Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Characterization of the senescent CD4 + T lymphocytes. ( a ) Flow cytometry gating strategy used to determine the purity of viable CD4 + T lymphocytes isolated from the spleen of wild-type C57 BL/6 mice. The following sequential gating strategy steps were used: First, cells were physically selected according to their cell size (FSC-A) and internal cell complexity (SSC-A) parameters. Second, only single cells were selected according to their FSC-H and FSC-A characteristics, discarding fused cells as duplets or triplets. Third, dead cells were excluded. Finally, the viable CD4 + T cells were selected for analysis. This cell characterization was performed separately for each animal. (b) Flow cytometry histogram and bar plot show the cell size quantification of viable CD4 + T lymphocytes exposed or non-exposed to oxidative stress (H 2 O 2 ). (c) Flow cytometry histogram and bar plot show the frequency of proliferating CD4 + lymphocytes. H 2 O 2 -exposed CD4 + T cells stimulated with CD3ε-CD28 (red) were compared with non-exposed CD4 + T cells stimulated with CD3ε-CD28 (gray). Non-exposed CD4 + T cells without CD3ε-CD28 stimulation were used as negative control (yellow). The dotted line indicates the reference point used to quantify the proliferating cells. (d) Relative quantification of the mRNA expression of the cell cycle inhibitors p16 Ink4a and p21 Cip1 in H 2 O 2 -exposed or non-exposed CD4 + T cells determined by RT-qPCR. (e) Western blot immunodetection of phospho-p38, total p38, and β-actin and quantification of the phospho-p38/total p38 band density in senescent and non-senescent CD4 + T lymphocytes. (f) Quantification of total p38/β-actin band density in senescent and non-senescent CD4 + T lymphocytes. Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To neutralize the p38 MAPK activity, the H 2 O 2 -exposed CD4 + T lymphocytes were cultured for 4 days in the presence of the p38 MAPK-specific inhibitor BIRB-796 500 nM (MedChemExpress), with DMSO used as the vehicle control.

Techniques: Flow Cytometry, Isolation, Negative Control, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Immunodetection

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: p38 MAPK in dysfunctional mitochondria accumulation and autophagy disruption in senescent CD4 + T lymphocytes. ( a ) Flow cytometry histogram and bar plot showing the MitoTracker dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. (b) Relative quantification of the mRNA expression of the fusion/fission markers MFN2 and DRP1 in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO determined by RT-qPCR. (c) Flow cytometry histogram and bar plot showing the MitoTracker dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796, BIRB-796 plus Bafilomycin (autophagy inhibitor), Rapamycin (autophagy inducer), or DMSO. The histogram blue peak corresponds to senescent CD4 + T lymphocytes exposed to BIRB-796, the pink peak to senescent CD4 + T lymphocytes exposed to BIRB-796 plus Bafilomycin, the grey peak to senescent CD4 + T lymphocytes exposed to Rapamycin, the red peak to senescent CD4 + T lymphocytes exposed to DMSO, and the yellow peak to the negative control (MitoTracker dye MFO). (d) The same analysis described in (c) using the frequency of MitoTracker high senescent CD4 + T lymphocytes. (e) Flow cytometry histogram and bar plot showing the frequency of MitoSox-positive cells and MitoSox dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. (f) Flow cytometry histogram and bar plot showing the H2DCFDA dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. In all flow cytometry histograms, the blue peak corresponds to senescent CD4 + T lymphocytes exposed to BIRB-796, the red peak to senescent CD4 + T lymphocytes exposed to DMSO, the grey peak to non-senescent CD4 + T lymphocytes, and the yellow peak to the negative controls (MitoTracker, MitoSox, and H2DCFDA dye MFO, respectively). Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test, when comparing two groups, or the one-way ANOVA and Tukey post hoc tests, when comparing more than two experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Baf, bafilomycin; DMSO, dimethyl sulfoxide; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate reagents; MFI, mean fluorescence intensity, MFO: fluorescence minus one; Rap, rapamycin

Article Snippet: To neutralize the p38 MAPK activity, the H 2 O 2 -exposed CD4 + T lymphocytes were cultured for 4 days in the presence of the p38 MAPK-specific inhibitor BIRB-796 500 nM (MedChemExpress), with DMSO used as the vehicle control.

Techniques: Disruption, Flow Cytometry, Comparison, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Negative Control, Fluorescence

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Role of p38 MAPK in the Th17-type SASP release in senescent CD4 + T lymphocytes. ( a ) Quantification of the secreted levels of IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-23, and GM-CSF in senescent and non-senescent CD4 + T lymphocytes determined by ELISA-Multiplex. (b) Relative quantification of the mRNA expression of RORγt, Tbx 21, STAT-4, and CCL4 in senescent and non-senescent CD4 + T lymphocytes determined by RT-qPCR. (c) Quantification of the secreted levels of IL-6, IL-17A, and GM-CSF in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. (d) Relative quantification of the mRNA expression of RORγt and Tbx 21 in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. (e) Flow cytometry and bar plots showing the number and frequency of senescent CD4 + T lymphocytes exposed to BIRB-796, exposed to DMSO, and non-senescent CD4 + T lymphocytes expressing IL-17A (CD4 + IL-17A + T cells). Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. CCL, chemokine ligand; DMSO, dimethyl sulfoxide; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; ROR, retinoid-related orphan receptor; SASP, senescence-associated secretory phenotype; STAT, signal transducer and activator of transcription; Tbx, T-box transcription factor

Article Snippet: To neutralize the p38 MAPK activity, the H 2 O 2 -exposed CD4 + T lymphocytes were cultured for 4 days in the presence of the p38 MAPK-specific inhibitor BIRB-796 500 nM (MedChemExpress), with DMSO used as the vehicle control.

Techniques: Enzyme-linked Immunosorbent Assay, Multiplex Assay, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Graphic summary that contextualizes the main findings of the present study. Activation of p38 MAPK drives cellular senescence in CD4 + T lymphocytes. Particularly, p38 MAPK activation causes mitophagy disruption, accumulation of dysfunctional mitochondria, and production of an SASP enriched in Th17-type mediators. These cell-senescent phenomena were inhibited when BIRB-796 was used to neutralize the p38 MAPK activation

Article Snippet: To neutralize the p38 MAPK activity, the H 2 O 2 -exposed CD4 + T lymphocytes were cultured for 4 days in the presence of the p38 MAPK-specific inhibitor BIRB-796 500 nM (MedChemExpress), with DMSO used as the vehicle control.

Techniques: Activation Assay, Disruption

Journal: iScience

Article Title: Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase.

doi: 10.1016/j.isci.2022.105730

Figure Lengend Snippet: Figure 5. Sequence analysis of p38 MAPK and the levels of p38 mapk transcripts at different developmental stages of I. scapularis (A) Pie-chart represents percent identify of I. scapularis p38 MAPK amino acid sequence with ortholog proteins from Homo sapiens (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), Culex quinquefasciatus (Cq) and Aedes aegypti (Aa). (B) Number of predicted post-translational sites in p38 MAPK protein were analyzed at PROSITE. (C) Schematic representation of I. scapularis p38 MAPK protein structure is shown. Domain analysis of I. scapularis p38 MAPK primary amino acid sequence showing presence of serine/threonine protein kinases catalytic domain (21–305 aa). (D) Sequence analysis of I. scapularis p38 MAPK amino acid sequence with other orthologs is shown. The phylogenetic tree was generated in DNASTAR using the Neighbor-Joining (BIONJ) method with BIONJ algorithm. (E) qPCR analysis showing expression of p38 MAPK at different tick developmental stages in uninfected unfed ticks. Each data point in nymphs and adult samples represents transcript levels noted in an individual tick. For larval samples, each data point represents transcript levels noted in 5 pooled larvae. Horizontal lines in the graphs represents mean of the data points. The mRNA levels are normalized to tick beta-actin mRNA levels. Statistical significance was calculated using ANOVA analysis.

Article Snippet: Tick cell line experiments with BIRB796, the p38 MAPK inhibitor Stock (10mM) of BIRB796 (Tocris, USA) was made in dimethyl sulfoxide (DMSO) solution.

Techniques: Sequencing, Generated, Expressing

Journal: iScience

Article Title: Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase.

doi: 10.1016/j.isci.2022.105730

Figure Lengend Snippet: Figure 6. Anaplasma phagocytophilum upregulates p38 MAPK levels in unfed nymphal ticks and tick cells (A and B) Quantitative PCR analysis showing expression of p38 mapk in unfed uninfected or A. phagocytophilum-infected ticks (A) or tick cells (B). Open circles represent data from uninfected (UI) and closed circles represent data from infected (I) ticks or tick cells. Each circle represents data from one tick (A) or samples collected from one culture plate well in a plate (B). Horizontal lines in the graphs represents mean of the data points. The mRNA levels of these genes were normalized to tick beta-actin mRNA levels. p value from Student’s t test is shown. (C) Immunoblotting analysis showing phosphorylated and total p38 MAPK levels in uninfected (UI) or A. phagocytophilum-infected (I) unfed nymphal ticks. (D) Immunoblotting analysis showing phosphorylated p38 MAPK levels in uninfected (UI) or A. phagocytophilum-infected (I) tick cells. Coomassie blue stained gel image for total protein profile serves as loading control in the immunoblotting analysis. M indicates Precision Plus protein all blue marker from BioRad, USA.

Article Snippet: Tick cell line experiments with BIRB796, the p38 MAPK inhibitor Stock (10mM) of BIRB796 (Tocris, USA) was made in dimethyl sulfoxide (DMSO) solution.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot, Staining, Control, Marker

Journal: Cell & Bioscience

Article Title: FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

doi: 10.1186/s13578-022-00816-4

Figure Lengend Snippet: FANCC deficiency induces microglial pyroptosis by regulating p38-MAPK and inflammasome NLRP3. A Western blotting of FANCC protein levels in the spinal cord at 7 days post-injury; n = 3. B Bar graph showing a quantitative analysis of FANCC expression; n = 3. The error bars represent the SD. *p < 0.05 vs. Sham group by t-test (*p < 0.05, **p < 0.01, and ***p < 0.001). C Western blotting of FANCC in LPS-stimulated primary microglia for 24 h; n = 3. β-actin was used as the control. D Bar graph showing the densitometry analysis of FANCC expression. The error bars represent the SD. *p < 0.05 vs. PBS group by t-test (*p < 0.05, **p < 0.01, and ***p < 0.001). E Representative immunofluorescence labeling images for IBA-1 (green) and FANCC (red) in LPS-activated microglia after transfection with OE-FANCC and KD-FANCC; Scale bar = 50 μm. F Western blotting for p-p38, p38, NLRP3, ASC, GSDMD and GSDMD-N in LPS-activated microglia after transfection with OE-FANCC and KD-FANCC; n = 3. G Bar graph showing the ratio analysis of p-p38/p38. H Densitometric analysis of NLRP3 expression. I Densitometric analysis of ASC expression. J Densitometric analysis of GSDMD expression. K Densitometric analysis of GSDMD-N expression. L , M ELISAs performed for the IL-1β and IL-18 in culture medium obtained at 24 h in LPS-activated microglia after transfection with OE-FANCC and KD-FANCC (n = 5). N Representative flow cytometry performed for the distinction of iNOS + &F4/80 + microglia transfected OE-FANCC and KD-FANCC post 24 h LPS inducement. O Bar graph showing the quantitative analysis of the percent; n = 4. The error bars represent the SD. *p < 0.05 vs. NC-PBS group, #p < 0.05 vs. NC-LPS group by one-way ANOVA followed by Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001). NC-PBS microglia were treated with PBS for control, NC-LPS microglia were treated with LPS for 24 h, OE-LPS microglia were transfected with OE-FANCC before LPS stimulation for 24 h, KD-LPS microglia were transfected with KD-FANCC before LPS stimulation for 24 h

Article Snippet: Microglia were pretreated with the p38 MAPK inhibitor BIRB 796 (500 nM, Axon Medchem, Groningen, The Netherlands) for 6 h or the NLRP3 inhibitor glyburide (50 mM, Sigma-Aldrich) for 2 h, after which LPS (1 μg/mL, Sigma-Aldrich) was administered to activate microglial inflammation for 24 h.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Labeling, Transfection, Flow Cytometry

Journal: Cell & Bioscience

Article Title: FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

doi: 10.1186/s13578-022-00816-4

Figure Lengend Snippet: FANCC downregulation of inflammation in microglia is p38/NLRP3 dependent. A Western blotting performed for the proteins including p-p38, p38, NLRP3, pro-Caspase-1, cleaved-Caspase-1, pro-IL-1β and cleaved-IL-1β in LPS-activated microglia pretreated with BIRB 796 and glyburide after transfection with OE-FANCC; n = 3. β-actin was used as the control. B Bar graph showing the ratio analysis of p-p38/p38. C Densitometric analysis of NLRP3 expression. D Densitometric analysis of pro-Caspase-1expression. E Densitometric analysis of cleaved -Caspase-1expression. F Densitometric analysis of pro-IL-1β expression. G Densitometric analysis of cleaved -IL-1β expression. H , I ELISAs performed for the IL-1β and IL-18 in culture medium obtained at 24 h in LPS-activated microglia pretreated with BIRB 796 and glyburide after transfection of OE-FANCC (n = 5). J Representative flow cytometry performed for the distinction of iNOS + &F4/80 + microglia pretreated with BIRB 796 or glyburide and transfected OE-FANCC post 24 h LPS inducement. K Bar graph showing the quantitative analysis of the percent; n = 4. The error bars represent the SD. *p < 0.05 vs. NC-PBS group, #p < 0.05 vs. NC-LPS group, &p vs. OE-LPS group by one-way ANOVA followed by Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001)

Article Snippet: Microglia were pretreated with the p38 MAPK inhibitor BIRB 796 (500 nM, Axon Medchem, Groningen, The Netherlands) for 6 h or the NLRP3 inhibitor glyburide (50 mM, Sigma-Aldrich) for 2 h, after which LPS (1 μg/mL, Sigma-Aldrich) was administered to activate microglial inflammation for 24 h.

Techniques: Western Blot, Transfection, Control, Expressing, Flow Cytometry

Journal: Cell & Bioscience

Article Title: FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

doi: 10.1186/s13578-022-00816-4

Figure Lengend Snippet: Silencing FANCC exacerbates SCI-induced pyroptosis and NLRP3-depended neuroinflammation. A Western blotting of FANCC, p-p38, p38, ASC, GSDMD and GSDMD-N protein levels at 3 dpi in NC-sham, NC-SCI and KD-SCI mice. B Bar graph showing a quantitative analysis of FANCC expression; n = 3. C Bar graph showing the ratio of p-p38/p38; n = 3. D Densitometric analysis of ASC expression. E Densitometric analysis of GSDMD expression. F Densitometric analysis of GSDMD-N expression. G Double immunofluorescence of Caspase-1 (green) and NLRP3 (red), obtained from longitudinal sections centered around central canal at 3 dpi in Sham, SCI and KD-SCI mice. Scale bar = 100 μm. H Double immunofluorescence labeling of microglia for IBA-1(green) and iNOS (red), obtained from longitudinal Sects. 1 mm caudal to the lesion site at 3 dpi in Sham, SCI and KD-SCI mice. As shown in the KD-SCI mice, iNOS was increased in the microglia. Scale bar = 100 μm. I , J ELISAs performed for the IL-1β and IL-18 expressions in injured tissue obtained at 3 dpi, showing significantly increased levels of IL-1β and IL-18 in KD-SCI group; n = 5. The error bars represent the SD. *p < 0.05 vs. NC-sham group, #p < 0.05 vs. NC-SCI group by one-way ANOVA followed by Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001). NC-sham mice were performed laminectomy only, NC-SCI mice were performed spinal cord contusion, KD-SCI mice were transfected with KD-FANCC before SCI

Article Snippet: Microglia were pretreated with the p38 MAPK inhibitor BIRB 796 (500 nM, Axon Medchem, Groningen, The Netherlands) for 6 h or the NLRP3 inhibitor glyburide (50 mM, Sigma-Aldrich) for 2 h, after which LPS (1 μg/mL, Sigma-Aldrich) was administered to activate microglial inflammation for 24 h.

Techniques: Western Blot, Expressing, Immunofluorescence, Labeling, Transfection

Journal: Cell & Bioscience

Article Title: FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

doi: 10.1186/s13578-022-00816-4

Figure Lengend Snippet: Pyroptosis mediated by the pore-forming protein GSDMD is a necessary step of neuroinflammation and subsequent neuronal apoptosis after SCI. We demonstrated that deficiency of FANCC, a member of the FA gene family, can aggravate microglial pyroptosis and neuronal apoptosis via p38/NLRP3 signaling pathway

Article Snippet: Microglia were pretreated with the p38 MAPK inhibitor BIRB 796 (500 nM, Axon Medchem, Groningen, The Netherlands) for 6 h or the NLRP3 inhibitor glyburide (50 mM, Sigma-Aldrich) for 2 h, after which LPS (1 μg/mL, Sigma-Aldrich) was administered to activate microglial inflammation for 24 h.

Techniques: