Journal: Journal of Cancer

Article Title: Clinicopathological Significance and Antitumor Effect of MPHOSPH1 in Testicular Germ Cell Tumor

doi: 10.7150/jca.25279

Figure Lengend Snippet: The efficacy of MPHOSPH1 knockdown using small-interfering RNA (siRNA). A . Protein was extracted from the cell lines at 24 h and 48 h post-transfection. Knockdown of MPHOSPH1 was confirmed by Western blotting ( A ). MPHOSPH1 knockdown significantly reduced cell migration ( B ) and cell invasion ( C ) in both NEC8 and NEC14 ( p <0.001 for both). MPHOSPH1 knockdown also significantly inhibited cell proliferation ( D ) and colony formation ( E ) in both of the cell lines ( p <0.001 for both).

Article Snippet: The human embryonal carcinoma cell lines NEC8 and NEC14 were purchased from the Riken Bioresource Center (Tsukuba, Japan) and cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS).

Techniques: Knockdown, Small Interfering RNA, Transfection, Western Blot, Migration

Journal: Journal of Translational Medicine

Article Title: Cold atmospheric plasma drives USP49/HDAC3 axis mediated ferroptosis as a novel therapeutic strategy in endometrial cancer via reinforcing lactylation dependent p53 expression

doi: 10.1186/s12967-025-06449-8

Figure Lengend Snippet: CAP impedes the proliferation, migration, and invasion of endometrial cancer cells in vitro. ( A , B ) CCK-8 assay detects cell viability in HEC-1B, Ishikawa, and EEC cells. ( C - F ) colony formation ( C , D ) and EdU assays ( E , F ) were conducted to assess the proliferative effects of CAP therapy during the specified duration (0s, 20s, 40s, and 60s) on HEC-1B and Ishikawa cells. ( G - L ) Wound-healing ( G, H ) and transwell ( I - L ) assay showed how CAP affects HEC-1B and Ishikawa cell invasion and migration. Data are shown as means ± standard deviation (SD). *: p < 0.05; **: p < 0.01; ***: p < 0.001

Article Snippet: Procell Life Science & Technology Company from Wuhan, China, supplied the HEC-1B and Ishikawa human endometrial carcinoma cell lines.

Techniques: Migration, In Vitro, CCK-8 Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Cold atmospheric plasma drives USP49/HDAC3 axis mediated ferroptosis as a novel therapeutic strategy in endometrial cancer via reinforcing lactylation dependent p53 expression

doi: 10.1186/s12967-025-06449-8

Figure Lengend Snippet: CAP facilitates ferroptosis signal pathway in endometrial cancer cells. ( A ) Significantly altered protein expression in HEC-1B cells is depicted by a volcano plot (blue is down-regulated, orange is down-regulated). ( B ) Heatmap illustrating distinct proteins following CAP treatment. ( C , D ) GO and KEGG analyses were conducted on the differential proteins obtained by proteomics. ( E ) GSEA revealed CAP treatment group enrichment of the ferroptosis signalling pathway. ( F ) A Venn diagram depicting the probable targets of ferroptosis related to CAP therapy alongside FerrDb, an extensive ferroptosis database. ( G , H ) Fluorescence images of JC-1 stained HEC-1B and Ishikawa cells following different treatments: control, CAP, RSL3, and CAP + RSL3. Red JC-1 aggregates show normal mitochondrial membrane potentials, while green JC-1 monomers indicate depolarized membrane potentials. The fluorescence images were used to determine JC-1’s red/green fluorescence ratios. ( n = 3). ( I ) The MDA test was used to measure the lipid peroxidation levels of HEC-1B and Ishikawa cells treated with CAP. ( J , K ) lipid ROS was assessed by BODIPY-C11 staining and statistical analysis. Oxidized (red fluorescence, Ex = 540–580 nm, Em = 600–660 nm) and non-oxidized (green fluorescence, Ex = 450–490 nm, Em = 500–550 nm). The ratio of red (oxidized) to green (non-oxidized) fluorescence determines the degree of lipid peroxidation. ( L , M ) Fluorescence images of JC-1 stained HEC-1B and Ishikawa cells following different treatments: control, CAP, and CAP + Fer-1. Data are shown as means ± standard deviation (SD). *: p < 0.05; **: p < 0.01; ***: p < 0.001

Article Snippet: Procell Life Science & Technology Company from Wuhan, China, supplied the HEC-1B and Ishikawa human endometrial carcinoma cell lines.

Techniques: Expressing, Fluorescence, Staining, Control, Membrane, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Cold atmospheric plasma drives USP49/HDAC3 axis mediated ferroptosis as a novel therapeutic strategy in endometrial cancer via reinforcing lactylation dependent p53 expression

doi: 10.1186/s12967-025-06449-8

Figure Lengend Snippet: The deubiquitinase USP49 interacts with HDAC3 and enhances its stability. ( A ) Mass spectrometry revealed that HDAC3 was linked to USP49. ( B ) CAP-treated HEC-1B cells were used for Co-IP experiments. ( C ) Co-localization of HDAC3 and USP49 in CAP-treated HEC-1B nuclei was observed by laser confocal microscopy (scale bar: 10 μm). ( D ) Molecular docking analysis of binding between HDAC3 and USP49. ( E ) USP49 mRNA was measured by RT-qPCR after CAP treatment. ( F ) Protein expression levels of USP49 detected by Western blot after CAP treatment. ( G , H ) HDAC3 mRNA and protein levels were determined in HEC-1B and Ishikawa cell lines transfected with USP49-shRNA or overexpressing USP49. ( I , J ) The connection between USP49 mRNA and HDAC3 mRNA in RNA-seq study using the GEO database GSE115810 ( I ) and GSE56026 ( J ). ( K , L ) Degradation of the HDAC3 protein was measured after 100 µg/ml CHX treatment in Ishikawa cell. ( M , N ) Western blot analysis was conducted to assess HDAC3 expression following 12-hour treatment with 10 or 20 µM MG132 in Ishikawa stable cell lines, with data presented as a fold-change compared to the control. ( O ) HDAC3 ubiquitination was examined by immunoprecipitation using an anti-Flag antibody, then detected with anti-HA and anti-Flag antibodies through immunoblotting. This procedure was conducted in HEC-1B cells that had been transfected with the specified constructs. Data are shown as means ± standard deviation (SD). *: p < 0.05; **: p < 0.01; ***: p < 0.001

Article Snippet: Procell Life Science & Technology Company from Wuhan, China, supplied the HEC-1B and Ishikawa human endometrial carcinoma cell lines.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Confocal Microscopy, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection, shRNA, RNA Sequencing, Stable Transfection, Control, Ubiquitin Proteomics, Immunoprecipitation, Construct, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Cold atmospheric plasma drives USP49/HDAC3 axis mediated ferroptosis as a novel therapeutic strategy in endometrial cancer via reinforcing lactylation dependent p53 expression

doi: 10.1186/s12967-025-06449-8

Figure Lengend Snippet: Induction of p53 expression by CAP involves lactylation of histone H3K18 by HDAC3. ( A , B ) Western blot assay was performed using pan-lactylation and pan-acetylation antibodies ( A ) and acetylation ( B ) levels in HEC-1B and Ishikawa cells after CAP treatment (0s, 20s, 40s, and 60s). ( C ) Western blot was used to detect the levels of H3K18 lactylation and acetylation at the indicated times. ( D , E ) Western blot ( D ) and RT-qPCR ( E ) were used to measure p53 expression levels at the indicated times. ( F - H ) ChIP-qPCR was conducted to detect the enrichment of H3K18la on the p53 promoter region. ( I - K ) The expression of H3K18la and p53 were assessed via western blot and RT-qPCR analysis following transfection with the specified plasmids. ( L ) H3K18la and p53 protein levels were assessed using Western blot assays after CAP treatment or HDAC3 overexpression. ( M ) p53 mRNA levels were measured by RT-qPCR following CAP treatment or HDAC3 overexpression. ( N ) Western blot analysis was performed to examine the expression of USP49, HDAC3, H3K18la, and p53 in tumor tissues from endometrial cancer. Data are shown as means ± standard deviation (SD). *: p < 0.05; **;: p < 0.01; ***: p < 0.001

Article Snippet: Procell Life Science & Technology Company from Wuhan, China, supplied the HEC-1B and Ishikawa human endometrial carcinoma cell lines.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Transfection, Over Expression, Standard Deviation