Journal: Translational Cancer Research

Article Title: Quetiapine inhibits the oxidative phosphorylation in head and neck squamous cell carcinoma through suppressing NAT10-mediated ac4C modification

doi: 10.21037/tcr-2025-1-2683

Figure Lengend Snippet: Quetiapine could regulate NAT10-mediated ac4C modification in HNSCC. (A) The mainly potential drugs identified by L1000FWD database. (B) The chemical structure of quetiapine. (C) Molecular docking of NAT10 and quetiapine. (D) The binding sensorgram of the interactions between NAT10 and quetiapine. (E) The protein expression of NAT10 was detected through western blotting (left) and quantitatively analyzed (right); n=3. From left to right, P=0.001, <0.001, and 0.002, respectively. (F) The mRNA expression of NAT10 in HNSCC cells; n=3. From left to right, P=0.03, 0.002, and 0.08, respectively. (G) The protein expression of NAT10 after SCC-15 cells were treated with quetiapine was detected through western blotting (left) and quantitatively analyzed (right); n=3. P<0.001. (H) The mRNA expression of NAT10 after SCC-15 cells were treated with quetiapine; n=3. P<0.001. (I) Dot blot assay was conducted to assess the ac4C level. P<0.001. Data are presented as mean ± SD. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001 by unpaired t -test. EGFR, epidermal growth factor receptor; HDAC, histone deacetylase; HNSCC, head and neck squamous cell carcinoma; MEK, methyl ethyl ketone; MOA, mechanism of action; mRNA, messenger RNA; NF-κB, nuclear factor kappa-B; PARP, poly ADP-ribose polymerase; PLK, polo-like kinase; RAF, Raf kinase; RU, response unit; SD, standard deviation.

Article Snippet: The human oral keratinocytes (HOK) and three HNSCC cell lines, namely WSU-HN6, SCC-15 and CAL-27, were acquired from the American Type Culture Collection.

Techniques: Modification, Binding Assay, Expressing, Western Blot, Dot Blot, Histone Deacetylase Assay, Standard Deviation

Journal: Translational Cancer Research

Article Title: Quetiapine inhibits the oxidative phosphorylation in head and neck squamous cell carcinoma through suppressing NAT10-mediated ac4C modification

doi: 10.21037/tcr-2025-1-2683

Figure Lengend Snippet: Quetiapine treatment inhibited HNSCC malignant phenotypes in vitro . (A) CCK-8 assay showed that quetiapine treatment inhibited the proliferation of SCC-15 cells; n=5. P<0.001. (B) Representative images of SCC-15 clones treated with quetiapine using crystal violet solution staining. (C) The quantitation of colony number for SCC-15 cells treated with quetiapine; n=3. P=0.001. (D,E) Apoptosis assay results of SCC-15 cells treated with quetiapine; n=3. P=0.02. (F) Representative imaged of SCC-15 transwell migration treated with quetiapine using crystal violet solution staining. (G) Quantified of transwell migration in SCC-15 cells treated with quetiapine; n=3. P=0.003. (H,I) Wound healing assays showed the treatment of quetiapine inhibited migration of SCC-15 cells. The migration distance was quantified at 24 hours after scratching; n=3. P<0.001. *, P<0.05; **, P<0.01; ***, P<0.001 by unpaired t -test. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; HNSCC, head and neck squamous cell carcinoma; PI, propidium iodide.

Article Snippet: The human oral keratinocytes (HOK) and three HNSCC cell lines, namely WSU-HN6, SCC-15 and CAL-27, were acquired from the American Type Culture Collection.

Techniques: In Vitro, CCK-8 Assay, Clone Assay, Staining, Quantitation Assay, Apoptosis Assay, Migration, Cell Counting

Journal: Translational Cancer Research

Article Title: Quetiapine inhibits the oxidative phosphorylation in head and neck squamous cell carcinoma through suppressing NAT10-mediated ac4C modification

doi: 10.21037/tcr-2025-1-2683

Figure Lengend Snippet: Quetiapine treatment inhibited the process of OXPHOS. (A) The mRNA expression of OXPHOS related genes after SCC-15 cells were treated with quetiapine; n=3. From left to right, P values <0.001, <0.001, <0.001, 0.02, <0.001, 0.003, 0.004, <0.001, and <0.001, respectively. (B) The protein expression of OXPHOS related proteins after SCC-15 cells were treated with quetiapine were detected through western blotting (left) and quantitatively analyzed (right); n=3. From left to right, P=0.002, <0.001, <0.001, 0.01, and 0.003, respectively. (C) OCR, which reflects mitochondrial respiration, was decreased in quetiapine treated SCC-15 cells. (D) Statistical chart of OCR various parameters about SCC-15 cells after treated with quetiapine; n=3. From left to right, P values <0.001, 0.002, 0.64, <0.001, and 0.07, respectively. ns, no significance; *, P<0.05, **, P<0.01; ***, P<0.005 by unpaired t -test. mRNA, messenger RNA; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation.

Article Snippet: The human oral keratinocytes (HOK) and three HNSCC cell lines, namely WSU-HN6, SCC-15 and CAL-27, were acquired from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: TNFAIP8 variants are differentially expressed in cancer cells. (a) A heat map showing significant changes in variant 1 (v1) and variant 2 (v2) expression levels in tumors compared with normal tissue of the same origin. *P<0.05; #P<0.01. Patient-level tumor/normal tissue pairings (same patient) plus unpaired (different patients) samples were analyzed to maximize statistical power. Intrapatient paired analysis alone yielded similar results, except that v1 expression was not significantly different in breast carcinoma and lung adenocarcinoma. (b) Relative levels of TNFAIP8 v1 and v2 mRNA in the indicated cell lines/types normalized to β-actin. THP-1, acute monocytic leukemia; Raji, B-cell lymphoma; ‘190' and ‘191' are primary human macrophages from two donors; HSF, human skin fibroblast. (c) Compared with v2, v1 includes additional 11 amino acids. Immunoblot of TNFAIP8 and β-actin (control) was performed in the indicated cell types. ‘MAC' refers to primary human macrophages. A549 cells were transduced with either TNFAIP8 shRNA (TP8i) or scrambled control shRNA (scri). Results are representative of three or more independent experiments

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Variant Assay, Expressing, Western Blot, Control, Transduction, shRNA

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: p53 regulates DOX-induced TNFAIP8 v2. (a) v2 and p21 mRNA expression in A459 cells after nutlin-3 or DMSO (vehicle) treatment (24 h). (b) v2 and p21 expression after DOX treatment in A549 cells stably expressing scramble shRNA (‘scri') or p53 shRNA (‘p53i'). Fold change is displayed as the ratio of DOX-treated over nontreated (NT) cells. (c) Immunoblotting of p53 and actin (loading control) in parental (‘−'), scri, and p53i A549 cells following no treatment (NT) or DOX. (d) DOX-induced v2 mRNA expression in U2OS cells stably expressing scri or p53i. (e) v2 and p53 protein levels are shown in untreated and DOX-treated U2OS cells stably expressing scramble control shRNA (‘cont') or two different p53-directed shRNAs (‘p53i-55' and ‘p53i-56'). (f) v2 and p21 mRNA expression in untreated or DOX-treated p53-proficient and p53-null HCT116 cells. (g) v2 and p21 mRNA expression was measured with Nanostring technology in indicated cell types, untreated (‘NT') or treated with DOX for durations shown. Results are representative of three or more independent experiments. *P<0.05

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Expressing, Stable Transfection, shRNA, Western Blot, Control

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: p53 binds to an intragenic enhancer region in TNFAIP8. (a) ChIP-seq analysis in U2OS cells untreated or treated with DOX shows p53 enrichment in the v2 intronic region of TNFAIP8. v1 and v2 genomic locations are shown below (exons depicted as vertical ticks). The p53 binding peak is 50 kb downstream and 30 kb upstream from the v2 and v1 transcriptional start sites, respectively. ‘p53 Scan' software analysis reveals a predicted functional p53 response element (RE), as indicated. Lowercase letter indicates mismatches compared with p53RE consensus (RRRCWWGYYYnRRRCWWGYYY).16 (b) Validation of p53 binding to v2 intronic region was tested by p53 ChIP-PCR (or IgG control) in untreated and DOX-treated U2OS cells (depicted as % of total input DNA). p53 binding to the p21 promoter is shown as positive control. (c) Luciferase activity was measured in untreated or DOX-treated U2OS cells stably expressing scramble (‘scri') or p53-directed shRNA (‘p53i') and transfected with firefly luciferase reporter constructs without (‘empty') or with a 600 bp region encompassing the intronic p53RE region. ‘p53+' is a p53RE-driven luciferase construct (positive control). (d) H3K4me1, H3K27ac, and H3K4me3 ChIP-PCR is shown for the intronic p53RE region (‘v2 p53RE') and the v2 promoter (‘V2P') in untreated and DOX-treated U2OS cells (‘Ab' (antibody)). (e) Schematic showing the 6 EcoRI sites that were used for a three-dimensional chromosome looping assay, as they relate to the v2 promoter region and the intronic p53RE. PCR was performed using the indicated primer pairs in untreated U2OS cells. Results are representative of three or more independent experiments. *P<0.05

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: ChIP-sequencing, Binding Assay, Software, Functional Assay, Biomarker Discovery, Control, Positive Control, Luciferase, Activity Assay, Stable Transfection, Expressing, shRNA, Transfection, Construct

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: TNFAIP8 v2 depletion induces p53 binding and p53 target expression. (a) Immunoblot of v2 and actin (control) in whole-cell lysates of A549 cells treated with TNFAIP8 (TP8i) or scrambled (scri) shRNA. (b) Immunoblot of p53 from nuclear and whole-cell extracts of cells under indicated treatments. (c) Immunoblot of acetylated p53-K382 (‘p53-ac') and total p53 in chromatin-bound nuclear extracts of p53+ or p53-silenced A549 cells treated with scri or TP8i. Histone 3 (‘H3') and actin are loading controls. (d) p53 ChIP-PCR of promoter regions of indicated target genes in TP8i and scri cells. IgG serves as negative control. (e and f) mRNA expression of p21 and Gadd45a (RT-PCR) (e) and FAS and 14-3-3σ (Nanostring) (f) was measured in p53+ or p53-silenced A549 cells treated with scri or TP8i. (g) v2 mRNA expression (normalized to GusB) and (h) p53 binding to the indicated target genes was measured with RT-PCR and ChIP-PCR, respectively, in A549 cells transiently transfected with an empty vector (‘empty') or a v2 expression vector (‘v2'). Results are representative of three or more independent experiments. *P<0.05

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Binding Assay, Expressing, Western Blot, Control, shRNA, Negative Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: TNFAIP8 v2 depletion stalls DNA replication and induces p53-dependent cell cycle arrest. (a) TNFAIP8 v2 mRNA levels were quantified in p53+ and p53-silenced (p53i) A549 cells untransduced (‘−') or transduced with scramble (‘scri') or TNFAIP8-directed (‘TP8i') shRNA. At right, an immunoblot of v2 under the same conditions is shown. (b) 5-Bromo-2'-deoxyuridine (BrdU) incorporation and 7AAD staining were quantified using flow cytometry in p53+ and p53i A549 cells transduced with scri or TP8i shRNA. BrdU+ cells (S phase), yellow; G1, green; G2, purple. (c) Quantification of BrdU intensity of cells in (b). (d) Cell cycle profile based on 7AAD staining of cells in (a). G2-phase cells (purple) are defined as cells with double the nuclear 7AAD stain intensity compared with G1-phase cells (green). S-phase cells are BrdU+ (yellow). (e and f) mRNA levels of PCNA (RT-PCR) (e) and cyclins D1, E1, and E2 (Nanostring) (f) of cells under indicated treatments. (g) p21 quantified by RT-PCR and immunoblot in p53+ A549 cells transduced with either scri or TP8i shRNA, followed by transfection of scramble (‘scri') or p21 (‘p21i') siRNA. (h) Cell cycle analysis of cells described in (g). Results are representative of three or more independent experiments. *P<0.05

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Transduction, shRNA, Western Blot, BrdU Incorporation Assay, Staining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Cycle Assay

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: TNFAIP8 v2 depletion enhances damage-induced apoptosis. (a) Annexin V and propidium iodide (PI) staining were quantified by flow cytometry in p53+ and p53i A549 cells transduced with a scramble (‘Scri') or TNFAIP8-directed (‘TP8i') shRNA and untreated or treated with DOX. (b and c) The percentage of Annexin V+ cells was quantified in untreated and DOX-treated cells (b), and in DMSO-treated cells (vehicle control) and staurosporine (‘Stauro')-treated cells (c). (d) The fold change in caspase 3/7 activity is shown in the cells described in (b and c) after DOX or Stauro treatment. DOX and Stauro values are normalized to untreated or DMSO-treated values, respectively. Results are representative of three or more independent experiments. *P<0.05

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Staining, Flow Cytometry, Transduction, shRNA, Control, Activity Assay

Journal: Cell Death and Differentiation

Article Title: The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

doi: 10.1038/cdd.2016.130

Figure Lengend Snippet: Proposed scheme of interactions of TNFAIP8 v2 with p53 in cancer. Under basal conditions in A549 cancer cells, v2 promotes DNA replication, possibly by maintaining PCNA levels, and inhibits cell cycle arrest through restraining p53-dependent expression of p53 target genes including p21. Failed downregulation of cyclins may also contribute to S-phase stalling. In response to DNA damage (e.g., DOX), p53 promotes v2 induction. v2, in turn, suppresses p53-dependent pro-apoptotic responses. In this manner, basal overexpression of TNFAIP8 v2 may promote tumorigenesis (by facilitating DNA replication and preventing cell cycle arrest), whereas p53-induced v2 in the setting of DNA-damaging agents may also promote resistance to cancer therapy (by suppressing apoptosis)

Article Snippet: For v2 overexpression, empty (pCMV-entry, Origene, Rockville, MD, USA) or v2 expression vector (RC220669, Origene) were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA; 24 h).

Techniques: Expressing, Over Expression