Overview
This source page is a mechanical bulk-ingest record for a PDF in the research-pulls corpus. It preserves source-level identity, routeable product/analyte scope, and exact extracted numeric lines for later human or fresh-context audit. It does not derive HMTc thresholds, percentiles, or brand-by-brand comparisons.
Key numbers
The worker extracted the full PDF text with layout preservation twice and compared extraction hashes before commit. The following lines are copied from numeric/table-bearing regions of the PDF and retain the source units and wording where legible:
- disrupt higher-order chromatin structures.23,24 Studies have line, were cultured in RPMI-1640 medium supplemented with 10%
- methylation sites on a global scale, likely via interference with mL streptomycin at 37 °C in a 5% CO2 and 100% humidified
- 37 °C in a 5% CO2 and 100% humidified atmosphere. Passages 45−
- reagents were obtained from Cell Signaling Technology (Leiden, For sample preparation, the cells were dissolved in 500 μL of 30%
- Netherlands) and Santa Cruz Biotechnology (Dallas, TX, USA). H2O2 and 96% HNO3 (1:1, v/v) and heated stepwise to 95 °C. After
- dissolved in 0.2% HNO3. An external calibration was performed using
- Supporting Information Table S1, and primer sequences were trypsinization (at protein and peptide levels) at reactive free amines of
- cross-linked with 1% formaldehyde for 10 min, neutralized with Details of the LC gradient (Buffer A (0.1% FA in H2O); Buffer B
- glycine for 5 min. After washing twice, fixed cells were scraped into (ACN with 0.08% v/v FA)) and MS parameters are summarized in
- ice-cold PBS with protease inhibitor cocktail (PIC) and pelleted by Supporting Information Tables S4 and S5, respectively.
- Ultrasonics, Danbury, USA) at 10% energy input with 1 s pulses (on/ (standing for the sum of a methyl and a propionyl on the same lysine
- running an aliquot of the fragmented lysates on a 2% agarose gel. modifications. Mass tolerance on peptides and fragments was set to
- Sonicated lysates were diluted (1:5 ratio) in ChIP-Buffer with PIC, 5 and 20 ppm, respectively. Identification result DAT files were
- and a 2% input was aliquoted for each sample. Specific hPTM imported into Proline software33 for identification validation (Mascot
- Detection System (Bio-Rad, Feldkirchen, Germany) with Sso Fast The data was visualized as bar plots representing the mean of 3
- positive and negative controls (GAPDH, MB, MYOD1, and RPL30). interval at 0.95%. Data analysis and visualization were carried out
- Primer sequences are listed in Supporting Information Table S3. The using in-house written scripts (https://github.com/HijaziHassan/
- Histones were extracted using the Core Histone Isolation Kit (Sigma- samples were further sonicated using a probe sonicator at 10%
- nonfat milk in 0.1% TBS-T. Primary and secondary antibody were calculated relative to those of unexposed control cells.
- qPCR analyses. Statistical analyses were performed using one-way frequently exceed the WHO limit of 10 μg/L. In some areas,
- reports of our group,12,29 A549 cells were exposed to NaAsO2 15 μM range, no signs of cytotoxicity were observed. However,
- study, the cells were subsequently cultured in arsenite-free detected, though the levels remained above 80% of control at
- as a marker of cytotoxicity, were determined to identify approximately 60% in both setups. Thus, postincubation did
- and occupationally high exposure acute scenarios and was no changes (Figure 1C). The values remained near 100%, both
- decreased significantly. At 5 μM, levels were barely measurable. Information Table S2 and Figure S1.
- These correspond to a reduction to approximately 1.2% and and excluding postincubation, a trend toward effect intensifi-
- 0.9%, respectively. This pronounced decline indicates efficient cation was observed with extended postincubation time
- range, irrespective of postincubation (Figure 1E/F). recovery time. Furthermore, the number of relevant gene
- To analyze the time-dependent transcriptional response of changes of <1, corresponding to at least a 50% reduction
- Figure 3. Gene expression profiles of arsenite-treated and postincubated A549 cells. Cells were exposed to NaAsO2 in the dose range of 1 μM to 25
- H3K18ac at 20 μM and (D) at 5 μM and 10 μM. (E) Displays the ChIP-qPCR data of H3K9ac in the whole dose range. As positive and negative
- reduction to below 50% of their original transcript levels. Such chromatography coupled with tandem mass spectrometry
- 50% after recovery at the highest dose. The repression of TET3 activating marks (H3K4me3, H3K18ac, H3K9ac) and one
- was particularly detectable 48 h after treatment at the highest gene-repressive mark (H3K27me3). Our study expanded upon
- respective loading controls (Ponceau, histone H3, and β-Actin) and averaged. Shown are mean values ± SD from at least three independent
- (BER), MLH1 and MSH2 (MMR), XPA and XPC (NER), as 20% (5 μM) and 27% (10 μM). This pattern aligns with our
- investigated, indicating active transcription of these genes loci at the highest dose. Significant H3K9ac declines of 54%
- (Figure 4A). Acute exposure to 20 μM NaAsO2 resulted in no and 50% were observed at MPG and MLH1 promoters after
- MPG, MLH1, MSH2, and XPA (Figure 4C). In detail, in a notable increase in H3K4me3 abundance of 45%
- H3K18ac levels decreased by 33% at MPG, 29% at MLH1, compared to the control (Figure 5A). At lower concentrations
- 36% at MSH2, and 27% at XPA, respectively. Further (5 μM and 10 μM), no significant effects were detected,
- concentrations exhibited an upward trend, a significant 57%
Methods (brief)
- μM NaAsO2, reflecting low to high acute exposure scenarios, followed
- arsenite-induced cellular stress and after recovery, tracking the Logarithmically growing A549 cells were treated with NaAsO2 at low
- data. Moreover, we performed an antibody-based and unbiased with 2.5−10 μM NaAsO2. For postincubation studies, treated cells
- Logarithmically growing cells were treated with NaAsO2. For
- Penicillin−Streptomycin from Sigma-Aldrich (Burlington, MA, USA). collected in fresh medium, and cell numbers were measured using the
- from Applied Biosystems (Waltham, MA, USA), Bio-Rad (Munich, furnace-AAS (PinAAcle 900 T, PerkinElmer, Rodgau, Germany).
- reagents were obtained from Cell Signaling Technology (Leiden, For sample preparation, the cells were dissolved in 500 μL of 30%
- AAS elemental standard solutions to ensure accurate measurements.
- and logarithmically growing cells were exposed to NaAsO2 and Proteomic Analysis of H3 PTMs
- enabled simultaneous analysis of 96 samples for the expression of treated as stated before. Histones were acid-extracted and analyzed by
- ΔΔCq-method, with results expressed as log2-fold changes compared stopped by adding a few drops of pure TFA. The samples were then
- and a 2% input was aliquoted for each sample. Specific hPTM imported into Proline software33 for identification validation (Mascot
- ChIP control H3 antibody (10 μL) were added to the samples and free quantification by using the MS1 intensity at the chromatographic
- digestion were included (according to gene expression profiling RA =
- Histones were extracted using the Core Histone Isolation Kit (Sigma- samples were further sonicated using a probe sonicator at 10%
- Figure 1. Assessment of arsenite cytotoxicity, uptake, and impact on cell cycle progression in A549 cells. Cells were treated with NaAsO2 for 24 h,
- uptake of arsenic was quantified by AAS (D). Moreover, the cell cycle distribution was analyzed by flow cytometry using DAPI staining (E,F).
- significance of treated cells relative to the control. For AAS, ANOVA 200-fold above the WHO limit.36 To confirm that the selected
Implications
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Verification notes
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Update history
The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.