Journal: Aging Cell

Article Title: Aging Triggers an Intestinal Energy Crisis and HDL3 Deficiency Disrupting Gut–Liver Axis Homeostasis

doi: 10.1111/acel.70445

Figure Lengend Snippet: Mitochondrial dysfunction‐mediated ATP deficiency suppresses HDL3 synthesis in aging intestinal cells. (a) Representative images (scale bar: 5 μm) of intestinal cell microstructure measured by TEM, n = 18 images from n = 3 independent experiments; (b) ileum ATP levels, n = 5; (c) representative images (scale bar: 5 μm) of IME microstructure measured by TEM, n = 18 images from n = 3 independent experiments; (d) IME ATP levels, n = 5; (e) glycolysis assay measured as cytoplasmic acidification, the fluorescence signal was enhanced with the increase of acidification degree, n = 4; (f) oxygen consumption, as mitochondrial respiration depletes the oxygen within the assay medium, quenching of the fluorescent dye is reduced, and the fluorescence signal increases proportionately, n = 4; (g, h) OXPHOS protein expression levels in the ileum, n = 3; and (i) exogenous ATPγS‐AM (50 μM) partially restored HDL3 synthesis in senescent IME cells, whereas native ATP (50 μM) had no significant effect, n = 5. Data are express as the mean ± SEM. * p < 0.05, ** p < 0.01. D‐Gal: D‐galactose; NC, normal control.

Article Snippet: In the normal control groups (10 replicates), the medium was replaced with complete DMEM, whereas in the model control groups (10 replicates), the medium was replaced with D‐galactose (200 mM, dissolved in complete DMEM) and cultured for 24 h. After successful establishment of the aging model, the medium was discarded, and the model control group (D‐Gal) (replaced with complete DMEM containing ApoA1 10 μg/mL), ATP intervention group (D‐Gal‐ATP) was replaced with complete DMEM containing ApoA1 10 μg/mL and ATP 50 μM, ATPγS‐AM intervention group (D‐Gal‐ATPγS‐AM) was replaced with complete DMEM containing ApoA1 10 μg/mL and ATPγS‐AM 50 μM, NMN intervention group (D‐Gal‐NMN) was replaced with complete DMEM containing ApoA1 10 μg/mL and NMN 5 μM, the agonist CS‐6253 (MedChem Express, Shanghai, China) intervention group (D‐GAL‐CS‐6253) was replaced with complete DMEM containing ApoA1 10 μg/mL and CS‐6253 1 μM; and the NMN and CS‐6253 synergistic group (D‐Gal‐NMN‐CS‐6253) was replaced with complete DMEM containing ApoA1 10 μg/mL, NMN 5 μM, and CS‐6253 1 μM.

Techniques: Fluorescence, Expressing, Control

Journal: Aging Cell

Article Title: Aging Triggers an Intestinal Energy Crisis and HDL3 Deficiency Disrupting Gut–Liver Axis Homeostasis

doi: 10.1111/acel.70445

Figure Lengend Snippet: ABCA1 downregulation limits HDL3 synthesis in aging. (a) Relative mRNA expression of ABCA1 , ApoA1 , LPL , and ANGPTL3 in ileum, n = 5; (b, c) representative images (scale bar: 50 μm) and quantitative analysis of ABCA1, ApoA1, LPL, and ANGPTL3, measured by IHC staining, n = 3; and (d) activation of ABCA1 expression combined with ATPγS‐AM supplementation enhances cellular HDL3 synthesis capacity n = 5. Data are express as the mean ± SEM. ** p < 0.01. ABCA1, ATP‐binding cassette transporter 1; ANGPTL3, angiopoietin‐like3; CS‐6253, ABCA1 activators; D‐Gal, D‐galactose; HDL3, high‐density lipoprotein 3; IHC, immunohistochemistry; IME, intestinal mucosa epithelial; LPL, lipoprotein lipase; NC, normal control.

Article Snippet: In the normal control groups (10 replicates), the medium was replaced with complete DMEM, whereas in the model control groups (10 replicates), the medium was replaced with D‐galactose (200 mM, dissolved in complete DMEM) and cultured for 24 h. After successful establishment of the aging model, the medium was discarded, and the model control group (D‐Gal) (replaced with complete DMEM containing ApoA1 10 μg/mL), ATP intervention group (D‐Gal‐ATP) was replaced with complete DMEM containing ApoA1 10 μg/mL and ATP 50 μM, ATPγS‐AM intervention group (D‐Gal‐ATPγS‐AM) was replaced with complete DMEM containing ApoA1 10 μg/mL and ATPγS‐AM 50 μM, NMN intervention group (D‐Gal‐NMN) was replaced with complete DMEM containing ApoA1 10 μg/mL and NMN 5 μM, the agonist CS‐6253 (MedChem Express, Shanghai, China) intervention group (D‐GAL‐CS‐6253) was replaced with complete DMEM containing ApoA1 10 μg/mL and CS‐6253 1 μM; and the NMN and CS‐6253 synergistic group (D‐Gal‐NMN‐CS‐6253) was replaced with complete DMEM containing ApoA1 10 μg/mL, NMN 5 μM, and CS‐6253 1 μM.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Binding Assay, Control

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Chemical structure of ′(3′)-O-(N-methyl-anthraniloyl) ATPγS (mant-ATPγS). (B) ATPγS is not hydrolyzed by nsP2. Under the conditions tested, 50 nM nsP2 converted ∼80% of 1 mM ATP to ADP within 30 minutes, whereas no detectable hydrolysis of 1 mM ATPγS was observed after 120 minutes. No luminescence signal was detected in reactions containing ATP or ATPγS in the absence of enzyme (data not shown). (C) Representative tryptophan to mant FRET emission spectra collected using excitation at 80 nm. nsP2 alone (1 μM) exhibits an emission peak at 350 nm, whereas mant-ATPγS alone (10 μM) shows weak emission at 445 nm under 280-nm excitation. Addition of mant-ATPγS to nsP2 products an increase in 445-nm emission, consistent with FRET arising from formation of the nsP2·mant-ATPγS complex. Data in panels D-G were generated by subtracting mant-ATPγS-only emission at 445 nm from spectra collected in the presence of nsP2. (D) Direct binding of mant-ATPγS to nsP2. ns2P (0. 25 μM) was titrated with 0.002 5 μM mant-ATPγS. Data represent mean ± SD ( n = 3). (E-G) Competitive binding experiments. nsP2 (0. 5 μM) was incubated with 0.1 μM mant-ATPγS and increasing concentrations of unlabeled competitor. ATPγS (E; 0-10 μM), ADP (F; 0-9 mM), or inorganic phosphate (Pi) and tripolyphosphate (TPP) (G; 0-40 mM) were added as indicated. Fluorescence data in panels E and F were normalized to percent relative fluorescence, with the signal in the absence of competitor defined as 100%. Data were fit by nonlinear regression, and IC₅₀ values were converted to inhibition constants ( K i ) using the Cheng-Prusoff equation.

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Generated, Binding Assay, Incubation, Fluorescence, Inhibition, Fluorescence Spectroscopy

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Exp rimental design for ATPγS association kinetics. nsP2 was rapidly mixed with mant-ATPγS under stopped-flow conditions, and binding was monitored by tryptophan-to-mant FRET. (B) ATPγS association kinetics. Representative fluorescence time courses following rapid mixing of mant-ATPγS (0.1 μM) with increasing concentrations of nsP2 (0.5-3 μM). (C) Observed rate constants ( k obs ) extracted from single-phase fits to the association traces in panel B were replotted as a function of nsP2 concentration (n = 3 independent experiments). Linear regression was used to determine the second-order association rate constant ( k ₒₙ). (D) Experimental design for ATPγS dissociation kinetics. Pre-formed nsP2·mant-ATPγS complexes were rapidly mixed with excess unlabeled ATPγS to initiatw ligand displacement. (E) ATPγS dissociation kinetics. Time-dependent loss of sensitized Mant fluorescence following competition with unlabeled ATPγS. Traces were fit to a two-phase exponential decay, revealing fast and slow dissociation components ( k off,fast and k off,slow ). (F) Experimental design for ATP dissociation in the presence of inhibitor. Pre-formed nsP2·mant-ATP complexes were rapidly mixed with excess unlabeled ATP in the presence of the nsP2 inhibitor RA-NSP2- (5 μM). (G) ATP dissociation kinetics in the presence of inhibitor. Representative fluorescence decay trace fit to a single-phase exponential model, yielding the apparent ATP dissociation rate constant ( k off ).

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Binding Assay, Fluorescence, Concentration Assay, Association Rate Constant, Dissociation Rate Constant, Fluorescence Spectroscopy

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

Article Title: Differential Regulation of ATP- and UTP-Evoked Prostaglandin E 2 and IL-6 Production from Human Airway Epithelial Cells.

doi: 10.4049/jimmunol.2100127

Figure Lengend Snippet: FIGURE 6. ERK1/2 activation is essential for both PGE2 synthesis and IL-6 secretion. (A) A Western blot illustrating ATP-mediated (100 mM) induction of ERK1/2 phosphorylation in NHBE cells (15 min following agonist treatment). This is abolished by the selective P2Y2 receptor antagonist AR-C (10 mM). Cells were pretreated with AR-C for 1 h. (B) Densitometry analysis of the Western blot data. Data are mean ± SEM of n 5 34 samples. (C) The MEK1/2 inhibitor U0126 (20 mM) abolishes ATP-induced (50 mM) PGE2 synthesis. Data are mean ± SEM of n 5 45 samples. (D) Likewise, UTP-induced (50 mM) PGE2 synthesis is abolished by U0126 (20 mM). Cells were pretreated with U0126 for 1 h. Data are mean ± SEM of n 5 45 samples. (E) ATP (100 mM, 2 h) induces a low level of COX-2 upregulation, which is not inhibited by MEK1/2 inhibitor U0126 (20 mM). (F) Densitometry analysis of the Western blot data illustrated in (E). Data are mean ± SEM of n 5 7 samples. (G) The ATPgS-induced (100 mM) IL-6 secretion is abolished by the MEK1/2 inhibitor U0126 (20 mM). Data are mean ± SEM of n 5 4 samples. **p < 0.01, ***p < 0.001, ****p < 0.0001. P-ERK, phospho-ERK; T-ERK, total ERK.

Article Snippet: Pharmacological tools used in the study were: UTP (U6875; Sigma-Aldrich), SLIGKV-NH2 (4153; Tocris Bioscience), FK-506 (3631; Tocris Bioscience), BTP2 (203890; Sigma-Aldrich), ATP (A6419; Sigma-Aldrich), diclofenac (4454; Tocris Bioscience), apocynin (4663; Tocris Bioscience), NAC (106425; Sigma-Aldrich), U0126 (1144; Tocris Bioscience), ATPgS (4080; Tocris Bioscience), AACOCF3 (1462; Tocris Bioscience), AR-C 118925XX (4890; Tocris), NF546 (3892; Tocris Bioscience), S3QEL 2 (5735; Tocris Bioscience), ADPbS (A8016; SigmaAldrich), UDP (U4125; Sigma-Aldrich), NF157 (2450; Tocris Bioscience), Apyrase (A6410 Grade VI, High ATPase/ADPase activity; Sigma-Aldrich), TNP-ATP (2464; Tocris), 5-BDBD (3579; Tocris), A740003 (3701; Tocris Bioscience), Suramin (AC328540500; Acros Organics-Fisher), and PPADS (0625; Tocris Bioscience).

Techniques: Activation Assay, Western Blot, Phospho-proteomics

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

Article Title: Differential Regulation of ATP- and UTP-Evoked Prostaglandin E 2 and IL-6 Production from Human Airway Epithelial Cells.

doi: 10.4049/jimmunol.2100127

Figure Lengend Snippet: FIGURE 7. Distinct Ca21-sensing enzymes are necessary for PGE2 and IL-6 induction. (A) The calcineurin inhibitor FK-506 (1 mM) blocks ATPgS- induced IL-6 secretion. Data are mean ± SEM of n 5 56 samples. (B) By contrast, FK-506 does not inhibit ATP-induced (100 mM) PGE2 synthesis. Data are mean ± SEM of n 5 56 samples. (C) ATP-induced (100 mM) PGE2 synthesis is inhibited with pretreatment of cells with cPLA2 inhibitor AACOCF3 (5 mM). Data are mean ± SEM of n 5 5 samples. (D) IL-6 release induced by ATPgS (100 mM) is not affected by AACOCF3 (5 mM). Data are mean ± SEM of n 5 5 samples. (E) The P2Y2 receptor antagonist AR-C (10 mM) blocks ATP-induced enrichment of cPLA2 in the nuclear fraction. (F) Densitometry analysis of the Western blot data illustrated in (E). Data are mean ± SEM of n 5 4 samples. (G and H) The CRAC channel inhibitors CM4620 (1 mM, referred to as “CM”) and BTP2 (1 mM) block ATP-induced (100 mM) enrichment of cPLA2 in the nuclear fraction. Data are mean ± SEM of n 5 45 sam- ples. (I) Likewise, CM4620 (1 mM) also blocks UTP-induced (100 mM) enrichment of cPLA2 in the nuclear fraction. (J) Densitometry to quantify experi- ments shown in (I). Data are mean ± SEM of n 5 3 samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Pharmacological tools used in the study were: UTP (U6875; Sigma-Aldrich), SLIGKV-NH2 (4153; Tocris Bioscience), FK-506 (3631; Tocris Bioscience), BTP2 (203890; Sigma-Aldrich), ATP (A6419; Sigma-Aldrich), diclofenac (4454; Tocris Bioscience), apocynin (4663; Tocris Bioscience), NAC (106425; Sigma-Aldrich), U0126 (1144; Tocris Bioscience), ATPgS (4080; Tocris Bioscience), AACOCF3 (1462; Tocris Bioscience), AR-C 118925XX (4890; Tocris), NF546 (3892; Tocris Bioscience), S3QEL 2 (5735; Tocris Bioscience), ADPbS (A8016; SigmaAldrich), UDP (U4125; Sigma-Aldrich), NF157 (2450; Tocris Bioscience), Apyrase (A6410 Grade VI, High ATPase/ADPase activity; Sigma-Aldrich), TNP-ATP (2464; Tocris), 5-BDBD (3579; Tocris), A740003 (3701; Tocris Bioscience), Suramin (AC328540500; Acros Organics-Fisher), and PPADS (0625; Tocris Bioscience).

Techniques: Western Blot, Blocking Assay

Journal: Scientific reports

Article Title: Epithelial wound healing in Clytia hemisphaerica provides insights into extracellular ATP signaling mechanisms and P2XR evolution.

doi: 10.1038/s41598-023-45424-5

Figure Lengend Snippet: Figure 3. eATP promotes closure of epithelial wounds. (a) Microinjection of 1 mM ATP (n = 11) or 1 mM ATPγS (n = 9) 5 min before wounding increases wound closure rate in comparison to ASW-injected controls (n = 9). One-way analysis of variance (ANOVA) at each time point: P < 0.01 (**); P < 0.05 (*). (b) Microinjection of 10U of apyrase, an ATP scavenger (n = 22), 30–60 min before wounding slows healing rate in comparison to ASW-injected controls (n = 13). Unpaired two-tailed T-test at each time point: P < 0.05 (*); P < 0.01 (**); P < 0.001 (***). n = number of wounds analyzed in independent animals. Data is presented as mean percent of original wound area ± SEM.

Article Snippet: ATPγS tetralithium (ATPγS, Tocris, Bristol UK) was dissolved in MQ water to make a 50 mM stock.

Techniques: Microinjection, Comparison, Injection, Two Tailed Test