Journal: bioRxiv

Article Title: Loss of EIF4G2 Mediates Aggressiveness in Distinct Human Endometrial Cancer Subpopulations with Poorer Survival Outcome in Patients

doi: 10.1101/2023.09.14.557672

Figure Lengend Snippet: A-C Flow cytometry analysis of cell-surface expression of CD133 (A) and CD44 (B, C) in control and EIF4G2 KD HEC-1A cells ( A, B ) and control and EIF4G2 KD RL95-2 cells ( C ). Quantification of results is presented as mean±SEM, n>5 for both overall mean florescent intensity (MFI) and as percent of total population. P -vals were calculated using two-tailed t-test (*: p <0.05, **: p <0.01, ****: p <0.0001). A representative flow cytometry plot is shown for each. D Control and EIF4G2 KD HEC-1A and RL95-2 cells were separated for CD133 and CD44 expression, respectively. Separated populations were treated with 2.5 nM Taxol or DMSO as control for 4 d and cell viability assayed by CellTiter-Glo. Luminescence was normalized to DMSO treated cells. Left graph, HEC-1A cells, right graph RL95-2 cells. E Cell viability of HEC-1A (left) and RL95-2 (right) CD133+ and CD133-control and EIF4G2 KD separated populations 4 d after exposure to 16Gy (HEC-1A) or 8Gy (RL95-2) X-ray irradiation. For ( D, E ), results are presented as mean values±SEM of n>4 independent experiments. Significance was determined by two-way ANOVA. (*: p <0.05, **: p <0.01, ***: p <0.001, ****: p <0.0001). F, G Total cell lysates form HEC-1A (F) and RL95-2 (G) CD133+ and CD133-control and EIF4G2 KD separated populations were subjected to western blot analysis for γH2AX and GAPDH, as loading control 4 d following irradiation. γH2AX signal was normalized to GAPDH and quantification results are presented as individual data points and also as mean values of 4 independent experiments. Statistical significance was determined for log 2 transformed mean values by two-way ANOVA (*: p <0.05).

Article Snippet: Separations were performed on Control and EIF4G2KD HEC-1A cells using magnetic beads for CD133 (CD133 Microbead Kit, Miltenyi Biotec, 130-097-049), or CD44 (CD44 Microbeads Kit, Miltenyi Biotec, 130-095-194) for RL95-2 cells, on LS-columns (Miltenyi Biotec, 130-042-401) according to the manufacturer’s recommendations.

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test, Irradiation, Western Blot, Transformation Assay

Journal: bioRxiv

Article Title: Loss of EIF4G2 Mediates Aggressiveness in Distinct Human Endometrial Cancer Subpopulations with Poorer Survival Outcome in Patients

doi: 10.1101/2023.09.14.557672

Figure Lengend Snippet: Control and EIF4G2 KD HEC-1A cells were FACS sorted by CD133 marker expression and subjected to RNA-seq and MS analysis. A Heat map showing hierarchical clustering of gene expression levels of all DEGs identified following RNA-seq analysis of CD133- and CD133+ sorted control and EIF4G2 KD populations. B Heat map showing hierarchical clustering of all proteins with significantly changed abundance in MS analysis of CD133- and CD133+ sorted control and EIF4G2 KD HEC-1A cells. C Venn diagram showing overlap of all differentially abundant proteins among the four comparisons. Numbers at the edges of the diagram represent the overall number of proteins with differential abundance in each comparison. D Volcano plot of the Log 2 (Fold Ratio) of the abundance of the detected proteins in CD133+ EIF4G2KD / CD133+ Control comparison, vs. their significance expressed as log 10 p -value. Proteins with significant increased abundance are indicated in orange, and decreased abundance in purple. E Total cell lysates from separated control and EIF4G2 KD HEC-1A populations were subjected to western blot analysis for ALDH1A1 and GAPDH as loading control. Shown is a representative blot of 3 independent experiments. F High scoring significant pathways identified by GeneAnalytics pathway analysis of the set of proteins with increased abundance in the CD133+ EIF4G2KD / CD133+ Control comparison. Score numbers on X-axis indicate significance, numbers at right represent the number of proteins identified in the dataset out of the total number of proteins within the given pathway. G Protein expression of KLC1, KLC2 and KIF5 based on the abundance detected by the MS analysis. Data presented as mean of Log 10 (Intensity), n=4. Statistical significance was determined by two-way ANOVA (*: p <0.05, ***: p <0.001, ****: p <0.0001). Representative western blots of HEC-1A separated CD133- and CD133+ control and EIF4G2 KD cells validating the MS results for KLC1, KIF5B, and KLC2. GAPDH was used as a loading control. Shown is one representative blot of n=3.

Article Snippet: Separations were performed on Control and EIF4G2KD HEC-1A cells using magnetic beads for CD133 (CD133 Microbead Kit, Miltenyi Biotec, 130-097-049), or CD44 (CD44 Microbeads Kit, Miltenyi Biotec, 130-095-194) for RL95-2 cells, on LS-columns (Miltenyi Biotec, 130-042-401) according to the manufacturer’s recommendations.

Techniques: Control, Marker, Expressing, RNA Sequencing, Gene Expression, Comparison, Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-195 inhibits epithelial-mesenchymal transition by targeting G protein-coupled estrogen receptor 1 in endometrial carcinoma

doi: 10.3892/mmr.2019.10652

Figure Lengend Snippet: Overexpression of miR-195 exerts an inhibitory effect on AN3-CA and Hec1A cell viability. To examine the effects of miR-195 on EC development, the miR-195 mimics vector was transfected into human EC (AN3-CA and Hec1A) cells. (A and C) The transfection efficiency was determined by RT-qPCR. (B and D) Cell viabilities at 12, 24 and 48 h of each experimental group were detected using the CCK-8 kit. Each value represents the mean ± SEM (n=3). U6 was considered as an internal control. a P<0.05, aa P<0.01 vs. Control group; b P<0.05, bb P<0.01 vs. Mock group. EC, endometrial carcinoma.

Article Snippet: Human endometrial cancer cell lines (AN3-CA and Hec1A) were obtained from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, CCK-8 Assay, Control

Journal: Scientific Reports

Article Title: Integrative network toxicology and experimental evidence reveal mechanisms underlying diethyl phthalate-induced initiation and progression of endometrial cancer

doi: 10.1038/s41598-026-39325-6

Figure Lengend Snippet: DEP inhibited proliferation and induced ROS accumulation in EC cells. ( A , B ) CCK-8 assay showing the effect of different concentrations of DEP on proliferation of HEC-1-A ( A ) and Ishikawa ( B ) cells at 0, 24, and 48 h (n = 3). ( C , D ) Representative EdU staining images of HEC-1-A ( C ) and Ishikawa ( D ) cells treated with DEP at the indicated concentrations (DMSO, DEP at 10 nM, 1 μM, and 10 μM). Nuclei were counterstained with Hoechst 33,342 (blue), and EdU-positive cells are shown in red. Scale bar = 100 μm. ( E ) Quantification of EdU-positive cells in HEC-1-A and Ishikawa cell lines (n = 3). ( F ) Representative fluorescence images of ROS staining in HEC-1-A and Ishikawa cells following DEP treatment. White arrows indicate cells with increased ROS levels, and blue arrows indicate cells with lower ROS levels (n = 3). Scale bar = 20 μm. Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. (* P < 0.05, *** P < 0.001, **** P < 0.0001).

Article Snippet: The human EC cell lines HEC-1-A and Ishikawa were obtained from GeneChem Gene Technology Co., Ltd. (Shanghai, China).

Techniques: CCK-8 Assay, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Integrative network toxicology and experimental evidence reveal mechanisms underlying diethyl phthalate-induced initiation and progression of endometrial cancer

doi: 10.1038/s41598-026-39325-6

Figure Lengend Snippet: DEP induced cell cycle arrest and modulated the PI3K/AKT signaling pathway in EC cells. ( A ) Flow cytometry analysis of cell cycle distribution in Ishikawa and HEC-1-A cells treated with DEP at the indicated concentrations (DMSO, DEP at 10 nM, 1 μM, and 10 μM). ( B ) Quantification of the proportion of cells in G0/G1, S, and G2/M phases (n = 3). ( C ) WB analysis of PI3K, P-PI3K, AKT, P-AKT, ERK, P-ERK, CDK4, Cyclin D1, GSK3β, and P-GSK3β expression in Ishikawa and HEC-1-A cells after DEP treatment. ( D ) Densitometric quantification of protein expression levels normalized to GAPDH or β-actin (n = 3). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The human EC cell lines HEC-1-A and Ishikawa were obtained from GeneChem Gene Technology Co., Ltd. (Shanghai, China).

Techniques: Flow Cytometry, Expressing