Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: (A and B) BMH-21-caused nucleolar stress is independent of ATM pathway activation. A375 cells were pretreated with ATM inhibitor (ATMi) KU55933 (10 μM) for 30 min as indicated, followed by treatment with BMH-21 (1 μM) or IR (2 Gy) and incubation for 3 h. Cells were stained for (A) S1891-phosphorylated ATM (PATM, green ) or (B) NCL ( green ) and counterstained for DNA ( blue ). (C) Parent DLD and DLD cells with ATR-knock in mutation (DLD Seckel cells) were treated with BMH-21 for 6 h followed by staining for NPM ( green ). Merged images with DNA ( blue ) are shown. (D) Inhibition of DDR pathways does not affect BMH-21-mediated RPA194 degradation. A375 cells were pretreated for 30 min with the following: KU55933 (10 μM), caffeine (2 mM), wortmannin (10 μM), NU7441 (5 μM) followed by addition of BMH-21 (1 μM) and incubation for 2 h. Cells were stained for RPA194 ( green ), UBF ( red ) and counterstained for DNA ( blue ) Arrowheads , nucleolar caps. (E) A375 cells were pretreated with KU55933 (10 μM) for 1 h as indicated, followed by IR (2 Gy) and incubation for the indicated times. BMH-21 (1 μM) was added for the final 3 h as indicated. Cell lysates were analyzed by western blotting for RPA194 and GAPDH was used as a loading control. (F) A375 cells were pretreated with NU7441 (10 μM) for 1 h as indicated, followed by addition of ActD (50 ng/ml) or BMH-21 (1 μM) and incubation for 3 h. Cell lysates were analyzed by western blotting for RPA194, NCL and GAPDH was used as a loading control. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Staining, Knock-In, Mutagenesis, Inhibition, Western Blot, Control

Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: U2OS cells were treated with LI-216 (10 μM) for 3 h in the presence or absence of NU7441 (10 μM). Cells were fixed and stained for (A) PATM, (B) γH2AX, (C) PKAP1, (D) PDNA-PK and counterstained for DNA. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Staining

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

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