Journal: Experimental & molecular medicine

Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction.

doi: 10.1038/s12276-024-01354-4

Figure Lengend Snippet: Fig. 6 Immune-epithelial interaction in aged colon tissues. a The schematic image depicts the effect of senescent immune cells on the surrounding microenvironment of tissues. b A dot plot illustrates the incoming and outgoing strength (interaction count) in each cell type in GSE178341. c The ingoing and outgoing interaction strength across 18 different signaling pathways in each cell type. A blue box indicates the interaction strength of p16INK4A- T cells, while a red box indicates the interaction strength of p16INK4A+ T cells. The y axis of the top bar graph indicates the average number of interactions or connections for each cell type within the signaling network. d The interaction strength of PARs signaling network is displayed. e The strength of sender, receiver, mediator, and influencer in the PARs signaling pathway network were examined in each cell type. f The IHC analysis of PAR1 (left panel) and PAR2 (right panel) in normal colon tissues from young and elderly individuals is shown. g The violin plot displays the mRNA expression level of GzmA in p16INK4A- and p16INK4A+ T cells from GSE178341 (left panel). The violin plot illustrates the mRNA expression of GzmA in T cells from young and old individuals (right panel). h The IHC analysis of GzmA was performed in colon tissues from young and old individuals, respectively (left panel). The right panel shows the quantification data. The data is presented as mean ± standard deviation. “Young” and “Old” indicate the young and the elderly individuals, respectively. The p-value is calculated using Mann–Whitney U test. i The multiplex IHC analysis shows the expression of CD3 (brown) and GzmA (red) (upper panel) and p16INK4A (brown) and GzmA (red) (lower panel) in old individuals, respectively. j The multiplex IHC analysis shows the expression of GzmA (brown) and PAR1 (red) (upper panel) and GzmA (brown) and PAR2 (green) (lower panel) in old individuals, respectively.

Article Snippet: The primary antibodies used for human samples were as follows: p16INK4A, predilution (805–4713, Roche, Basal, Switzerland); human Ki67, 1:3,000 (M7240, Dako, Glostrup, Denmark); H3K9me3, 1:500 (ab176916, Abcam, Cambridge, MA); CD3, 1:150 (ab135372, Abcam); CD19, 1:100 (ab227688, Abcam), CD68, 1:2,500 (NBP2-48923, Novus Biologicals, Centennial, CO); PAR1, 1:200 (ab233741, Abcam); PAR2, 1:100 (ab184673, Abcam); Granzyme A, 1:100 (ab209205, Abcam); Cleaved caspase-3, 1:400 (9661S, Cell Signaling Technology, Danvers, MA); IL8, 1:300 (27095-1-AP, Proteintech, Rosemont, IL) IFNα 1:200 (PA5-119649, Thermo Fisher Scientific, Waltham, MA); IFNβ 1:500 (PA5-102429, Thermo Fisher Scientific).

Techniques: Protein-Protein interactions, Expressing, Standard Deviation, MANN-WHITNEY, Multiplex Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 1. Neuroinflammation and PAR2 expression in AD brains. PAR2 transcript abundance (a) was decreased in brains from AD (n 6) compared with non-AD (n 6) patients. TNF- (b), IL-8 (c), and IL-10 (data not shown) mRNA levels were up-regulated, whereas IL-4 transcript levels (d) were suppressed in AD brains. There was a down- regulation of the ER chaperone, GRP58, mRNA levels in AD brains (e). In contrast, FPRL1, a potential receptor for A1– 42 in glial cells, was up-regulated in AD brains compared with non-AD brains (f). In non-AD patients, preactivated (g) and total (i) PAR2 immunoreactivity was chiefly present in cortical neurons (i, inset shows the staining with Ab absorbed with immunogen peptide), whereas in AD brains, preactivated (h) and total (j) PAR2 immunoreactivity was colocalized with CD45 demonstrating its expression predominantly in monocytoid cells in the cortex. Original magnification, 200 for main panels; 1000 for h and j insets. Data are mean SEM by Student’s t test; , p 0.05; , p 0.01; , p 0.001; RFC, relative fold change.

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 2. Neuronal PAR2 activation pro- tects neurons against A1–42 toxicity. a, Human cholinergic (LAN-2) and RFN cells expressed PAR2. Background represents the same condi- tions without primary Ab. b, Fibrillar A1–42 in- cubation for 36 h decreased the cytoskeletal iso- type III -tubulin immunoreactivity as a neuronal viability marker in RFNs; c, decreased PAR2 expression (normalized against -tubulin immunoreactivity) on LAN-2 cells in concentra- tion-dependent manners; d, PAR2 activation with SLIGRL increased the neuronal viability and protected RFNs against A1–42 toxicity, whereas the control peptide LSIGRL had no ef- fect; e, A1–42 also induced the expression of ER stress protein, GRP58 (normalized against -tubulin immunoreactivity) in RFNs; f, activa- tion of PAR2 with SLIGRL reversed A1–42- induced GRP58 expression. Data are mean SEM by Student’s t test (a), Dunnet (b, c, and e) and Tukey-Kramer multiple comparisons (d and f) tests; , p 0.05; , p 0.01; , p 0.001. All experiments were performed in qua- druplicate and background subtracted average intensities of fluorescence units (arbitrary) are used for quantification of immunoreactivity (IR).

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Activation Assay, Marker, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 3. PAR2 activation increases FPR2 expression on mouse glia. Activation of PAR2 with SLIGRL enhanced the expression of FPR2 in mouse primary macrophage (M; a) and astrocytes (Astro; b). Induction of FPR2 expression by TNF- was significantly reduced in PAR2 KO mac- rophages (c) and astrocytes (d) compare with WT cells. Data are mean SEM; Tukey-Kramer multiple comparisons test; , p 0.01; , p 0.001; n 5; RFC, relative fold change. FIGURE 4. Macrophage and astrocytic (Astro) PAR2 activation in- creases A1–42-induced neuroimmune activation and neurotoxicity. a, IL-8 expression in human macrophages was increased during A1–42 treatment, whereas PAR2 activation exerted an additive effect with A1–42. b, In human astrocytes, IL-8 expression is unchanged whereas PAR2 activation suppressed IL-4 expression. c, Supernatants (S/N) from A1–42-treated macrophages were toxic to human fetal neurons, whereas macrophage PAR2 activation augmented the toxicity. Astrocyte-derived supernatant had no toxic effects on human fetal neurons. Data are mean SEM; Dun- nett’s test; , p 0.05; , p 0.01; , p 0.001. Experiments per- formed in quadruplicate. (SLI, SLIGRL-NH2; LSI, LSIGRL-NH2).

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Activation Assay, Expressing, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 5. APP-Tg mice display early stages of neurodegenerative dis- ease. a, Western blot analysis showed no difference in the neuronal protein, MAP-2, or synaptophysin immunoreactivity between WT (n 3) and APP-Tg (n 4) groups. Brain PAR2 (b) mRNA levels were up-regulated in APP-Tg mice, without any change in trypsinogen (c) expression com- pared with WT controls. Transcript levels of the activated macrophage/ microglial gene, F4/80 (d), in brain was unchanged, in contrast to astrocytic gene, GFAP (e), which was up-regulated in APP-Tg mice. MIP-2 (f), the murine homolog of human IL-8, remained unchanged, whereas IL-4 (g) was induced in the brains of the APP-Tg group. There were no differences in FPR2 expression between groups (h). Data are mean SEM; Student’s t test; , p 0.05; RFC, relative fold change.

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 6. PAR2-deficient mice showed reduced neuroimmune re- sponses after A1–42 implantation. a, Decreased transcript levels of PAR2 were evident in A1–42-implanted mice. b, There was no significant change in trypsinogen transcript levels. Up-regulation of F4/80 (c) and GFAP (d) was evident in A1–42-implanted WT animals but not in implanted PAR2 KO littermates or in PBS-implanted animals. MIP-2 (e) mRNA levels were increased in A1–42-implanted WT animals, in contrast to IL-4 transcript levels, which were up-regulated in PAR2 KO animals receiving A1–42 implants (f). mRNA levels of ER stress gene GRP58 were also increased in A1–42-implanted PAR2 KO animals (g). PAR2 KO animals, regardless of whether they were A1–42 implanted or not, showed lower levels of FPR2 (h). Data are mean SEM; Tukey-Kramer multiple comparisons test; , p 0.05; , p 0.01; , p 0.001. n 6 in all four groups. RFC, relative fold change.

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 7. PAR2 deficiency is neuroprotec- tive against A1–42 implantation. Immunolabel- ing of PBS-implanted PAR2 WT (a and d), PBS-implanted PAR2 KO (data not shown), A1–42-implanted PAR2 KO (b and e), and A1–42-implanted PAR2 WT (c and f) mouse brains with anti-NeuN (green) and anti-Iba-1 (red) (a–c) or anti-GFAP (red; d–f) Abs re- vealed more severe microglial and actrocytic ac- tivation in dorsal hippocampi of WT (c and f) animals implanted with A1–42, as compared with A1–42-implanted PAR2 KO (b and e) and PBS-implanted WT (a and d) animals. Colocal- ization of IL-4 with GFAP immunoreactivity was evident (e, inset). Immunolabeling for cleaved caspase-3 showed higher immunoreac- tivity in A1–42-implanted WT animals (i) com- pared with A1–42-implanted PAR2 KO animals (h). Immunoreactivity for cleaved-caspase-3 was absent in PBS-implanted WT animals (g). Insets in g, h, and i show colocaliztion with the neuronal marker, NeuN. Original magnification, 630; 2 630 magnification for e, inset; j, A1–42-implanted PAR2 WT animals exhibited consistently delayed neurobehavioral responses during the Morris water maze. Data are mean SEM, Tukey-Kramer multiple comparisons test; , p 0.05; , p 0.01. n 6.

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Immunolabeling, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease.

doi: 10.4049/jimmunol.179.8.5493

Figure Lengend Snippet: FIGURE 8. Divergent effects of PAR2 on AD patho- genesis. In addition to the protective properties of neu- ronal PAR2 in the context of A1–42 neurotoxicity, A1–42-induced microglial activation, mediated by FPR2/FPRL1, was amplified by PAR2 coactivation, leading to the release of neurotoxins, inflammatory cy- tokines, and chemokines such as IL-8, and subsequently induces neuronal death. Although PAR2 mediated these proinflammatory effects, it also suppressed an important inhibitor of this cascade, astrocyte-derived IL-4. The overall consequence of PAR2 activation was to aug- ment the neuroinflammatory response, which over- whelmed the direct protective effects of neuronal PAR2.

Article Snippet: Cells were incubated with LI-COR Odyssey Blocking Buffer for 1.5 h before the addition of a mouse monoclonal anti- -tubulin isotype III (1/800; SigmaAldrich), a rabbit polyclonal Ab (B5) raised against rat PAR2 (1/500; 30GPNSKGR2SLIGRLDT46P-YGGC, coupled to keyhole limpet hemocyanin; 2 trypsin cleavage site, YGGC for conjugation) (8) or goat polyclonal anti-GRP58 (1/50; Santa Cruz Biotechnology) Ab in blocking buffer overnight at 4°C.

Techniques: Activation Assay, Derivative Assay

Journal: Journal of Cellular Signaling

Article Title: The Role of Membrane-embedded DUOX2 on Ectodomain Shedding via G protein-coupled Receptor Signaling

doi: 10.33696/signaling.2.033

Figure Lengend Snippet: Figure 7: Role of DUOX2 for shedding of membrane-bound fractalkine. Macrophages transfected with small interfering RNA for DUOX2 were stimulated with substance P (5 μM) for 6 hours, after which protein levels of membrane-bound fractalkine in whole-cell lysates (a) and soluble fractalkine in cell-culture supernatants (b) were measured by ELISA. Data were obtained by using samples from three individuals in each group and represent the mean (SE). **P<0.01; *P<0.05 (with Bonferroni’s correction). Protein levels of membrane-bound fractalkine were determined by western blotting. Data were obtained by using samples from three volunteers in each group, and a representative result is shown as arbitrary density units (c).

Article Snippet: The membranes were incubated with 0.2 × 103 μg/L mouse anti-human PAR2, fractalkine, and TGFβ1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, washed, and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) diluted to 1:5000.

Techniques: Membrane, Transfection, Small Interfering RNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Cellular Signaling

Article Title: The Role of Membrane-embedded DUOX2 on Ectodomain Shedding via G protein-coupled Receptor Signaling

doi: 10.33696/signaling.2.033

Figure Lengend Snippet: Figure 8: Role of nitric oxide (NO) for shedding of membrane-bound fractalkine. Human macrophages were pretreated with L-NAME (0, 2 and 5 nM/L) for 1 hour and stimulated with substance P (5 μM) for 6 hours, after which protein levels of membrane-bound fractalkine were measured by ELISA. Data were obtained by using samples from three individuals in each group and represent the mean (SE). **P<0.01; *P<0.05 (with Bonferroni’s correction) N.S.; Not Significant.

Article Snippet: The membranes were incubated with 0.2 × 103 μg/L mouse anti-human PAR2, fractalkine, and TGFβ1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, washed, and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) diluted to 1:5000.

Techniques: Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular Signaling

Article Title: The Role of Membrane-embedded DUOX2 on Ectodomain Shedding via G protein-coupled Receptor Signaling

doi: 10.33696/signaling.2.033

Figure Lengend Snippet: Figure 10: Effects of small interfering RNAs for β arrestin 2, G-protein coupled receptor kinase 2 (GRK2), tumor growth factor (TGF) β1/2/3 and TGF-β-activated kinase 1 (TAK-1) on production of membrane-bound fractalkine after exposure to substance P. Human macrophages transfected with small interfering RNAs for β arrestin 2, GRK2, TGFβ1/2/3 and TAK-1 were stimulated with substance P (5 μM) for 6 hours, which after protein levels of membrane-bound fractalkine were measured by ELISA (a). Data were obtained by using samples from three individuals in each group and represent the mean (SE). **P<0.01; *P<0.05 (with Bonferroni’s correction) N.S.; Not Significant.

Article Snippet: The membranes were incubated with 0.2 × 103 μg/L mouse anti-human PAR2, fractalkine, and TGFβ1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, washed, and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) diluted to 1:5000.

Techniques: Membrane, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular Signaling

Article Title: The Role of Membrane-embedded DUOX2 on Ectodomain Shedding via G protein-coupled Receptor Signaling

doi: 10.33696/signaling.2.033

Figure Lengend Snippet: Figure 11: Effect of TRIM28/TIF1β and Fli-1 on protein levels of TGFβ1, nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) and membrane-bound fractalkine after exposure to substance P. After co-transfection of human macrophages (on day 9 of culture) with specificity protein 1 (Sp1) small interfering RNA (siRNA) and TRIM28/TIF1β siRNA or Fli-1 siRNA, the macrophages were stimulated with substance P (5 µM) for 6 hours. TGFβ1 mRNA levels were assessed by reverse transcription quantitative polymerase chain reaction (RT- qPCR) (a). Protein levels of TGFβ1 were assessed by western blotting. The density of each band was normalized for that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were obtained by using samples from three volunteers in each group, and a representative result is shown as arbitrary density units (b). Protein levels of NOS2/ iNOS (c) and membrane-bound fractalkine (d) in whole-cell lysates were measured by ELISA, respectively. Data were obtained by using samples from three volunteers in each group and represent the mean (SE). **P <0.01; *P<0.05 (with Bonferroni’s correction); N.S.: Not Significant.

Article Snippet: The membranes were incubated with 0.2 × 103 μg/L mouse anti-human PAR2, fractalkine, and TGFβ1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, washed, and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) diluted to 1:5000.

Techniques: Membrane, Cotransfection, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay