Journal: Cell Death & Disease

Article Title: Enlarged PML-nuclear bodies trigger conflicting cell cycle signal-mediated cytotoxicity in leukemia cells

doi: 10.1038/s41419-025-07911-7

Figure Lengend Snippet: NB4 and HL60 cell lines were treated with 1 μM ATRA. a The expression of CD38 and CD11b on non-treated cells (black dots) and cells treated with ATRA for 24 h (blue dots) or 48 h (red dots) are shown. Representative data from three independent experiments are shown. b NBT staining of NB4 and HL60 cells 48 h after treatment with ATRA. DMSO-treated cells served as controls. Representative images and mean + SD of the percentage of NBT-positive cells, calculated from eight randomly selected fields across three independent experiments, are shown. c Cell growth in drug-free medium following prior incubation with ATRA for 24 or 48 h ( n = 4). DMSO-treated cells for 24 h served as controls. The fold change in cell number was calculated by dividing the values at each time point by the values at day 0. d Immunofluorescent staining for PML-NBs in NB4 and HL60 cells 48 h after treatment with ATRA. DMSO-treated cells were used as controls. Representative images and mean + SD of the number of PML-NBs per nucleus are shown ( n = 4). ** P < 0.01; N.S., no significant difference (two-sided Student’s t -test).

Article Snippet: HL60, MV4-11, and NB4 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Staining, Incubation

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 1. Susceptibility of NB4, U937 and THP-1 cells to ATO induced mitochondrial ROS formation.NB4, U937 or THP-1 (A-D) cells were pretreated for 5 min with the vehicle or 0.5 µM rotenone and incubated for 6 h with increasing concentrations of ATO (A, B), or NaAsO2 (C, D). After treatments, the cells were analyzed for MitoSOX red- (A, C) or DHR- (B, D) fluorescence, as detailed in the Materials and Methods section. In other experiments, U937 were incubated for 6 h with 5 µM ATO, alone or associated with rotenone, and analyzed for MitoSOX red- (E) or DHR- (F) fluorescence. Results represent the means ± SD calculated from three separate experiments. *P < 0.05, * *P < 0.01, compared with untreated cells. #P 0.05, ##P < 0.01 compared with ATO or NaAsO2 treated cells (one-way ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Incubation, Fluorescence

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 2. Clinically relevant concentrations of ATO stimulate phosphorylation of phospho p47phox in NB4 but not in U937 or THP-1 cells.NB4 (A, D), U937 (B, E) or THP-1 (C, F) cells were incubated for 6 h with 0.5 or 1 µM ATO. In other experiments, NB4 (G) or U937 (H) cells were treated for 6 h with 2.5 NaAsO2 or 5 µM ATO, respectively. PMA (0.162 µM, 15 min) was used as a positive control. After treatments, the cells were analyzed for phospho p47phox expression (A-H). The blot, representative of three separate experiments, was re-probed for p47phox. Representative micrographs providing immunocytochemical evidence of p47phox phos phorylation are also reported in panel D (NB4 cells), E (U937 cells) and F (THP-1 cells). After treatments, the cells were fixed and processed as detailed in Material and Methods. Scale bar represents 20 µm. Results represent the means ± SD calculated from three separate experiments. *P < 0.05, * *P < 0.01, compared with untreated cells (one-way ANOVA followed by Dunnett’s test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Incubation, Positive Control, Expressing

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 3. Pharmacological inhibition of NOX 2 activity suppresses mitochondrial ROS formation induced by ATO in NB4 cells.NB4 cells were pretreated for 5 min with the vehicle, 10 µM apocynin, or 1 µM DPI, and exposed for 15 min to 0.162 µM PMA. After treatments, the cells were analyzed for phospho p47phox expression (A) or DHR-fluorescence (B). NB4 cells pretreated as indicated above or with rotenone, were exposed for 6 h to 1 µM ATO. After treatments, the cells were analyzed for phospho p47phox expression (C), MitoSOX red- (D) or DHR- (E) fluorescence. In other experiments, the cells were pretreated with the vehicle, rotenone, apocynin or DPI, incubated for 6 h with 2.5 (F, G) or 5 µM (H-L) NaAsO2 and finally analyzed for MitoSOX red- (F, I) and DHR- (G, L) fluorescence, or for phospho p47phox

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Inhibition, Activity Assay, Expressing, Fluorescence, Incubation

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 4. ATO fails to promote mitochondrial ROS formation in NB4 cells with downregulated p47phox expression.NB4 control cells, transfected with scrambled or p47phox siRNA, were lysed and analyzed by Western blotting 48 h post-transfection using anti-p47phox antibody. The relative band intensity of p47phox is depicted in the top bar chart (A). GADPH was accounted for loading control. Results represent the means ± SD calculated from three separate experiments. *P < 0.05, compared with control cells (one-way ANOVA followed by Dunnett’s test).Cells transfected with scrambled or p47phox specific siRNA were pretreated for 5 min with the vehicle, apocynin, or DPI and exposed for either 15 min to PMA (B) or 6 h to 1 µM ATO (C, D). In some experiments, rotenone was added to the cultures 5 min before ATO. After treatments, the cells were analyzed for DHR- (B, C) or MitoSOX red- (D) fluorescence. Results represent the means ± SD calculated from three separate ex periments. *P < 0.05, * *P < 0.01, compared with untreated cells. #P < 0.05, ##P < 0.01 compared with ATO treated cells. $P < 0.05, $$P < 0.01 compared with treated scrambled siRNA cells (ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Transfection, Western Blot, Fluorescence

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 5. Low mitochondrial ROS formation induced by ATO in resistant cells is due to poor activation of NOX 2.U937 (A), THP-1 (B) or p47phox siRNA trans fected NB4 (C) cells were pretreated for 5 min with the vehicle, rotenone, apocynin or DPI and incubated for 6 h with 0.5 or 1 µM ATO alone or associated PMA. After treatments, the cells were analyzed for MitoSOX red fluorescence. Results represent the means ± SD calculated from three separate experiments. * *P < 0.01, compared with untreated cells. ##P < 0.01 compared with ATO/ PMA treated cells (ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Fluorescence

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 6. Clinically relevant concentrations of ATO promote mitochondrial permeability transition-dependent apoptosis in susceptible NB4 cells. NB4 control cells, and NB4 cells previously transfected with scrambled or specific p47phox siRNA, were pretreated for 5 min with the vehicle, rotenone, apocynin or 0.5 µM CsA and incubated for 6 (A), 9 (B, C) or 14 (D, E, F) h with 1 µM ATO. After treatments, the cells were analyzed for NAO-fluorescence (A) MitoTracker red CMXRos- fluorescence (B), cytochrome c (Cyt c) localization (C), caspase 3 activity (D) and apoptotic DNA fragmentation/condensation by both the Hoechst (E) and comet (F) assays. * *P < 0.01, compared with untreated cells. ##P < 0.01 compared with ATO treated cells. $$P < 0.01 compared with ATO treated scrambled siRNA cells (one-way ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Permeability, Control, Transfection, Incubation, Fluorescence, Activity Assay