human eoc cell lines a2780  (BioResource International Inc)

Journal: Open Medicine

Article Title: Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation

doi: 10.1515/med-2022-0422

Figure Lengend Snippet: SphK2 inhibition suppresses the adipocyte-induced EOC cell growth. The human EOC cell lines (a) SKOV3 and (b) A2780 were serum-starved overnight and then cultured with SFM or Adi-CM in the presence or absence of ABC294640 (10 μM) for 48 h. Cell proliferation was measured by CCK-8 assay. (c) Twenty-four hours after siRNA transfection, SphK2 mRNA levels were determined by RT-PCR. (d) Forty-eight hours after siRNA transfection, SphK2 protein levels were determined by using Western blot. Densitometric analysis of SphK2 (normalized to GAPDH) is shown on the right. (e) SKOV3 and (f) A2780 cells were transfected with the indicated siRNAs, followed by culture with SFM or Adi-CM for 48 h. Cell proliferation was measured by CCK-8 assays. Molecular weight of SphK2 is 69 kDa, and molecular weight of GAPDH is 36 kDa. Data are mean ± SD. *, P < 0.05 vs control; #, P < 0.05 vs Adi-CM alone.

Article Snippet: The human EOC cell line A2780 was obtained from the China Center for Type Culture Collection.

Techniques: Inhibition, Cell Culture, CCK-8 Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Molecular Weight, Control

Journal: Open Medicine

Article Title: Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation

doi: 10.1515/med-2022-0422

Figure Lengend Snippet: Adipocytes activate the SphK2/ERK pathway in EOC cells. (a) SKOV3 and A2780 cells were cultured with SFM or Adi-CM for 24 h. SphK2 phosphorylation was determined by using Western blot. Densitometric analysis of pSphK2 (normalized to total SphK2) is shown on the right. (b) SKOV3 and (c) A2780 cells were transfected with the indicated siRNAs and cultured with SFM or Adi-CM for 24 h. Total and phosphorylated ERK (pERK) levels were then determined by using Western blot. The right panel shows densitometric analysis of pERK (normalized to total ERK). Molecular weight of pSphK2 is 70 kDa, molecular weight of SphK2 is 69 kDa, molecular weight of GAPDH is 36 kDa, molecular weight of pERK is 42, 44 kDa, and molecular weight of ERK is 42, 44 kDa. Data are the mean ± SD. *, P < 0.05.

Article Snippet: The human EOC cell line A2780 was obtained from the China Center for Type Culture Collection.

Techniques: Cell Culture, Phospho-proteomics, Western Blot, Transfection, Molecular Weight

Journal: Open Medicine

Article Title: Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation

doi: 10.1515/med-2022-0422

Figure Lengend Snippet: Adipocyte-induced SphK2 activation is ERK dependent. (a) SKOV3 and (b) A2780 cells were serum starved overnight and pretreated with U0126 (5 μM) for 2 h. Cells were then cultured with SFM or Adi-CM for 24 h. Total and phosphorylated SphK2 (pSphK2) levels were then determined by using Western blot. Right panels show the densitometric analysis of pSphK2 (normalized to total SphK2) corresponding to the bands shown in the Western blots. Molecular weight of pSphK2 is 70 kDa, molecular weight of SphK2 is 69 kDa, and molecular weight of GAPDH is 36 kDa. Data are the mean ± SD. *, P < 0.05.

Article Snippet: The human EOC cell line A2780 was obtained from the China Center for Type Culture Collection.

Techniques: Activation Assay, Cell Culture, Western Blot, Molecular Weight

Journal: Open Medicine

Article Title: Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation

doi: 10.1515/med-2022-0422

Figure Lengend Snippet: Adipocyte-induced c-Myc expression is partly SphK2 dependent. (a) SKOV3 and A2780 cells were cultured with SFM or Adi-CM for 24 h. c-Myc expression level was determined by using Western blot. Densitometric analysis of c-Myc is shown on the right. (b) SKOV3 and (c) A2780 cells were transfected with the indicated siRNAs and cultured with SFM or Adi-CM for 24 h. c-Myc levels were then determined by using Western blot. Right panel shows the densitometric analysis of c-Myc. Molecular weight of c-Myc is 57 kDa and molecular weight of GAPDH is 36 kDa. Data are the mean ± SD. *, P < 0.05.

Article Snippet: The human EOC cell line A2780 was obtained from the China Center for Type Culture Collection.

Techniques: Expressing, Cell Culture, Western Blot, Transfection, Molecular Weight

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: Drug resistance identification and the resistance effect of SKOV3 cells on A2780 cells. (A) IC50 for DDP, PTX and CTX in SKOV3 cells and A2780 cells. (B) Schematic diagram of coculture. Cell viability (C) and IC50s for DDP (D) of A2780 cells and A2780 cells cocultured with SKOV3 cells. Apoptosis (E) and the related quantitative analysis of apoptosis (F) of A2780 cells and A2780 cells cocultured with SKOV3 cells.

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: SKOV3 cells enhance the chemoresistance of A2780 recipient cell exosomes. (A) Transmission electron micrography showed round-shaped vesicles with bilayered membranes ranging from 50 nm to 200 nm in diameter released by SKOV3 (SKOV3-EXO). Scale bar: 100 nm. (B) Nanosight particle tracking analysis (NTA) indicated that the dominant size of SKOV3-EXO was approximately 100 nm. (C) FCM analysis of exosomal marker proteins CD63 and CD81 in SKOV3-EXO. (D) Schematic diagram of A2780 cells cocultured with SKOV3-EXO. (E) Confocal microscopy showed exosome internalization by A2780 recipient cells after coincubation with PKH67-labeled (green fluorescence) SKOV3-EXO. DAPI was used to stain the nuclei of A2780 recipient cells with blue fluorescence. Scale bar = 10 µm. Cell viability (#: PBS vs SKOV3-EXO, *: CO-culture+DMSO vs CO-culture+GW4869) (F) and IC50s for DDP (G) of cocultured A2780+DMSO, cocultured A2780+GW4869, A2780+PBS, and A2780+SKOV3-EXO.

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Transmission Assay, Marker, Confocal Microscopy, Labeling, Fluorescence, Staining, Co-Culture Assay

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: Identification and screening of miRNAs in A2780 cells vs SKOV3 cells and A2780-EXO vs SKOV3-EXO. A. The heatmap showed that 16 miRNAs have higher expression levels and 17 miRNAs have lower expression levels in SKOV3 cells than in A2780 cells (left panel); 15 miRNAs have higher expression levels and 2 miRNAs have lower expression levels in SKOV3-EXO compared with those in A2780-EXO (right panel). B. The Venn diagram shows three miRNAs (miR-429, miR-200b-5p and miR-1269b) with higher expression in both comparison groups. C. Real-time qRT-PCR revealed that the levels of miR-429, miR-200b-5p and miR-1269b were higher in SKOV3 cells than in A2780 cells and that the levels were higher in SKOV3-EXO than in A2780-EXO.

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Expressing, Comparison, Quantitative RT-PCR

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: Chemoresistant effect of miR-429 on A2780 cells by CASR/STAT3 pathway. (A) After transfection with the agomirs of miR-429, miR-200b-5p and miR-1269b, the A2780 cells were treated with different concentrations of DDP for 48 h. DDP resistance was significantly enhanced in A2780 miR-429 agomir cells compared with that in A2780 miR-200b-5p agomir cells and A2780 miR-1269b agomir cells. (B) SKOV3 cells transfected with the Cy3-miR-429 mimic (red fluorescence) were plated in the lower chamber and coincubated with A2780 cells seeded in the upper chamber in a coculture system with a 0.4-µm pore membrane. Red fluorescence was observed in the A2780 recipient cells under the fluorescence microscope. Scale bar = 10 µm. IC50 for DDP (C), fold change of CASR mRNA (D) and CASR, STAT3, p-STAT3 protein levels (E) in A2780 miR-429 agomir, A2780 miR-429 agomir NC, A2780 miR-429 antagomir, and A2780 miR-429 antagomir NC. (F) RIP assays using an anti-CASR antibody showed that CASR interacts with miR-429 in A2780 cells, followed by qRT-PCR analysis of miR-429 in immunoprecipitated RNAs. The CASR, STAT3, p-STAT3 protein levels (G) (The original uncropped gels of Western blot assay were shown in Figure S6) and IC50s for DDP (H) of A2780 miR-429 antagomir NC, A2780 miR-429 antagomir, A2780 miR-429 antagomir+siCASR-NC, A2780 miR-429 antagomir+siCASR.

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Transfection, Fluorescence, Membrane, Microscopy, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: Effect of exosomal miR-429 in DDP resistance in vivo. Exosomes derived from SKOV3 cells were transfected with agomir or antagomir (SKOV3-EXO-agomir-NC, SKOV3-EXO-agomir, SKOV3-EXO-antagomir-NC, and SKOV3-EXO-antagomir). A2780 cells were subcutaneously injected into BALB/c nude mice, and potential tumors were allowed to grow for a week. Next, exosomes (50 μg) or PBS was intratumorally injected into the xenograft tumors three times per week for one week (n = 6). All the groups were administered DDP (5 mg/kg) by intraperitoneal injection three times a week for 2 weeks and were sacrificed. A. Flow chart of the animal experiment. B. Representative images of the excised tumors on day 35 after tumor cell injection. C. Tumor volumes of the excised tumors on day 35 after tumor cell injection. D. Immunohistochemistry analyses for KI-67 and CASR staining were carried out on A2780 xenograft tumor sections. Representative staining is shown (×200 magnification). E. The expression of miR-429 was higher in A2780+SKOV3-EXO-agomir cells than in A2780+PBS cells. The expression of miR-429 was lower in A2780+SKOV3-EXO-antagomir cells than in A2780+PBS cells. F. The level of CASR, STAT3, p-STAT3 proteins were downregulated in the A2780+SKOV3-EXO-agomir group, and the level of CASR protein was upregulated in the A2780+SKOV3-EXO-antagomir group, as assessed by Western blotting in mouse xenograft tumor tissues. The original uncropped gels of Western blot assay was shown in Figure S6. *P < 0.05 and **P < 0.01. ***P < 0.001.

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: In Vivo, Derivative Assay, Transfection, Injection, Immunohistochemistry, Staining, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

doi:

Figure Lengend Snippet: NF-κB promotes the transcription of miR-429 to mediate drug resistance. After treatment with different concentrations of PDTC for 24 h, NF-κB p65 protein expression in SKOV3 and A2780 cells were measured by Western blotting (A). The original uncropped gels of Western blot assay were shown in Figure S7. Additionally, the expression of miR-429 was detected by real-time qRT-PCR (B). Cell viability and IC50s for DDP of SKOV3 cells (C) and A2780 cells (D) treated with 0 or 200 μM PDTC (E). Cell viability and IC50s for DDP of A2780 cells cocultured with SKOV3 cells treated with 0 or 200 μM PDTC (F). Cell viability and IC50s for DDP of A2780 cells cocultured with SKOV3 transfected with miR-429 antagomir. The chrome immunoprecipitations (CHIP) were performed by using specific anti-NF-κB-p65 (G).

Article Snippet: Cell culture The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection

Journal: Journal of Ovarian Research

Article Title: Activation of SphK1 by adipocytes mediates epithelial ovarian cancer cell proliferation

doi: 10.1186/s13048-021-00815-y

Figure Lengend Snippet: Inhibition of SphK1 suppressed adipocyte-induced EOC cell proliferation. SKOV3 ( a ) and A2780 ( b ) cells were serum starved overnight and cultured with serum-free medium (SFM) or adipocyte-conditioned medium (Adi-CM) in the presence or absence of PF543 (10 μM) for 48 h. Cell proliferation was measured using CCK-8 assay. c SphK1 mRNA was measured by quantitative RT-PCR 24 h after siRNA transfection. d SphK1 protein levels were determined by western blot analysis 48 h after siRNA transfection. SKOV3 ( e ) and A2780 ( f ) cells were transfected with the indicated siRNAs, followed by culture with SFM or Adi-CM for 48 h. Cell proliferation was measured by CCK-8 assay. The data are presented as the means±SD. *, P < 0.05 vs control; #, P < 0.05 vs Adi-CM alone

Article Snippet: The A2780 human EOC cell line was obtained from the China Center for Type Culture Collection and cultured in DMEM supplemented with 10% FBS.

Techniques: Inhibition, Cell Culture, CCK-8 Assay, Quantitative RT-PCR, Transfection, Western Blot, Control

Journal: Journal of Ovarian Research

Article Title: Activation of SphK1 by adipocytes mediates epithelial ovarian cancer cell proliferation

doi: 10.1186/s13048-021-00815-y

Figure Lengend Snippet: Adipocytes activated SphK1/ERK signal in EOC cells. a Serum-starved SKOV3 and A2780 cells were cultured with SFM or Adi-CM for 24 h. SphK1 phosphorylation was determined by western blot analysis. Densitometric analysis of pSphK1 (normalized to total SphK1) is shown on the right. b SKOV3 cells were transfected with the indicated siRNAs and cultured with SFM or Adi-CM for 24 h. Levels of total and phosphorylated ERK (pERK) were determined by western blot analysis. The right panel shows densitometric analysis of pERK (normalized to total ERK). The data are presented as the means±SD. *, P < 0.05

Article Snippet: The A2780 human EOC cell line was obtained from the China Center for Type Culture Collection and cultured in DMEM supplemented with 10% FBS.

Techniques: Cell Culture, Phospho-proteomics, Western Blot, Transfection

Journal: Journal of Ovarian Research

Article Title: Activation of SphK1 by adipocytes mediates epithelial ovarian cancer cell proliferation

doi: 10.1186/s13048-021-00815-y

Figure Lengend Snippet: Adipocyte-induced SphK1 activation was ERK dependent. SKOV3 ( a ) and A2780 ( b ) cells were serum starved overnight, pretreated with U0126 (5 μM) for 2 h and then cultured with SFM or Adi-CM for 24 h. Levels of total and phosphorylated SphK1 (pSphK1) were determined by western blot analysis. Right panels show densitometric analysis of pSphK1 (normalized to total SphK1) corresponding to the bands shown in the western blots. The data are presented as the means±SD. *, P < 0.05

Article Snippet: The A2780 human EOC cell line was obtained from the China Center for Type Culture Collection and cultured in DMEM supplemented with 10% FBS.

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: Oncotarget

Article Title: Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy

doi: 10.18632/oncotarget.14663

Figure Lengend Snippet: A . ZFAS1 expression levels was determined by qRT-PCR in 66 EOC tissues and 10 normal epithelial tissues samples ( p <0.001). B . Relative expression levels of ZFAS1 in seven paired primary and metastatic EOC tissues by qRT-PCR ( p <0.05). C . Relative expression levels of ZFAS1 in 6 EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780, and COV644), as compared to those in human ovarian surface epithelial (HOSE cells, * p <0.05). D . Kaplan-Meier survival curves in high and low ZFAS1 expression groups ( p <0.001).

Article Snippet: The human EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780, and COV644) and normal Human Ovarian Surface Epithelial (HOSE) cells were supplied by China Center for Type Culture Collection (CCTCC).

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy

doi: 10.18632/oncotarget.14663

Figure Lengend Snippet: A . The putative binding sites of ZFAS1 and miR-150-5p. B . The luciferase reporter plasmid containing ZFAS1 was co-transfected with miR-150-5p or miR-Control in Caov3 and SKOV3 cells. Luciferase activities were determined at 48 h after transfection. The relative luciferase activities normalized to Renilla activity.* p <0.05 compared with control cells. C . Relative expression of miR-150-5p in Caov3 and SKOV3 cells transfected with ZFAS1 siRNA. * p <0.05 compared with control cells. D . Expression levels of miR-150-5p in 66 EOC tissues relative to 10 normal epithelial tissues samples ( p <0.001). E . The correlation of the expression levels of ZFAS1 and miR-150-5p ( r =0.613; p <0.001).

Article Snippet: The human EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780, and COV644) and normal Human Ovarian Surface Epithelial (HOSE) cells were supplied by China Center for Type Culture Collection (CCTCC).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Transfection, Control, Activity Assay, Expressing

Journal: Oncotarget

Article Title: Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy

doi: 10.18632/oncotarget.14663

Figure Lengend Snippet: A . The putative binding sites of miR-150-5p in the Sp1 3′-UTR regions. B . Luciferase activities in Caov3 and SKOV3 cells 48 h after co-transfected with Sp1 3′-UTRluciferase reporter plasmids and miR-150-5p, miR-Control, or their inhibitors, respectively. * p <0.05 compared to control cells. C . mRNA expression levels of Sp1 in Caov3 and SKOV3 transfected miR-150-5p, miR-Control, anti-miR-150-5p or anti-miR-Control, respectively. * p <0.05 compared to control cells. D . Levels of Sp1 protein was determined by Western blot in Caov3 and SKOV3 cells transfected with miR-150-5p, miR-Control, anti-miR-150-5p or anti-miR-Control, respectively. E . Immunofluorescent staining of Sp1 in EOC tissues (Scale bar, 50 μm).MiR-150-5p expression in low and high Sp1 expression groups ( p <0.001).

Article Snippet: The human EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780, and COV644) and normal Human Ovarian Surface Epithelial (HOSE) cells were supplied by China Center for Type Culture Collection (CCTCC).

Techniques: Binding Assay, Luciferase, Transfection, Control, Expressing, Western Blot, Staining