Journal: Integrative Medicine Research

Article Title: Frankincense ameliorates endometriosis via inducing apoptosis and reducing adhesion

doi: 10.1016/j.imr.2023.100947

Figure Lengend Snippet: Fr reduced human endometriotic cell proliferation. Fr was dissolved in DMSO and diluted in DMEM-F12 medium. Indicated concentrations of Fr was treated to human endometriotic epithelial (12Z) cells. After 24 h (A) and 48 h (B) , the cell viability was analyzed by measuring at a wavelength of 450 nm using MTT. Data shown as mean ± SEM of quintuplicated and are representative of three independent experiment. Statistical analysis was conducted using a Dunnet one-way ANOVA (*; p < 0.05, **; p < 0.01, ***; p < 0.001).

Article Snippet: Human pleural mesothelial Met-5A cells and immortalized human endometriotic epithelial 12Z cells were obtained from American Type Culture Collection (#CRL-9444; Manassas, VA) and Applied Biological Materials (#T0764; Richmond, Canada), respectively.

Techniques:

Journal: Integrative Medicine Research

Article Title: Frankincense ameliorates endometriosis via inducing apoptosis and reducing adhesion

doi: 10.1016/j.imr.2023.100947

Figure Lengend Snippet: Fr activates the apoptosis-death signaling pathway in endometriotic cells. For analysis of frequency of apoptotic or dead cell populations, 12Z cells were incubated with the increasing doses of Fr for 24 h, and then the percentage of Annexin + or PI + cells were measured at excitation (Ex) 494/emission (Em) 525 nm for Annexin V and Ex 535/Em 617 nm for PI (A, B) . Mitochondria-relative apoptosis molecules, Bax and Bcl-2 (C) and the cleavage forms of caspase −3 and −9, and PARP, main intracellular apoptosis signaling proteins (D) , were detected 24 hrs after Fr treatment. GAPDH was used as control, and the specific molecular weight is represented on the left.

Article Snippet: Human pleural mesothelial Met-5A cells and immortalized human endometriotic epithelial 12Z cells were obtained from American Type Culture Collection (#CRL-9444; Manassas, VA) and Applied Biological Materials (#T0764; Richmond, Canada), respectively.

Techniques: Incubation, Control, Molecular Weight

Journal: Integrative Medicine Research

Article Title: Frankincense ameliorates endometriosis via inducing apoptosis and reducing adhesion

doi: 10.1016/j.imr.2023.100947

Figure Lengend Snippet: Fr decreased the ability of endometrial cells to adhere to mesothelial cells. 12Z cells were stained with CMFDA after 24 h of Fr treatment and then gently transferred onto a red fluorescent Met-5A cell monolayer. After 90 min with gentle shaking at 20 rpm, unbound 12Z cells were removed, and cells were then visualized using a fluorescent microscope (200 × magnification) (A) . The number of 12Z cells bound to Met-5A cells was manually counted in four randomly chosen areas for statistical analysis (B) . Data are representative of one of five independent experiments and values are expressed in mean ± SEM. Statistical analysis was conducted using a Dunnet one-way ANOVA (***; p < 0.001).

Article Snippet: Human pleural mesothelial Met-5A cells and immortalized human endometriotic epithelial 12Z cells were obtained from American Type Culture Collection (#CRL-9444; Manassas, VA) and Applied Biological Materials (#T0764; Richmond, Canada), respectively.

Techniques: Staining, Gentle, Microscopy

Journal: Integrative Medicine Research

Article Title: Frankincense ameliorates endometriosis via inducing apoptosis and reducing adhesion

doi: 10.1016/j.imr.2023.100947

Figure Lengend Snippet: RNA-sequencing analysis suggests possible mode of action (MoA) underlying anti-endometriosis effect of Fr. The 12Z cells were treated with Fr (70 μg/ml) for 12 h. The RNA-sequencing dataset supplied by commercial service was applied to GSEA. The results from hallmark and KEGG analysis were visualized by bubble plot. The major pathways related with endometriosis was indicated by red (upregulated) or blue (downregulated) characters (A, B) . The data from GO analysis was described by Cytoscape with enrichment map visualization. The cutoff value was set as p > 0.001 and q > 0.5 (C) . The DEG analysis visualized as log2 fold change and -log10 p -value. Genes that were noticeably up- or down-regulated were denoted by red or blue characters, respectively. The genes participating in endometriosis-related pathways, as shown in the A and B panels, were indicated by a symbol for each gene (D) . Heatmap analysis was used to display the relative expression levels of genes connected to endometriosis-related pathways (E) . A graphical scheme was used to outline the potential mechanisms of action (MoA) underlying the relieving effects of Fr on endometriosis (F) .

Article Snippet: Human pleural mesothelial Met-5A cells and immortalized human endometriotic epithelial 12Z cells were obtained from American Type Culture Collection (#CRL-9444; Manassas, VA) and Applied Biological Materials (#T0764; Richmond, Canada), respectively.

Techniques: RNA Sequencing, Expressing

Journal: Cancer Research

Article Title: MEX3A Mediates p53 Degradation to Suppress Ferroptosis and Facilitate Ovarian Cancer Tumorigenesis

doi: 10.1158/0008-5472.CAN-22-1159

Figure Lengend Snippet: MEX3A promotes cell survival and tumorigenesis in ovarian cancer cells expressing WT p53. A, qRT-PCR analysis of MEX3A level in HOEC, IHEC, PA-1, and OCCC cells. RNA18S5 was used as an internal control. Three independent experiments were performed and data are means ± SD from one representative experiment ( n = 3). *, P < 0.05; ***, P < 0.001. n.s., not significant. Significant differences are based on unpaired t test. B, IB of MEX3A and p53 protein expression with TUBULIN as a loading control. Blots shown are from one representative experiment of three replicates. C, Cell growth assays using control (sh-Ctrl) or MEX3A-depleted (sh-MEX3A #1 or #2) PA-1 and TOV21G cells. Data are shown as mean ± SD with P value based on two-way ANOVA test ( n = 3). ***, P < 0.001. The experiments were repeated 3 times. D, Clonogenic assays using sh-Ctrl or sh-MEX3A PA-1 and TOV21G cells. Data are shown as mean ± SD with P value based on unpaired t test ( n = 3). **, P < 0.01. The experiments were repeated 3 times. E and F, Orthotopic xenograft models in NOD/SCID mice using sh-Ctrl or sh-MEX3A PA-1 cells. Five mice were used for each group. Data are means ± SD, significant difference is based on unpaired T-test of the tumor size 8 weeks after the intra bursa injection. G, Cell growth assays using control or MEX3A overexpressing (oe) OVISE and RMG-1 cells. Data are shown as mean ± SD with p value based on two-way ANOVA-test ( n = 3). ***, P < 0.001. The experiments were repeated 3 times. H, Clonogenic assays using control or MEX3A oe OVISE and RMG-1 cells. Data are shown as mean ± SD with p value based on unpaired t test ( n = 4). ***, P < 0.001. The experiments were repeated 3 times. I and J, Subcutaneous xenograft models in NOD/SCID mice using control ( n = 5) or MEX3A oe ( n = 4) RMG-1 cells. Photographs and 3D reconstruction from stereographic tumor images are shown. Data are means ± SD, significant difference is based on unpaired t test of the tumor size 3 weeks after the injection. K, Annexin V/ PI staining assays using sh-Ctrl or sh-MEX3A PA-1 and TOV21G cells. Data are shown as mean ± SD with P value based on unpaired t test ( n = 3). *, P < 0.05; **, P < 0.01. The experiments were repeated 3 times.

Article Snippet: Human primary ovarian surface epithelial cells (HOEC, T4198) and immortalized human endometriotic cell line (IHEC) (12Z, T0764) from Applied Biological Materials were grown on Type I collagen coated plates in Prigrow X series (TM4198) and Prigrow III (TM003) medium, respectively.

Techniques: Expressing, Quantitative RT-PCR, Control, Injection, Staining

Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: (A) Chromatin state model generated by ChromHMM . A total of 18 states were identified through genomic profiling of 7 chromatin features in ARID1A wild-type and knockdown 12Z cells: total RNA-seq, ATAC-seq, and H3K27me3, H3K4me3, H3K4me1, H3K18ac, and H3K27ac ChIP-seq. Genome was segmented into 200-bp intervals based on state classifications. Darker heatmap colors indicate higher relative enrichment for each chromatin feature in that state. Right-side labels are inferred biological functions of each state based on combinatorial chromatin features and genome ontology annotation. (B) Heatmap displaying chromatin state adjacency frequencies (how often 2 chromatin states neighbor each other). The darker color indicates more frequent state neighboring. (C) Percentage of genome coverage for each chromatin state. (D) Total RNA quantification of each chromatin state as reads per kilobase per million mapped reads (RPKM) per 200-bp genomic interval. Left, linear scale; right, log 2 scale. (E) Percentage of genome coverage per chromatin state for all other measured chromatin features. The statistic is hypergeometric enrichment compared to whole genome. (F) Percentage of genome coverage per chromatin state for other genomic features. Active SEs and TEs are distal H3K27ac peaks marked by ATAC, as defined in . The statistic is hypergeometric enrichment. (G) Percentage of genome coverage per chromatin state for ARID1A binding. The statistic is hypergeometric enrichment. (H) Genome-wide association between ARID1A binding and profiled histone modifications. Enrichments are displayed as fold-enrichment, per genomic base pair. The statistic is hypergeometric enrichment. Pairwise enrichment statistics computed by the chi-square test. (I) Association between ARID1A binding and TEs versus SEs, per H3K27ac peak, as defined in . The statistic is 2-tailed Fisher’s exact test. ***p < 0.001.

Article Snippet: The 12Z cells were provided by the laboratory of Asgi Fazleabas, and cell line validation was performed by IDEXX BioResearch, finding the result that the 12Z cell line has a unique profile not found in the current public databases.

Techniques: Generated, Knockdown, RNA Sequencing, ChIP-sequencing, Binding Assay, GWAS

Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: (A) Genomic annotation of replicate-overlapping P300 ChIP broad peaks in wild-type 12Z (FDR < 0.05, n = 25,096 peaks). (B) Enrichment for P300 binding (control cells) among chromatin states compared to whole genome. The statistic is hypergeometric enrichment. (C) Proportional Euler diagram displaying overlap between distal regions bound by ARID1A (n = 42,165) and P300 (wild-type cells, n = 17,812). (D) ARID1A binding among accessible P300-bound sites. P300 bound sites (wild-type cells) were first segregated by promoter versus distal status, then filtered for accessibility (ATAC). The statistic is the 2-tailed Fisher’s exact test. (E) Differential P300 ChIP-seq following ARID1A loss. At left is an MA plot revealing differential binding, with significant sites (FDR < 0.05) highlighted in red. The x axis is signal abundance quantified as log 2 counts per million (log 2 CPM), and the y axis is the log 2 FC difference of P300 binding in shARID1A versus control conditions (n = 2 ChIP replicates). At right is the ratio of tested sites binned by differential binding significance. Further analyses of P300 binding use the control condition data (F–R). (F) Signal heatmap displaying chromatin accessibility (ATAC), H3K27ac, and binding of ARID1A and P300 at enhancers (n = 18,050), centered on H3K27ac peak ± 3 kb. Enhancers were ranked by total H3K27ac signal. (G) Proportion of active enhancers (n = 18,050) bound by ARID1A, P300, both, or neither. (H) H3K27ac ChIP peak signal (fold enrichment, FE) relative to input at active enhancers segregated by ARID1A and P300 binding. The statistic is the 2-tailed, unpaired Wilcoxon test. (I) H3K27ac ChIP peak width at active enhancers segregated by ARID1A and P300 binding. The statistic is the 2-tailed, unpaired Wilcoxon test. (J) Ratio of enhancers (n = 18,050) displaying differential H3K27ac following ARID1A loss (left), and further segregation by ARID1A and P300 binding status (n = 4,681) (right). (K) Proportion of differential H3K27ac regions among enhancers bound by ARID1A, P300, both, or neither. The statistic is the 2-tailed Fisher’s exact test. (L) P300 ChIP signal at distal SE and TE H3K27ac peaks. The x axis is the distance to the H3K27ac peak center. The y axis is signal as ChIP – Input RPM per base pair per peak. (M) Proportion of distal SE and TE H3K27ac peaks bound by P300. The statistic is the 2-tailed Fisher’s exact test. (N) Proportion of P300-bound SE and TE regions displaying differential H3K27ac upon ARID1A loss. The statistic is the 2-tailed Fisher’s exact test. (O) Proportion of increasing versus decreasing H3K27ac at differential SE and TE regions bound by P300. The statistic is the 2-tailed Fisher’s exact test. (P–R) Violin plots (left) of ChIP signal for P300 (P), ARID1A (Q), and H3K27ac (R) at distal H3K27ac peaks in SE and TE regions further binned by P300 binding. Peak n’s from left to right: 415, 1,015, 3,508, and 13,112. The statistic is the 2-tailed, unpaired Wilcoxon test. Meta peak profiles (right) for P300 (P), ARID1A (Q), and H3K27ac (R) at P300-bound SE (entire SE region, n = 329) and P300-bound TE (n = 3,508). *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The 12Z cells were provided by the laboratory of Asgi Fazleabas, and cell line validation was performed by IDEXX BioResearch, finding the result that the 12Z cell line has a unique profile not found in the current public databases.

Techniques: Binding Assay, Control, ChIP-sequencing

Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: (A) Western blot analysis as indicated in 12Z cells, representative of 2 independent experiments. (B) Invasion of 12Z following indicated treatments. Representative images and total invaded cell numbers are shown (scale bar, 500 μm). Means ± SDs, n = 3. Unpaired, 2-tailed t test. (C) Survival of mice based on time until vaginal bleeding. LtfCre 0/+ ; (Gt)R26Pik3ca * H1047R ; Arid1a fl/fl (n = 16) median (μ 1/2 ) 107 days. LtfCre 0/+ ; (Gt) R26Pik3ca * H1047R ; Arid1a fl/fl ; Ep300 fl/fl (n = 12) median 143 days (p = 0.0018, Mantel-Cox test). LtfCre 0/+ ; Ep300 fl/fl mice were aged to 187 days, and no phenotypes were observed (n = 6). (D) Histology and IHC using indicated antibodies (n ≥ 2 mice) in endometrium (scale bar, 200 μm). KRT8 was a positive control for endometrial epithelium. The arrowheads indicate epithelia. (E) Quantification of H3K27ac and H3K18ac IHC, ratio of H-scores of epithelia to stroma. Means ± SDs, n = 4–8 mice, unpaired, 2-tailed t test. (F) Western blot of H3K27ac following A-485 treatment of 12Z for 24 h and densitometry of H3K27ac relative to H3, normalized to control (vehicle). Means ± SDs, n = 3–5 independent replicates per condition. Unpaired, 2-tailed t tests were performed in comparison to the vehicle treatment condition. Irrelevant lanes were removed from the image; see . (G) Viability assay for cells treated with A-485, normalized cell counts relative to vehicle control. Raw data are presented in . Half-maximal inhibitory concentration (IC 50 ) values were not significantly different between 12Z untreated and control shRNA, or between control shRNA and shARID1A (unpaired, 2-tailed t test). Means ± SDs, n = 4. (H) Invasion of 12Z following indicated cell treatments. Representative images and total invaded cell numbers are shown (scale bar, 500 μm). Means ± SDs, n = 4. Unpaired, 2-tailed t tests performed in comparison to siARID1A + vehicle. (I) Caspase 3/7 activity of indicated cell treatments. Means ± SDs, n = 3. Unpaired, 2-tailed t test. (J) Ratio of dead to live cells after 16 h in Matrigel. Means ± SDs, n = 6. Unpaired, 2-tailed t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The 12Z cells were provided by the laboratory of Asgi Fazleabas, and cell line validation was performed by IDEXX BioResearch, finding the result that the 12Z cell line has a unique profile not found in the current public databases.

Techniques: Western Blot, Positive Control, Control, Comparison, Viability Assay, Concentration Assay, shRNA, Activity Assay

Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: (A) ROSE ranking of active SEs (n = 413). The SERPINE1 SE locus is ranked 20 out of 413 based on H3K27ac levels. (B) Genomic snapshot of ChIP and ATAC signals alongside differential H3K27ac and chromatin state annotations at the SERPINE1 SE locus. For signal tracks, the y axis represents assay signal-to-noise presented as log-likelihood ratio (logLR) as reported by MACS2 , and small bars below the tracks represent replicate-overlapping peaks. H3K27ac log 2 FC colored bars denote significant differential H3K27ac regions (FDR < 0.05 for ChIP, FDR < 0.01 for CUT&RUN). ROSE active SE locus is represented by the black bar. (C) Significance (log 10 FDR, y axis) of DE genes following ARID1A loss, ranked by FDR value (x axis). SERPINE1 is the most significantly upregulated gene (arrow). (D) Expression of SERPINE1 (RNA-seq) following indicated 12Z cell treatments. Means ± SDs, n = 3. The statistic is DESeq2 FDR. (E) Western blot analysis as indicated in 12Z cells, representative of 2 independent experiments. (F) Relative expression of SERPINE1 by RNA-seq. Means ± SDs, n = 3 control mice and n = 4 mutant mice. The statistic is DESeq2 FDR. (G) IHC of SERPINE1 in endometrium of indicated genotypes; n = 4–5 mice per condition. (H) Quantification of IHC staining, ratio of H-scores of epithelia to stroma. Means ± SDs, n = 4–5 mice, unpaired, 2-tailed t test. (I) Western blot analysis as indicated in 12Z cells, representative of 2 independent experiments. (J) Invasion of 12Z following indicated treatment. Representative images and total invaded cell numbers are shown (scale bar, 500 μm). Means ± SDs, n = 5, unpaired, 2-tailed t test. (K) Measurement of cell growth following indicated treatments. Means ± SDs, n = 4. No significant differences, unpaired, 2-tailed t test. (L) Caspase-Glo assay of 12Z in suspension following indicated treatments. Means ± SDs, n = 5, unpaired, 2-tailed t test. (M) Ratio of dead to live cells after 24 h in Matrigel. Means ± SDs, n = 6, unpaired, 2-tailed t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The 12Z cells were provided by the laboratory of Asgi Fazleabas, and cell line validation was performed by IDEXX BioResearch, finding the result that the 12Z cell line has a unique profile not found in the current public databases.

Techniques: Expressing, RNA Sequencing, Western Blot, Control, Mutagenesis, Immunohistochemistry, Caspase-Glo Assay, Suspension

Journal: Cell reports

Article Title: ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation

doi: 10.1016/j.celrep.2020.108366

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The 12Z cells were provided by the laboratory of Asgi Fazleabas, and cell line validation was performed by IDEXX BioResearch, finding the result that the 12Z cell line has a unique profile not found in the current public databases.

Techniques: Recombinant, Blocking Assay, Staining, Plasmid Preparation, Protease Inhibitor, Transfection, Titration, Chromatin Immunoprecipitation, Magnetic Beads, Bicinchoninic Acid Protein Assay, Caspase-Glo Assay, Library Quantification, Flow Cytometry, Gene Expression, shRNA, In Vivo, Mutagenesis, Control, Software, Membrane, Cell Culture