Journal: iScience

Article Title: The role of epithelial progesterone receptor isoforms in embryo implantation

doi: 10.1016/j.isci.2021.103487

Figure Lengend Snippet: Key resources table

Article Snippet: In vitro HEC1A cell culture The human endometrial cancer cell line HEC1A was purchased from the ATCC (HTB-112, ATCC).

Techniques: Immunohistochemistry-IF, Western Blot, RNAscope, Plasmid Preparation, Recombinant, Modification, Electrophoresis, Multiplex Assay, Bicinchoninic Acid Protein Assay, Staining, Reverse Transcription, SYBR Green Assay, RNA Amplification, Purification, Generated, Software, Magnetic Cell Separation

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: Short-form thymic stromal lymphopoietin (sfTSLP), but not long-form thymic stromal lymphopoietin (lfTSLP), was expressed in the human cell lines of ovarian, endometrial and cervical cancer. ( a ) Schematic representation of the human TSLP locus (5q22.1) from the UCSC Genome Browser (hg18). Primers for RT-PCR of TSLPv1 (NM_033035), TSLPv2 (NM_138551) and TSLPv3 (NR_045089) are labelled as blue arrows; primers and probes of TaqMan expression assay for TSLP (Hs00263639_m1) are labelled as red arrows. A CpG island located at the promoter region and exon 1 of TSLPv2 gene is labelled as green bar. ( b ) RT-PCR of TSLP isoforms (TSLPv1, TSLPv2 and TSLPv3) and TSLPR in 5 non-malignant human immortalised ovarian surface epithelial (IOSE), 2 non-malignant immortalised human fallopian tube secretory tube epithelial (FTSEC) and 16 human ovarian cancer (OvCa) cell lines. β-actin (ACTB) served as house-keeping gene controls. The ACTB controls for IOSE, FTSEC and OvCa cell lines in b,d are identical since they are part of the same original RT-PCR experiments with different primer sets of interest. The figures are organized as shown to better align with the data presented in the Result Section; NTC: No template controls. ( c ) Real-time quantitative RT-PCR (qRT-PCR) of TSLPv2 in IOSE, FTSEC and OvCa cell lines was performed by SYBR green assay with TSLPv2-specific primers (upper panel) and qRT-PCR of TSLP was performed by TaqMan expression assay for TSLP (Hs00263639_m1; lower panel). ( d ) RT-PCR of lfTSLP (TSLPv1) and sfTSLP (TSLPv2) in IOSE, FTSEC and OvCa cell lines. Primers specific for lfTSLP and sfTSLP were designed by Fornasa et al. and Biancheri et al. [ , ]. ( e ) mRNA expression of TSLP isoforms (TSLPv1, TSLPv2 and TSLPv3) and TSLPR in 6 human endometrial cancer cell lines (EndoCa) and an immortal human myometrial stromal cell line (hTERT-HM) was examined by RT-PCR (left panel) and qRT-PCR (middle panel: SYBR green assay for TSLPv2; right panel: TaqMan expression assay for TSLP). ( f ) mRNA expression of TSLP isoforms (TSLPv1, TSLPv2 and TSLPv3) and TSLPR in 4 human cervical cancer cell lines (CerxCa) was examined by RT-PCR (left panel) and qRT-PCR.

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, SYBR Green Assay

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: sfTSLP was expressed in human epithelial ovarian cancer and endometrioid endometrial cancer. ( a ) mRNA expression of sfTSLP in 15 tumour tissues of human epithelial ovarian cancer (EOC) was examined by qRT-PCR (SYBR green assay for TSLPv2). ( b ) mRNA expression of sfTSLP in 23 tumour tissues of human endometrioid endometrial cancer (EEC) was examined by qRT-PCR (SYBR green assay for TSLPv2). ( c ) mRNA expression of lfTSLP and sTSLP in selected human EOC and EEC tissues was examined by BaseScope Duplex RNA in situ hybridisation assay. Designed BA-Hs-TSLPv1-2zz-st-C2 (red) targeting TSLPv1 and BA-Hs-TSLPv2-3zz-st-C1 (green) targeting TSLPv2 were applied (left column). Images at 1000× magnification for TSLPv1 and TSLPv2-specific probes (scale bar 20 μm). Blue circles highlighted positive green signals of TSLPv2-specific probe. Positive control probes (for PPIB in green and POLR2A gene in red; middle column) and negative control probes (for bacterial DapB gene in green and red; right column) were also applied. Images at 1000× magnification (scale bar 20 μm). An inserted picture showed higher magnification in of a part of the tissue.

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Expressing, Quantitative RT-PCR, SYBR Green Assay, In Situ, Hybridization, Positive Control, Negative Control

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: Epigenetic regulation of sfTSLP transcription in human ovarian and endometrial cancer cells. ( a ) mRNA expression of sfTSLP in TSLP-negative human ovarian cancer cell lines (A2780, IGROV-1, KURAMOCHI and TOV21G) after treatment of 5-aza-2′-deoxycytidine (AZA; 1 μM), Trichostatin A (TSA; 300 nM) or BIX01294 (BIX; 5 μM) for 72 h was examined by qRT-PCR (upper panel: SYBR green assay for TSLPv2; bottom panel: TaqMan expression assay for TSLP). DMSO served as vehicle controls. ( b ) Chromatin immunoprecipitation (ChIP) was performed in cell lines with high levels of sfTSLP expression (IOSE20C2, IOSE25C2) and with low/no levels of sfTSLP expression (IGROV-1, TOV21G and TOV112D), using antibodies for acetylated H3K9/14 (H3K9/14ac; left panel) and acetylated H4K5 (H4K5ac; middle panel). Immunoprecipitated DNA was analysed by qPCR using primers flanking promoter region of TSLPv2 (sfTSLP) gene. Expression of sfTSLP transcript in IOSE20C2, IOSE25C2, IGROV-1, TOV21G and TOV112D was shown for reference (right panel, excerpt from c). ( c ) CpG content in the promoter region of TSLPv2 gene was analysed using MethPrimer website. A region of genomic DNA (including 500 base pairs upstream of the transcription start site and whole exon 1) was evaluated for the percentage of GC content and individual CpG dinucleotides. Nucleotide position was indicated along the X -axis and GC content was graded on the Y -axis. A CpG island was found and indicated by shading. Total 29 CpG nucleotides was contained in the CpG island. Each CpG dinucleotide was indicated by a hash mark below the nucleotide numbering, and the transcription start site was indicated by a red arrow. Amplification regions for bisulfite sequencing primers were indicated by blue arrows (upper panel). Bisulfite sequencing of TSLPv2 (sfTSLP) promoter was performed in a sfTSLP-expressing IOSE cell line (IOSE20C2) and two sfTSLP-negative ovarian cancer cell lines (IGROV-1 and TOV21G). ≥10 sequencing were analysed in each cell lines (≥10 rows of the circles; 29 circles in a row); each circle represented a CpG dinucleotide; dark circle represented methylation of the CpG dinucleotide, whereas empty circle represented unmethylation of the CpG dinucleotide. ( d ) mRNA expression of sfTSLP in TSLP-negative/low human endometrial cancer cell lines (HEC1A, RL95-2 and KLE) after treatment of AZA (1 μM), TSA (300 nM) or BIX (5 μM) for 72 h was examined by qRT-PCR (upper panel; SYBR green assay for TSLPv2; bottom panel: TaqMan expression assay for TSLP). DMSO served as vehicle controls. ( e ) Bisulfite sequencing of TSLPv2 (sfTSLP) promoter were performed in sfTSLP-negative endometrial (HEC1A, RL95-2 and KLE). The percentage of methylation at each CpG dinucleotide (total 29 CpG dinucleotides) in the CpG islands was analysed in the cancer cell lines with or without AZA treatment. ( f ) Bisulfite sequencing of TSLPv2 (sfTSLP) promoter were performed in sfTSLP-negative ovarian cancer cell lines (A2780 and KUMRAMOCHI) after treatment of DMSO and AZA (1 μM) for 72 h. Data shown ( a,b,d,e ) are expressed as mean ± S.E.M. from three independent replicates (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Expressing, Quantitative RT-PCR, SYBR Green Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Gene Expression, Amplification, Methylation Sequencing, Sequencing, Methylation

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: Overexpression of sfTSLP promoted tumour growth of ovarian and endometrial cancers in vitro. ( a ) mRNA expression of sfTSLP in human ovarian (A2780 and IGROV-1) and endometrial (HEC1A) cancer cells with stable transfection of sfTSLP-expression vector (pZERO-sfTSLP) or empty-expression vector (pZERO-mcs) was examined by qRT-PCR (TaqMan expression assay for TSLP (Hs00263639_m1)). ( b ) Cell viability of ovarian (A2780 and IGROV-1) and endometrial (HEC1A) cancer cells with sfTSLP overexpression or empty-vector expression was determined by cell viability assay after 24, 48 and 72 h incubation. ( c ) Cancer organoid formation of ovarian (A2780 and IGROV-1) and endometrial (HEC1A) cancer cells with sfTSLP overexpression or empty-vector expression was captured after a few days of incubation. Scale bar 200 μm. ( d ) Tumour invasion ability of ovarian (A2780 and IGROV-1) and endometrial (HEC1A) cancer cells with sfTSLP overexpression or empty-vector expression was evaluated by transwell invasion assay. Data shown ( a – d ) are expressed as mean ± S.E.M. from three independent replicates (* p < 0.1, ** p < 0.01, **** p < 0.0001).

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Over Expression, In Vitro, Expressing, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Viability Assay, Incubation, Transwell Invasion Assay

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: Overexpression of sfTSLP activated intracellular kinases in ovarian cancer cells. ( a ) Human Phospho-Kinase Array was performed in human ovarian (A2780 and IGROV-1) and endometrial (HEC1A) with stable transfection of sfTSLP-expression vector (pZERO-sfTSLP) or empty-vector expression (pZERO-mcs) (upper panel). ( b ) Phosphorylation of GSK3α/β, STAT2 and p53 in A2780 ovarian cancer cells and phosphorylation of AKT1/2, ERK1/2 and Src in IGROV-1 ovarian cancer cells was examined by Western blotting (lower panel). ( c ) To demonstrate clearly the signals of AMPKα1, GSK3α/β, p53, AKT1/2, ERK1/2 and Src that had been activated in response to sfTSLP overexpression in A2780 and IGROV-1 ovarian cancer cell lines, the band intensities of the Western blots were measured and analysed. The band intensity of particular phosphorylated or total protein was normalised to that of their corresponding β-actin expression. The whole Western blots showing all bands and molecular weight markers are included in . Data shown ( c ) are expressed as mean ± S.E.M. from three independent replicates (* p < 0.1, ** p < 0.01, *** p < 0.001).

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Western Blot, Molecular Weight

Journal: Cancers

Article Title: Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth

doi: 10.3390/cancers13050980

Figure Lengend Snippet: EFNB2 and PBX1 were commonly downregulated in human gynaecologic cancer cells with sfTSLP overexpression. ( a ) The volcano plot showed the upregulated (red dots) and downregulated (green dots) differentially expressed genes (DEGs) of A2780 ovarian cancer cells with sfTSLP overexpression (left top); the top 10 gene ontology (GO) enrichment terms of upregulated DEGs (grey bars) and downregulated DEGs (black bars) were shown (left bottom). The upregulated and downregulated DEGs of IGROV-1 and their top 10 GO enrichment of ovarian cancer cells with sfTSLP overexpression (middle top and bottom). The upregulated and downregulated DEGs and their top 10 GO enrichment of HEC1A endometrial cancer cells with sfTSLP overexpression (right top and bottom). ( b ) The Venn diagram shows relations of DEGs expression by sfTSLP overexpression in A2780 cells (ATvsAM: A2780 cells with stable transfection of pZERO-sfTSLP vs. A2780 cells with stable transfection of pZERO-mcs), IGROV-1 cells (ITvsIM: IGROV-1 cells with stable transfection of pZERO-sfTSLP vs. IGROV-1 cells with stable transfection of pZERO-mcs) and HEC1A cells (HTvsHM: HEC1A cells with stable transfection of pZERO-sfTSLP vs. HEC1A cells with stable transfection of pZERO-mcs). ( c ) Heatmap of the mRNA expression levels of 11 DEGs in three cancer cell lines (A2780, IGROV-1 and HEC1A) with sfTSLP overexpression. ( d ) mRNA expression of TSLPv2, EFNB2 and PBX in ovarian (A2780 and IGROV-1) and endometrial (HEC1A) cancer cells with stable transfection of pZERO-sfTSLP or pZERO-mcs was examined by TaqMan expression assays. Data shown ( d ) are expressed as mean ± S.E.M. from three independent replicates (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Five non-malignant immortalised human ovarian surface epithelial cell lines (IOSE20C2, IOSE20C5, IOSE21C21, IOSE25C2 and IOSE25C26; [ ]), 2 non-malignant immortalised human fallopian tube secretory epithelial cell lines (FTSEC190 and FTSEC194; [ ]), 16 human ovarian cancer cell lines (IGROV-1, PEO1, SKOV3, TOV21G, TOV112D, CaOV4, COV318, COV362, KURAMOCHI, OVKATE, OVSAHO, SNU119, SNU251, TYK-nu, UWB1.289 and A2780), 6 human endometrial cancer cell lines (AN3CA, HEC1A, HEC1B, KLE, Ishikawa and RL95-2), 4 human cervical cancer cell lines (C4-1, Caski, HeLa and HT3) and 1 immortalised human myometrial stromal cell line (hTERT-HM; [ ]) were used according to culture methods provided from ATCC or indicated references.

Techniques: Over Expression, Expressing, Stable Transfection

Journal: BMC Cancer

Article Title: G-protein coupled receptor 64 (GPR64) acts as a tumor suppressor in endometrial cancer

doi: 10.1186/s12885-019-5998-1

Figure Lengend Snippet: Effect of GPR64 loss on cell growth in human endometrial cancer cells . a and b The expression of GPR64 mRNA and protein in Ishikawa ( a ) and HEC1A ( b ) cells transfected with non-targeting pool (NT) siRNA or GPR64 siRNA was examined by RT-qPCR and Western blot analysis, respectively. c and d Colony formation assay of Ishikawa ( c ) and HEC1A ( d ) cells transfected with NT siRNA or GPR64 siRNA. Samples from each treatment were transferred to flat-bottomed 24-well plates and incubated. Cells were fixed and stained with crystal violet. The average colony formation number was quantified with crystal violet stained cells. e and f Cell proliferation assay of Ishikawa ( e ) and HEC1A ( f ) cells transfected with NT siRNA or GPR64 siRNA. The results represent the mean ± SEM. *, p < 0.05; **, p < 0.01; and ***, p < 0.001

Article Snippet: Human endometrial adenocarcinoma Ishikawa and HEC1A cells were maintained in phenol red–free DMEM/F12 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin streptomycin (P/S; Gibco).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Colony Assay, Incubation, Staining, Proliferation Assay

Journal: BMC Cancer

Article Title: G-protein coupled receptor 64 (GPR64) acts as a tumor suppressor in endometrial cancer

doi: 10.1186/s12885-019-5998-1

Figure Lengend Snippet: Ability of cell migration associated with GPR64 expression in endometrial cancer . The wound healing assay was performed in Ishikawa ( a ) and HEC1A ( b ) cells transfected with NT siRNA or GPR64 siRNA. After scratch, cells were incubated for 48 h to determine the cell migration ability. Representative result of wound healing assay and quantification of cell migration by wound healing assay during in vitro culture. The results represent the mean ± SEM. *, p < 0.05

Article Snippet: Human endometrial adenocarcinoma Ishikawa and HEC1A cells were maintained in phenol red–free DMEM/F12 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin streptomycin (P/S; Gibco).

Techniques: Migration, Expressing, Wound Healing Assay, Transfection, Incubation, In Vitro

Journal: BMC Cancer

Article Title: G-protein coupled receptor 64 (GPR64) acts as a tumor suppressor in endometrial cancer

doi: 10.1186/s12885-019-5998-1

Figure Lengend Snippet: An increase of cell invasion by GPR64 loss in endometrial cancer cells . Representative result of transwell invasion assays of Ishikawa ( a ) and HEC1A ( b ) cells transfected with NT siRNA or GPR64 siRNA. Quantification of invasion through matrigel and transwell membrane in Ishikawa and HEC1A cells with or without GPR64 siRNA treatment. The results represent the mean ± SEM. **, p < 0.01

Article Snippet: Human endometrial adenocarcinoma Ishikawa and HEC1A cells were maintained in phenol red–free DMEM/F12 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin streptomycin (P/S; Gibco).

Techniques: Transfection, Membrane