after CLP operation. n = 3 technical repetitions. (B) Serum cytokine levels in the Flox and cKO groups were measured by ELISA after CLP operation. n = 4 technical repetitions. (C and E) Proportion of CD3 + T lymphocytes in the peripheral blood of mice in different groups detected by flow cytometry. n = 3 technical repetitions. (D and F) The proportion of CD3 + CD4 + T lymphocytes in the peripheral blood of mice in different groups was detected by flow cytometry. n = 3 technical repetitions. (G and H) The ratio of Th1/Th2 of splenic CD4 + T cells was detected by flow cytometry. n = 3 technical repetitions. (I and J) Percentage of Tregs of splenic CD4 + T cells detected by flow cytometry. n = 3 technical repetitions. (K and L) Percentage of Th17 cells of splenic CD4 + T cells detected by flow cytometry. n = 3 technical repetitions. (M to Q) H&E staining assessment of various organ lesions, including heart (M), lung (N), liver (O), and kidney (P) in different groups of mice. The scale bar represents 50 μm. n = 3 biological independent samples. (R) One-week survival curves of different groups of mice (** P < 0.01 compared with the sham group; # P < 0.05 compared with the Flox-CLP group). n = 10 biological independent samples. Data were expressed as means ± SEM. A 2-way ANOVA test was applied to test the statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the Flox group. ## P < 0.01, ### P < 0.001 compared with the cKO-sham group.

Article Snippet: CD4 + T lymphocyte-specific NUFIP1 cKO mice ( Cd4 cre Nufip1 fl/fl ) and their corresponding control mice ( Nufip1 fl/fl ) were generated and raised to 6 to 8 weeks of age by Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Activity Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: Research

Article Title: NUFIP1-Mediated Ribophagy Alleviates PANoptosis of CD4 + T Lymphocytes in Sepsis via the cGAS-STING Pathway

doi: 10.34133/research.0895

Figure Lengend Snippet: NUFIP1 deficiency disrupts ribophagy and ribosomal collision to trigger cGAS-STING-dependent PANoptosis in septic CD4 + T lymphocytes. (A) Differentially expressed protein GO pathway enrichment maps of Jurkat T cells in the normal control and KD groups stimulated with LPS. (B) Differentially expressed protein KEGG pathway enrichment map of Jurkat T cells in the normal control and KD groups stimulated with LPS. (C and D) Expression of cGAS-STING signaling-related proteins in different groups of Jurkat T cells under LPS stimulation by WB. n = 3 technical repetitions. (E and F) Expression of cGAS-STING signaling-related proteins in splenic CD4 + T cells of the Flox and cKO groups under LPS stimulation by WB. n = 3 technical repetitions. (G) Ribosomal collision events of Jurkat T cells assessed by polysome profiling under PBS or LPS. (H) Ribosomal collision events of CD4 + T cells assessed by polysome profiling under sham or CLP. (I) The effects of NUFIP1 gene interference on ribosomal collisions within Jurkat T cells were assessed by polysome profiling. (J) The interaction between NUFIP1 and STING in Jurkat T cells was detected by Co-IP. Data were expressed as means ± SEM. A 2-way ANOVA test was applied to test the statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the empty-PBS/Flox-PBS group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the empty-LPS/Flox-LPS group.

Article Snippet: CD4 + T lymphocyte-specific NUFIP1 cKO mice ( Cd4 cre Nufip1 fl/fl ) and their corresponding control mice ( Nufip1 fl/fl ) were generated and raised to 6 to 8 weeks of age by Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Control, Expressing, Co-Immunoprecipitation Assay