amppnp  (Millipore)

Journal: bioRxiv

Article Title: Dual Pathways of Extracellular ATP Action in Cancer Cells: Purinergic Signaling–Driven Senescence and Macropinocytic ATP Internalization

doi: 10.64898/2026.04.23.720363

Figure Lengend Snippet: (A–B) Short-time interval ATP concentration assay (minutes). Intracellular ATP (iATP, % of control) for A549 (A) and A549STC1KO (B) cells measured at the indicated induction times between 30-120 minutes. (C–D) Long-time interval ATP concentration assay (hours). iATP (%) for A549 WT (C) and STC1-KO (D) across induction times between 2-4 hours. (E) Immunofluorescence/confocal imaging of macropinocytosis. A549 WT, A549 STC1-KO (two independent clones), and NL20 control cells were stained with NHF-ATP (green; ATP analog), High molecular weight dextran (red-macropinosome marker), and DAPI (blue; cell nuclei). Merged images show colocalization (yellow). STC1-KO cells display visibly reduced FITC-dextran uptake relative to WT based on gene expression reduction, while NL20 shows minimal uptake. Scale bars: 20 µm (A549 and STC1-KO), 10 µm (NL20). Data is representation for one set of biological replicates as mean ± SD for (n ≥ 5 technical replicates). Colors denote the eATP dose as indicated in the in-plot legend. One-way ANOVA with post-hoc multiple-comparisons; *P < 0.05,**P < 0.01,***P < 0.001, ****P < 0.0001.

Article Snippet: For fluorescence microscopy, NHF-ATP was obtained from Jena Bioscience (NU-810-488), ProLong TM Gold Antifade Mountant with DAPI from Thermo Fisher and high molecular weight fluorescent TMR-dextran (HMWFD) 70,000 Daltons MW, Neutral (no electric charge) from Invitrogen (D818).

Techniques: Concentration Assay, Control, Immunofluorescence, Imaging, Clone Assay, Staining, High Molecular Weight, Marker, Gene Expression

Journal: bioRxiv

Article Title: Dual Pathways of Extracellular ATP Action in Cancer Cells: Purinergic Signaling–Driven Senescence and Macropinocytic ATP Internalization

doi: 10.64898/2026.04.23.720363

Figure Lengend Snippet: Tumor xenografts were established by subcutaneous injection of 1×10 –5×10 cells into the right flank of 3–4-week-old male NU/J nude mice. When tumors reached ∼200–500 mm³, tumors were injected intratumorally with DMEM control, HMWFD tracer, or HMWFD plus NHF-ATP (100 μM) in a total volume of 50 μL using 27G needles. Tumors were harvested within ∼7–8 minutes post-injection, embedded in OCT, and cryosectioned (10 μm). Sections were ethanol-fixed, PBS-washed, and mounted with antifade medium containing DAPI. Images were acquired using a Nikon NiU epifluorescence microscope with identical exposure settings across groups and processed using Nikon NIS-Elements software. A-H. NHF-ATP and HMWFD colocalize in HT29 (A), PANC1 (B), MCF7 (C), SK-HEP1 (D), HOP-92 (E), H1299 (F), and A549 (G) and A375 (H). respectively. Color: Blue:DAPI (nuclei), Red: HMWFD, Green: NHF-ATP, Yellow: Co-localization. Scale bar: 10 µm, 100x magnification.

Article Snippet: For fluorescence microscopy, NHF-ATP was obtained from Jena Bioscience (NU-810-488), ProLong TM Gold Antifade Mountant with DAPI from Thermo Fisher and high molecular weight fluorescent TMR-dextran (HMWFD) 70,000 Daltons MW, Neutral (no electric charge) from Invitrogen (D818).

Techniques: Injection, Control, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: A dynamic structural framework for the allosteric regulation of Hsp70 chaperones

doi: 10.1016/j.jbc.2025.110516

Figure Lengend Snippet: Conformational dynamics of DnaK on binding of ATP analogs. A , overview of the ATP analogs investigated in this study. B , total amplitudes ( left panel ) and weighted rates ( right panel ) for binding of ATP and ATP analogs to apoDnaK lid (HiLyte Fluor 488-labelled DnaK E430C,R547C ). Weighted rates were calculated by weighting the rate of the individual phases according to their contribution towards the total amplitude and are used to characterize the population average. Shown are the mean and standard deviation for at least three independent replicates. Statistical significance of the values was assessed with ordinary one-way ANOVA and Dunnett's multiple comparison; ns, not significant; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. C , fluorescence signal changes within DnaK linker (HiLyte Fluor 488-labeled DnaK E217C,L392C ) on binding and hydrolysis of ATP or its analogues (ATPαS, AMPPNP) in the absence or the presence of pep65. D , rates of ATP or ATPαS hydrolysis by DnaK linker in the absence or presence of pep65. Shown are the mean and standard deviation for three independent replicates.

Article Snippet: The stocks of 4 μM DnaK linker , 4 μM ATP (Carl Roth, Karlsruhe, Germany) 4 μM ATPαS (Jena BioScience, Jena, Germany), 4 μM AMPPNP (Jena BioScience, Jena, Germany) and 4 μM ATP-free ADP (as a negative control) were prepared using HKM-buffer (40 mM HEPES/KOH pH 7.6, 150 mM KCl, 5 mM MgCl 2 ) with 0.05% Triton X-100.

Techniques: Binding Assay, Standard Deviation, Comparison, Fluorescence, Labeling, Analogues

Journal: bioRxiv

Article Title: Salmonella effector kinase SteC is activated by host-mediated phosphorylation

doi: 10.1101/2025.03.01.640964

Figure Lengend Snippet: a) SteC 1-457 expressed in Sf9 cells was analysed by mass spectrometry. Peptide SVSLATR (S377-R383) was detected in S379-phosphorylated and unphosphorylated forms following fragmentation obtained for precursor ion at m/z 407.2. C-terminal (y) ions and N-terminal (b) ions are presented here. y ions at 627 and 726 and the b3 ion at 354 indicate presence of phosphorylation. 529 represents unphosphorylated y5 minus H 2 O. b) S379 phosphorylated / non-phosphorylated peptides ratios were calculated as determined by mass spectrometry for the indicated conditions. c) Quantification of triplicate biological repeats for radioactive kinase assays performed with 5 μM SteC 210-457 expressed in E. coli and Sf9 and 100 μM FMNL1 peptide (see also Figure S2c ). Quantification was performed with ImageJ. d) AlphaFold2 prediction of the SteC kinase domain. N lobe secondary structural elements are highlighted in green and C lobe in light orange. The putative activation segment is highlighted in teal from D364 of the DGD motif to αF, including β9 and αEF. S379, at the C-terminal end of the activation segment, is shown as a purple stick. e) Quantification of four biological repeats of radioactive kinase assays containing 5 μM of SteC 1-457 WT or associated mutants expressed in E. coli together with 100 μM FMNL1 peptide (see also Figure S2d ). f) Radioactive kinase assays with SteC 1-457 WT and mutants and MYL12A WT at 100 nM kinase and 5 μM MYL12A. Representative of 2 repeats. g) Fluorescence titrations of 500 nM mant-AMPPNP with SteC 210-429 WT and mutants expressed in E. coli . The increase in fluorescence at increasing protein concentrations was measured. Data are representative of 2 repeats for each construct. Fitting curves are shown as purple lines. See also Figure S2g . h) Radioactive kinase assays of SteC 210-429 WT and mutants expressed in E. coli at 5 μM with FMNL1 peptide at 100 μM or with 1 μL murine cell lysate (right). Statistical analysis used one-way ANOVA (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant) with Dunnett’s multiple comparisons test (C&E). Assumption of normally distributed data was accepted using a Shapiro-Wilk and Kolmogoro-Smirnov test.

Article Snippet: Mant-Adenylyl imidodiphosphate (AMPPNP) was acquired from Jena Bioscience GmbH.

Techniques: Mass Spectrometry, Activation Assay, Fluorescence, Construct

Journal: ACS Omega

Article Title: Structural Basis for the Improved RNA Clamping of Amidino-Rocaglates to eIF4A1

doi: 10.1021/acsomega.4c09421

Figure Lengend Snippet: Comparison of the structures of eIF4A1:inhibitor complexes. (A) eIF4A1:RocA:RNA:AMPPNP complex from 5ZC9 with the corresponding chemical structure of RocA (inset). (B) eIF4A1:silvestrol:RNA:AMPPNP complex from 9AVR with the corresponding chemical structure of silvestrol (inset). Only one of the two conformations of the ethanediol substituent of the A-ring dioxanyloxy group is shown for clarity. (C) eIF4A1: DMPatA:RNA:AMPPNP complex from 6XKI with the corresponding structure of des-methyl pateamine A (DMPatA; MZ-735, inset). Important residues for inhibitor binding are labeled. (D) RocA overlaid with DMPatA by the superposition of liganded structures.

Article Snippet: AMPPNP (MilliporeSigma) and poly(AG) 5 RNA (Horizon Discovery/Dharmacon) were purchased commercially.

Techniques: Comparison, Binding Assay, Labeling

Journal: ACS Omega

Article Title: Structural Basis for the Improved RNA Clamping of Amidino-Rocaglates to eIF4A1

doi: 10.1021/acsomega.4c09421

Figure Lengend Snippet: Structure of the eIF4A1: CMLD012824 :RNA:AMPPNP quaternary complex. eIF4A1 shown as off-white transparent ribbon with poly(AG) 5 (salmon) CMLD012824 (yellow stick with transparent spheres), AMPPNP (teal) shown as sticks, and Mg 2+ as green sphere.

Article Snippet: AMPPNP (MilliporeSigma) and poly(AG) 5 RNA (Horizon Discovery/Dharmacon) were purchased commercially.

Techniques: