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: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Nucleotide specificity of the hBrr2 cassettes. A and B , time courses of mant -nucleotide binding to 0.5 μM nucleotide-free hBrr2 NC ( A ) and hBrr2 CC ( B ) measured by FRET from Trp to mant . 1, mant -ADP (5 μM); 2, mant -ATPγS (5 μM); 3, mant -ATP (5 μM); 4, mant -AMPPNP (5 μM); 5, mant -GDP (5 μM); 6, mant -GTP (5 μM); 7, mant -GTPγS (5 μM). The hBrr2 cassettes bind mant -ADP and mant -ATPγS but do not interact with mant -AMPPNP or mant -G nucleotides.

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Kinetics of mant -ADP and mant -ATPγS interaction with hBrr2 NC and hBrr2 CC . A and B , time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 NC (1; 0.4 μM) and hBrr2 CC (2; 0.2 μM) measured by FRET from Trp to mant . Controls correspond to binding experiments with unlabeled ADP or ATPγS (3 and 4). C and D , individual nucleotide binding traces were fitted to single exponentials and the dependencies of the apparent rate constants on nucleotide concentration for hBrr2 NC ( C ) and hBrr2 CC ( D ) were fitted by a linear equation, k app = k 1 [mant-nucleotide]+ k −1 , in which k 1 is derived from the slope and k −1 from the y -axis intercept. Closed circles , mant -ADP; open circles , mant -ATPγS. Values represent means ± SD of at least three independent measurements. Error bars that are not visible are smaller than the symbols. E and F , dissociation of 5 μM mant -ADP ( E ) or man t-ATPγS ( F ) from hBrr2 NC (1; 0.4 μM hBrr2 NC ) and hBrr2 CC (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3) were carried out in the absence of unlabeled nucleotide (curves shown are for hBrr2 CC ).

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques: Binding Assay, Concentration Assay, Derivative Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Kinetics of nucleotide binding and release

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Affinities of hBrr2 variants for mant -ADP and mant -ATPγS

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Kinetics of mant -ADP and mant -ATPγS interaction with hBrr2 FL and hBrr2 T1 . A and B , time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) measured by FRET from Trp to mant . Controls were performed with unlabeled ADP or ATPγS ( , ). C and D , individual nucleotide binding traces were fitted to double exponential equations, and the dependence of the apparent rate constants of the NC ( circles ) and CC ( diamonds ) on nucleotide concentration were fitted by a linear equation. Open symbols , hBrr2 FL ; closed symbols , hBrr2 T1 . The cassettes of hBrr2 FL and hBrr2 T1 bind nucleotides with different velocities as observed for the variants containing either one of the cassettes, hBrr2 NC and hBrr2 CC . While the CC binds mant -ADP and man t-ATPγS with similar rates, the NC in hBrr2 T1 binds mant -ATPγS faster than the NC in hBrr2 FL . Values represent means ± SD of at least three independent measurements. Error bars that are not visible are smaller than the symbols. E and F , dissociation of 5 μM mant -ADP ( E ) or mant -ATPγS ( F ) from hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3 and 4) were carried out in the absence of unlabeled nucleotide.

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques: Binding Assay, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2

doi: 10.1016/j.jbc.2021.100829

Figure Lengend Snippet: Effects of hBrr2 T1 mutations on the kinetics of mant -ADP and mant -ATPγS binding. A and B , apparent rate constants ( k app NC and k app CC ) of mant -ATPγS binding to the respective cassettes in hBrr2 T1 and variants thereof. Binding of mant -ATPγS to either NC or CC is affected by all intercassette mutations tested. S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2 and was carried along as a negative control. C and D , apparent rate constants ( k app NC and k app CC ) of mant -ADP binding to the respective cassettes of hBrr2 T1 and variants thereof. While most intercassette mutants bind mant -ADP at similar rates as hBrr2 T1 , mant -ADP binding is almost completely abrogated at both NC and CC in R603A ( red ). hBrr2 T1 , black ; S1087L (control with residue exchange in the N-terminal HB ratchet helix), blue ; R603A (cassette interface NC), red ; R637A (cassette interface NC), light gray ; PPP1296-8AAA (linker), magenta ; H1548A (cassette interface CC), dark gray . Coloring as in A and B , S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2 and was carried along as a negative control. E and F , apparent rate constants ( k app NC ) of mant -ADP and mant -ATPγS binding to the NC of hBrr2 T1 ( closed circles ) and its GK1355-6QE variant (altered CC nucleotide binding pocket; open circles ). The time courses of nucleotide binding to GK1355-6QE were fitted by a single exponential indicating no detectable nucleotide binding at the CC, as expected due to the two residue exchanges in the CC-binding pocket. As GK1355-6QE only has an intact NC nucleotide-binding pocket, only the hBrr2 T1 k app NC is shown for comparison. GK1355-6QE shows reduced rates for nucleotide binding at the NC, suggesting long-range modulation of NC nucleotide binding by nucleotide binding at the CC in hBrr2 T1 . Values represent means ± SD of at least three independent measurements. Error bars that are not visible are smaller than the symbols.

Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant -ATPγS (Jena Bioscience) in reservoir solution.

Techniques: Binding Assay, Mutagenesis, Negative Control, Control, Residue, Variant Assay, Comparison

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