Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: Chou-Talalay synergy analysis for NPB (N) in combination with three PARP inhibitors, namely Olaparib (O), Rucaparib (R) or Talazoparib (T), in a panel of EC cells including KLE ( PTEN- proficient), AN3CA ( PTEN- deficient), Ishikawa ( PTEN- deficient) and RL95-2 ( PTEN- deficient). Cells were treated with the indicated concentration (log 10 scale) of NPB and mentioned PARP inhibitor for 6 days. The survival fraction was assessed using a total cell number assay. The logarithmic combination index (CI) value corresponding to cell fraction affected (Fa) was determined using the CompuSyn software ( http://www.combosyn.com ) as described in “Materials and methods”. CI value indicates: <1 synergism; =1 additive Synergy; >1 antagonism ( n = 3). Dose-response curves for a panel of cells treated with the indicated concentration of PARP inhibitors with or without 1 µM NPB in total cell number assays. Arrow indicates fold reduction in respective PARP inhibitor IC 50 in the presence of NPB ( n = 3).

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Concentration Assay, Software

Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: A EC cells, KLE, AN3CA, Ishikawa, and RL95-2 were incubated with the indicated drug concentrations of NPB (N) and Olaparib (O), Rucaparib (R) or Talazoparib (T) or combinations thereof for foci formation assays and stained with 0.2% crystal violet ( n = 3). B Microscopic visualization of calcein-AM (green) stained spheroids (live) and BOBO-3 Iodide (red) stained cell debris (dead) generated by EC cells (KLE, Ishikawa, RL95-2, and AN3CA) cultured in 3D Matrigel after exposure to NPB (N) and PARP inhibitors (Olaparib = O, Rucaparib = R, Talazoparib = T) and combinations thereof ( n = 3). Red-fluorescence (staining of ethidium homodimer-1 indicating loss of plasma membrane integrity) and decreased green-fluorescence (staining of calcein-AM to indicate intercellular esterase activity). Scale bars, 100 µm. Right: Cell fraction (SF) was evaluated using AlamarBlue® in EC cells treated with NPB (N), PARP inhibitors (Olaparib = O, Rucaparib = R, Talazoparib = T) and in combination with the indicated drug concentrations for 14 days in 3D Matrigel. ( n = 3). The white color arrow highlights the apoptotic cell death. Columns are mean of triplicate experiments; bars, ±SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Incubation, Staining, Generated, Cell Culture, Fluorescence, Clinical Proteomics, Membrane, Activity Assay

Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: A Western blot analysis was used to assess the expression of PTEN and downstream effectors PI3K, AKT, BAD, and CHK1 protein; and levels of pAKT, pBADS99, and pCHK1 were determined in KLE- WT, and PTEN-KO cells. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted as described in materials and methods. β-ACTIN was used as input control for cell lysates. The sizes of detected protein bands in kDa are shown on the left side Densitometries of protein bands were subsequently determined using ImageJ software ( https://imagej.nih.gov/ij/ ). B Dose-response curves for a panel of KLE-WT and KLE-PTEN-KO cells treated with the indicated concentration of Olaparib with or without a constant concentration of NPB (1μM) in total cell number assays. Arrow indicates fold reduction in respective PARP inhibitor IC 50 in the presence of NPB ( n = 3). C KLE-WT and KLE-PTEN-KO cells were incubated with the indicated concentrations of NPB (N), Olaparib (O), or a combination thereof for foci formation and stained with 0.2% crystal violet ( n = 3). D Western blot analysis was used to assess the expression of BAD, CHK1, cle-CASP, and cle-CASP7 protein and the level of pBADS99 in KLE-WT and KLE-PTEN-KO cells after treatment with NPB (N), Olaparib (O) or combination thereof. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted as described in materials and methods. β-ACTIN (ACTB) was used as input control for cell lysate. The sizes of detected protein bands in kDa are shown on the left side. E Representative immunofluorescent images of RAD51 in KLE-WT and KLE-PTEN-KO cells. Cells were treated with NPB (N), Olaparib (O), or a combination (N + O). Scale bars, 50 µm. Below: Quantification of RAD51 foci positive AN3CA cells treated with NPB (N), Olaparib (O), or a combination (N + O) are shown. Cells with more than 5 RAD51 foci per nucleus were counted as RAD51 positive cells. 100 cells were analyzed in 2–3 separate fields for each sample ( n = 3). Representative EC cells with positive RAD51 foci are highlighted using the white color arrow. Columns and points are the mean of triplicate determinations; bars, ±SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Western Blot, Expressing, SDS Page, Control, Software, Concentration Assay, Incubation, Staining

Journal: Frontiers in Immunology

Article Title: Pan-cancer multi-omics characterization of calcyphosine and its revealed links to the immune microenvironment and regulatory networks in endometrial carcinoma

doi: 10.3389/fimmu.2025.1688606

Figure Lengend Snippet: CAPS expression in EC and its impact on cell proliferation. (A) Western blot analysis of CAPS protein levels in EC tissues and paired adjacent non-tumor tissues. (B) Quantification of CAPS protein expression based on Western blot results. (C) Representative immunohistochemical (IHC) staining images of CAPS in EC and adjacent tissues at 20× and 40× magnification. (D) Statistical analysis of IHC scores (n = 12 per group). (E, F) CCK-8 assays evaluating cell viability in KLE and Ishikawa cells following CAPS knockdown. (G, H) EdU staining assays assessing proliferative capacity after CAPS knockdown in KLE and Ishikawa cells. (I, J) Quantification of EdU-positive cells. (K) CAPS knockdown efficiency validated by Western blot in Ishikawa and KLE cells. CAPS protein levels were markedly reduced after transfection with Si1-CAPS and Si3-CAPS compared with Si-NC. β-actin served as the loading control.(* P < 0.05, ** P < 0.01, **** P < 0.0001).

Article Snippet: The human EC cell lines KLE and Ishikawa were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Immunohistochemistry, CCK-8 Assay, Knockdown, Staining, Transfection, Control

Journal: Frontiers in Immunology

Article Title: Pan-cancer multi-omics characterization of calcyphosine and its revealed links to the immune microenvironment and regulatory networks in endometrial carcinoma

doi: 10.3389/fimmu.2025.1688606

Figure Lengend Snippet: CAPS knockdown promotes migration and invasion of EC cells. (A, B) Wound healing assays evaluating the effect of CAPS knockdown on wound closure in KLE and Ishikawa cells. (C, D) . Quantification of wound healing areas. (E, F) Transwell assays assessing the impact of CAPS knockdown on cell migration (E) and invasion (F) in KLE and Ishikawa cells. (G, H) Quantification of migrated (G) and invaded (H) cells in Transwell assays. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The human EC cell lines KLE and Ishikawa were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Migration

Journal: Discover Oncology

Article Title: PTBP1 functions as a suppressor of ferroptosis in endometrial carcinoma cells by stabilizing SLC7A11 mRNA

doi: 10.1007/s12672-025-04128-0

Figure Lengend Snippet: In vitro functional analysis of PTBP1 in endometrial cancer cells and in vivo validation using an Ishikawa xenograft model. A and B Transfection with shRNA knockdown vectors targeting PTBP1 significantly reduced both mRNA and protein levels in Ishikawa and KLE cell lines. C PTBP1 knockdown notably decreased cell viability in both Ishikawa and KLE cells. D – H Cell-derived xenograft (CDX) experiments in mice implanted with Ishikawa cells demonstrated that PTBP1 knockdown significantly reduced tumor volume and weight. Subsequent IHC staining of the xenograft tissues revealed a substantial decrease in Ki-67 protein levels and increase in 4-HNE level upon PTBP1 knockdown. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Ishikawa EC cells (#CL-0823, Procell, Wuhan, China) and KLE EC cells (#STCC10610P, Servicebio, Wuhan, China) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM , Procell) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin, in a 5% CO 2 atmosphere.

Techniques: In Vitro, Functional Assay, In Vivo, Biomarker Discovery, Transfection, shRNA, Knockdown, Derivative Assay, Immunohistochemistry

Journal: Discover Oncology

Article Title: PTBP1 functions as a suppressor of ferroptosis in endometrial carcinoma cells by stabilizing SLC7A11 mRNA

doi: 10.1007/s12672-025-04128-0

Figure Lengend Snippet: PTBP1 knockdown facilitates ferroptosis in endometrial cancer cells. A Measurement of ROS production using DCF-DA probes. B GSH, C MDA, and D Fe 2+ levels in Ishikawa and KLE EC cells transfected with PTBP1-shRNA (shPTBP1) or nontargeting control (shNC) vectors, measured by relevant assay kits. E– I Ishikawa and KLE EC cells transfected with shPTBP1 or shNC for 48 h were analyzed for mRNA expression of ACSL4 ( E ) and GPX4 ( F ), by RT-qPCR, as well as protein expression of ACSL4 ( G and H ) by Western blot, and GPX4 ( I ) by immunofluorescence. J and K The mRNA expression of SLC7A11 ( J ), as well as protein expression of SLC7A11, ACSL4, and GPX4 ( K ) in mice tumor tissues were determined by RT-qPCR and western blot assays. L The protein level of HSP27 in mice tumor tissues was tested by IHC. M Cell viability of Ishikawa and KLE cells transduced with shNC or shPTBP1 and treated with DMSO, Erastin (10 µM), or Erastin plus Ferrostatin-1 (Fer-1, 1 µM) for 24 h, measured by CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Ishikawa EC cells (#CL-0823, Procell, Wuhan, China) and KLE EC cells (#STCC10610P, Servicebio, Wuhan, China) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM , Procell) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin, in a 5% CO 2 atmosphere.

Techniques: Knockdown, Transfection, shRNA, Control, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Transduction, CCK-8 Assay

Journal: Discover Oncology

Article Title: PTBP1 functions as a suppressor of ferroptosis in endometrial carcinoma cells by stabilizing SLC7A11 mRNA

doi: 10.1007/s12672-025-04128-0

Figure Lengend Snippet: PTBP1 mediates SLC7A11 mRNA stability by binding to its 5’UTR. A Venn diagram displaying the overlap between differentially expressed genes (DEGs) (using criteria of p -adj < 0.05 and |log 2 FoldChange| >0.58) in PTBP1-KD Ishikawa EC cells versus shNC controls, ferroptosis-related genes, and predicted PTBP1 targets identified by the StarBase algorithm. B Transcriptomic analysis of TCGA revealed the mRNA expression levels of SLC7A11 and CBS in EC tissues compared to normal tissues. C Quantitative PCR analysis showing enrichment of SLC7A11 mRNA in anti-PTBP1 immunoprecipitates compared to anti-IgG controls. D Schematic representation of the SLC7A11 5’UTR, the predicted PTBP1 binding site (WT), and the mutated binding site (MUT). E Relative luciferase activity of WT or MUT reporters in shPTBP1- or shNC-transfected 293T cells. F DNA pull down-western blot showing PTBP1 enrichment in Bio-SLC7A11 5’UTR pull-downs compared to Bio-NC controls. G and H Quantification of SLC7A11 mRNA in shPTBP1- or shNC-transfected Ishikawa and KLE cells. I and J mRNA stability analysis using the actinomycin D exposure method, with RNA collected at 0, 1, 2, 3, and 4 h after exposure and quantified by PCR. K and L Measurement of SLC7A11 protein levels in PTBP1 KD and control Ishikawa and KLE cells. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. non-significant

Article Snippet: Ishikawa EC cells (#CL-0823, Procell, Wuhan, China) and KLE EC cells (#STCC10610P, Servicebio, Wuhan, China) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM , Procell) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin, in a 5% CO 2 atmosphere.

Techniques: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Western Blot, Control

Journal: Discover Oncology

Article Title: PTBP1 functions as a suppressor of ferroptosis in endometrial carcinoma cells by stabilizing SLC7A11 mRNA

doi: 10.1007/s12672-025-04128-0

Figure Lengend Snippet: PTBP1 disruption induces ferroptosis through downregulation of SLC7A11. A – F Western blot analysis of SLC7A11 protein ( A ), CCK8 assay for cell viability ( B ), ROS production measured using DCFH-DA probes and fluorescence analysis ( C ), GSH content ( D ), MDA expression ( E ), and Fe2 + levels ( F ) in Ishikawa and KLE EC cells transfected with shPTBP1, shPTBP1 + SLC7A11-ov, shPTBP1 + vec, or nontargeting control shNC. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Ishikawa EC cells (#CL-0823, Procell, Wuhan, China) and KLE EC cells (#STCC10610P, Servicebio, Wuhan, China) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM , Procell) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin, in a 5% CO 2 atmosphere.

Techniques: Disruption, Western Blot, CCK-8 Assay, Fluorescence, Expressing, Transfection, Control

Journal: Discover Oncology

Article Title: PTBP1 functions as a suppressor of ferroptosis in endometrial carcinoma cells by stabilizing SLC7A11 mRNA

doi: 10.1007/s12672-025-04128-0

Figure Lengend Snippet: Knockdown of SLC7A11 alone is sufficient to induce ferroptosis in endometrial cancer cells. A Western blot analysis validating the knockdown efficiency of SLC7A11 in Ishikawa and KLE cells transfected with shSLC7A11 or shNC. B Cell viability measured by CCK-8 assay in Ishikawa and KLE cells after SLC7A11 knockdown . C and D Intracellular ROS levels detected by DCFH-DA fluorescence in Ishikawa and KLE cells after SLC7A11 knockdown. E – G Assessment of key ferroptosis biochemical markers in Ishikawa and KLE cells after SLC7A11 knockdown, including E glutathione (GSH) levels,​ F ​ malondialdehyde (MDA) content, and G ferrous iron (Fe 2+ ) concentration. ** p < 0.01, *** p < 0.001

Article Snippet: Ishikawa EC cells (#CL-0823, Procell, Wuhan, China) and KLE EC cells (#STCC10610P, Servicebio, Wuhan, China) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM , Procell) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin, in a 5% CO 2 atmosphere.

Techniques: Knockdown, Western Blot, Transfection, CCK-8 Assay, Fluorescence, Concentration Assay