Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 3 MRE11:p.K464R mutation leads to Olaparib resistance in ovarian cancer cells. (A-B) The stable cell lines of SKOV3 (A) and A2780-MRE11WT/MRE11K464R (B) were constructed expressing MRE11WT and MRE11K464R protein and detected the MRE11 protein expression with West ern blot. (C-D) SKOV3 (C) and A2780 (D) MRE11WT /MRE11K464R cells were treated for 96 h with indicated dose of Olaparib and viability assessed by CCK8. (E-F) Representative pictures of colony formation assay in SKOV3 (E) and A2780 (F) MRE11WT /MRE11K464R cells treated with or without Olaparib for 10 days are in left. Relative colony formation rates of cell are presented as percent relative to DMSO (right). (G-H) SKOV3 (G) and A2780 (H) MRE11WT/MRE11K464R cells were treated with or without Olaparib for 48 h and then stained for γH2AX, MRE11, and DAPI (left), and quantified the γH2AX foci per cell (right). Scale bar, 25 μm. (I-J) SKOV3 (I) and A2780 (J) MRE11WT/ MRE11K464R cells were treated with or without Olaparib for 48 h and subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. Each group represents at least 100 cells counted. Data are presented as mean values ± SEM from three independent experiments. ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Mutagenesis, Stable Transfection, Construct, Expressing, Colony Assay, Staining, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 4 Interactions of MRE11:p.K464R with RAD50/RPS3. (A) Schematic diagram of IP-MS detection. (B) The directly-interacting proteins of MRE11 were screened in the String database according to the IP-MS data. (C-D) SKOV3- MRE11WT/MRE11K464R cells were treated with or without Olaparib for 24 h. The cells were then lysed and immunoprecipitation with MRE11 antibody (C) and the relative gray scale density of RAD50(up) and RPS3 (down) to MRE11 are presented in D.(E-F) SKOV3- MRE11WT/MRE11K464R cells were treated with Olaparib or DMSO. The cells were then lysed and immunoprecipitation with RPS3 antibody (E) and the relative gray scale density of RAD50 (up) and MRE11 (down) to MRE11 are presented in F. (G-H) Detection of MRE11-RAD50 (G) and MRE11-RPS3(H) interaction was carried out by PLA labeling in SKOV3 MRE11WT/MRE11K464R cells treated with or without Olaparib for 24 h. Rep resentative images are shown. Scale bars, 5 μm. The scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells from three independent experiments. Data are mean ± SEM. (I-J) ER-AsiSI Hela cells were transfected with empty vector or MRE11WT or MRE11K464R, and then treated with 4-OHT to induce DSBs. RAD50 (I) and RPS3 (J) accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Protein-Protein interactions, Immunoprecipitation, Labeling, Transfection, Plasmid Preparation, Generated, ChIP-qPCR, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 5 Effects of MRE11_K464R mutation on NHEJ pathway. (A) Expression of NHEJ key proteins in SKOV3 MRE11WT /MRE11K464R cells after treated with Olaparib for 48 h, detected by Western Blot with indicated antibodies. (B) MRE11WT or MRE11K464R virus were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (C) ER-AsiSI Hela cells were infected with MRE11WT or MRE11K464R virus, and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detect ed by ChIP qPCR. (D) A2780 MRE11WT /MRE11K464R cells after treated with Olaparib for 48 h. The chromatin fractions and the soluble nuclear fractions were analyzed with indicated antibodies by Western Blot. (E-F) A2780 MRE11WT /MRE11K464R cells were treated with or with Olaparib for 48 h, and co-stained with Ku70 (green) and γH2AX (red) antibodies. E The representative images of immunofluorescence are presented, F quantification of Ku70 and γH2AX co-localization ratio per cell. Each group represents at least 100 cells counted. Scale bar, 10 μm. (G) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (H) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into ER-AsiSI Hela cells and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. (I) SKOV3 (left) and A2780 (right) MRE11K464R cells were treated for 96 h with indicated doses of Olaparib or SCR7 (up) / AZD7648 (down) alone or combined for 96 h and viability assessed. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Mutagenesis, Expressing, Western Blot, Virus, Infection, Transfection, Plasmid Preparation, Generated, ChIP-qPCR, Staining, Immunofluorescence, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 6 RPS3/RAD50 knockdown sensitizes K464R mutant cells to Olaparib. (A-B) SKOV3 (A) and A2780 (B) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated for 96 h with indicated doses of Olaparib and viability was measured by CCK8. The expres sion of scramble siRNA (siNC) was used as control. (C-D) Cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated with or without Olaparib for 10 days. The expression of siNC was used as control. Representative pictures of clonogenic assay in A2780 MRE11K464R cells (C). The mean clones of ROI are presented (D). (E-F) SKOV3 (E) and A2780 (F) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated for 48 h and then subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. The expression of siNC was used as control. Each group represents at least 150 cells counted. (G-H) SKOV3 (G) and A2780(H) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated with or without Olaparib for 48 h and then stained for γH2AX (green) and DAPI (blue), and γH2AX foci-positive cells were quantified (below). Each group represents at least 150 cells counted. Scale bar, 20 μm. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Mutagenesis, Transfection, Control, Expressing, Clonogenic Assay, Clone Assay, Staining, Two Tailed Test

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 inhibits viability of OVCA cells. (A and B) ONC201 reduces cell viability of SKOV3, OV433, CaOV3, TOV112D, VOA4627, and VOA1312 cells in a dose and time‐dependent manner. Cells were treated with either vehicle or drug at the indicated concentration (0/1/10/25/50/100/200 µM) for (A) 48 h and (B) 72 h MTT assay results are shown. The absorbance was read at 540 nm using an automated microplate reader. The percentage of cell viability was calculated to compare the vehicle group. Data were expressed as the percent cell proliferation relative to the control as mean ± SD from triplicate wells. (C) ONC201 inhibits OVCA migration. Cells were treated by ONC201 (0/1/10/100 µM) and migration of cells was evaluated via wound healing assay. Cells were seeded and left overnight. The following day a “wound” was created using 200 µl pipet tips and treated with ONC201. After incubation with ONC201 for 8–24 h depending on the cell line, cells were washed by DPBS and were stained with crystal violet. Dose‐dependent decreased cell migration was noted in all cell lines. A representative image from three independent experiments is shown (image magnification ×200). A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Concentration Assay, MTT Assay, Control, Migration, Wound Healing Assay, Incubation, Staining

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 downregulates PI3K/AKT and ERK/MEK signaling pathway. ONC201 (20 µM) inhibits AKT and ERK in high‐ and low‐grade OVCA cells. Lysates were collected from SKOV3 and VOA4627 cell lines at 48, 72, and 96 h and western blot was completed with indicated antibodies. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Western Blot

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 activates ER stress. The expression of UPR‐related genes was evaluated by qPCR in SKOV3 (A), VOA1312 (B), OV433 (C), and CaOV3 (D). CHOP expression was induced 5–15 fold in all cell lines, suggesting that ONC201 prompts ER stress in human OVCA cells. (E) ONC201 (20 µM) upregulated protein expression of CHOP and ATF4. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 promotes apoptosis both in high‐ and low‐grade OVCA cells. (A) SKOV3 and VOA4627 cells treated with or without ONC201 (20 µM) were double‐stained with Annexin V and PI, and then analyzed by flow cytometry. The percentages of viable cells (compared to vehicle) are shown. (B) Representative flow diagram of Annexin V‐PI staining. (C) Caspase3/7 activity was measured by Caspase‐Glo 3/7 assay. SKOV3 and VOA4627 cells were treated with ONC201 (20 µM) for 48, 72 h. Caspase3/7 activity was increased from 48 to 72 h. (D) ONC201‐treated lysates were collected from SKOV3 and VOA4627 cell lines and blotted with Wee1 antibodies. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Staining, Flow Cytometry, Activity Assay, Caspase-Glo Assay

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 leads cells to intrinsic rather than TRAIL‐induced cell death. (A) ONC201 leads to a loss of mitochondrial membrane potential (MMP). MMP was measured by JC‐1 assay. JC‐1 monomers (green) were increased by ONC201 (20 µM) treatment over time both in SKOV3 and VOA4627 cells. (B) After treating with ONC201 (20 µM) for 48 and 72 h, cells are harvested and cell extracts were analyzed by western blotting to detect the expression of cleaved PARP, BIM, and Mcl‐1. (C and D) ONC201 (20 µM) led OVCA cells to death via TRAIL‐independent pathway. Lysates were collected from SKOV3 and VOA4627 cell lines and blotted with TRAIL antibodies. Whereas, the expression of DR5 gene was confirmed by qPCR in SKOV3, OV433, CaOV3, and VOA1312. (E) ONC201 does not activate Caspase‐8. Caspase‐8 (p18/p10) was found intact both in ONC201 treated and control SKOV3 cells. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Membrane, Western Blot, Expressing, Control

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 increases endoplasmic reticulum stress in both high‐grade and low‐grade ovarian cancer cells; overwhelming pro‐survival signals and activating the pro‐death arm of the unfolded protein response pathway. In addition, a weekly oral dose of ONC201 lowers the tumor burden in mice. This is a promising therapeutic agent in OVCA treatment and should be considered for clinical translation

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques:

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 promotes anchorage-independent growth of ovarian cancer cells. (A) CaOv3, OvCar3, and SKOv3 cells were incubated in serum-free media for 48 h. GDF15 concentration was then determined by ELISA in media from cell lines. (B) SKOv3, OvCar3, and CaOv3 ovarian cancer cells were plated in matrigel, and maintained in media containing 20 ng/mL rhGDF15 or vehicle control. Media was changed twice a week for 3–4 weeks. Representative photographs taken with 4× objective lens are shown. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) cell growth is shown for rhGDF15 group (GDF) versus control vehicle group (C) per line. (C) SKOv3 cells were stably transfected with empty vector control or GDF15 expression plasmid. Three GDF15 stable clones (G1, G3, G5) were selected and analyzed by real-time PCR for GDF15 transcript level versus control clone (C). Levels of GDF15 transcript were normalized to RPLPO internal control transcript. Values reflect the average fold change in normalized GDF15 transcript expression per stable clone relative to control clone. (D) Control clone (C) and two GDF15 stable clones (G3 and G5) were plated in matrigel. Media was changed twice a week for 3–4 weeks. Matrigel was then dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) growth is shown for the GDF15 clones relative to control clone. (E) Growth of SKOv3 parental cells treated with vehicle control or 20 ng/mL rhGDF15, or stable control clone or GDF15 clones G1 or G3 was assessed by Real-Time Cell Analysis. Cell index reflects growth over 24 h, and represents the average of triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Clone Assay, Real-time Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 promotes invasiveness of ovarian cancer cells. (A) SKOv3 cells were plated in Boyden chambers in the presence of 10% FBS plus vehicle control (C) or 20 ng/mL rhGDF15. (B) SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 cells were plated in Boyden invasion chambers in the presence of 10% FBS. For both (A) and (B), photos were taken after 24 h of invasion, and the number of invaded cells was counted in ten different fields per sample. Representative photos are shown. Values reflect the total number of invaded cells in triplicate cultures per group. (C) Representative photos of stable control and GDF15 clones are shown at 10× magnification. Western blotting for mesenchymal marker N-cadherin was performed twice; a representative blot is shown for total cell lysates from control and GDF15 stable clones. Quantification of N-cadherin was normalized to actin, and is shown relative to control clone. (D) SKOv3, SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 cells were plated in the presence of 5% FBS in the upper chamber of XCelligence CIM-plates. Medium containing 10% FBS, and 20 ng/mL rhGDF15 where indicated, was placed into the plate’s corresponding bottom chambers. Cell index reflects cell migration measured every 15 min for 10 h, and represents the average of triplicate cultures per group. (E) Real-time PCR was performed for invasion markers MMP-2, MMP-9, and VEGF in SKOv3 control and GDF15 stable clones 1, 3, and 5. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. (F) A tumor tissue array consisting of 122 tumor samples and 10 normal tissue samples was stained for MMP2 and MMP9. Photos were taken at 5× magnification under the microscope, and representative photos are shown. (G) SKOv3 control and GDF15 clones were plated in Boyden chambers, and treated for 24 h with DMSO (C), 1 µM (labeled “1”) or 10 µM (labeled “10”) of the pan-MMP inhibitor GM6001. The number of invaded cells was counted in ten different fields per sample. Values reflect the average number of invaded cells in triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Stable Transfection, Clone Assay, Western Blot, Marker, Migration, Real-time Polymerase Chain Reaction, Staining, Microscopy, Labeling

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: PI3K/mTORc1 and MAPK signaling are activated by GDF15 in ovarian cancer cells. (A) SKOv3 cells were serum starved overnight, and then stimulated with 20 ng/mL rhGDF15 for 2 or 5 min, or with the corresponding volume of vehicle control for 5 min. Western blots of total protein lysates were performed at least 3 times for p-Thr180/ Tyr182 p38MAPK, total p38, p-Thr202/Tyr204 p42/p44 Erk1/2, total Erk1/2, p-S473 Akt, and total Akt; representative blots are shown. Quantification is shown as a ratio of phospho-protein to total protein, and is shown relative to vehicle control. (B) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL rhGDF15, or GDF15 plus 1 µM PI3K inhibitor LY294002, 100 nM MEK inhibitor PD0325901, or 10 µM p38MAPK inhibitor SB203580. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the vehicle control group; ** p < 0.005 for GDF15-stimulated versus control vehicle, and for inhibitor + GDF15 groups versus GDF15 alone. (C) Total protein lysates from SKOv3 stable control and GDF15 clones 1, 3, and 5 were Western blotted at least twice for phosphorylated and total p38MAPK, Akt, and 4EBP1; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein, and is shown relative to control clone. (D) (Left) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with 10 nM or 100 nM of rapamycin, or with (right) 1, 5, or 10 µM TGF beta receptor type II inhibitor SB431542. Control groups (C) were treated with DMSO alone. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates. (E) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL GDF15, or GDF15 plus 100 nM rapamycin or 5 µM TGF beta receptor type II inhibitor SB431542. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the control group. (F) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with DMSO control (C) and the concentrations shown for LY294002, PD0325901, or SB203580. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: Rapamycin inhibits GDF15-mediated ovarian cancer cell invasion. (A) SKOv3 cells were plated in Boyden invasion chambers in the presence of vehicle control, 20 ng/ mL GDF15, or GDF15 plus 100 nM rapamycin (Rp). Photos were taken after 24 h of invasion (representatives shown), and the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (B) SKOv3 cells were treated with vehicle control, 20 ng/mL GDF15, or 20 ng/mL GDF15 plus 100 nM rapamycin (Rp). Real-time PCR was performed for MMP-9 and VEGF. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. (C) SKOv3 GDF stable clones 1 and 3 were plated in Boyden invasion chambers in the presence of vehicle control (C) or 100 nM rapamycin (Rp). After 24 h of invasion, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (D) SKOv3, SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 were plated in the presence of 5% FBS, and 100 nM rapamycin (Rp) where indicated, in the upper chamber of XCelligence CIM-plates. Medium containing 10% FBS, and 20 ng/mL rhGDF15 (G) where indicated, was placed into the plate’s corresponding bottom chambers. CIM-plates were monitored every 15 min for 10 h. Standardized cell index (CI) values were plotted linearly against time. The CI slope per hour was calculated and is shown here as a measure of migration. Values reflect the average of triplicates at each time point.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Stable Transfection, Migration

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: Neutralization of secreted GDF15 reduces invasion and growth of GDF15-overexpressing ovarian cancer cells. (A) Concentrations of secreted GDF15 were determined by ELISA in media from SKOv3 and Tov21 cell lines. Cells were incubated in serum-free media for 48 h prior to ELISA. Values reflect the average fold expression in three samples per group. (B) Tov21 cells were either untreated, or treated with 1 µg/mL control IgG or GDF15 mAb (147627; R&D Systems) for 24 h. The concentration of GDF15 in media was measured by ELISA in triplicates per group. Values reflect the average fold expression per group. (C) Tov21 cells were treated with 1 µg/mL control IgG or GDF15 mAb for 24 h. Real-time PCR was then performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and treated with 1 µg/mL control IgG or GDF15 mAb (147627); media was changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown relative to the control group. (E) Tov21 cells were plated in Boyden chambers and treated with 1 µg/mL control IgG or GDF15 mAb (147627). After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 knockdown reduces invasion and growth in association with reduced p-4EBP1 in GDF15-overexpressing ovarian cancer cells. (A) Real-time PCR was performed for GDF15 in SKOv3 and Tov21 cells, and normalized to the level of internal control transcript RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (B) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was then performed for GDF15 and RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (C) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and infected with lentiviral control or GDF15 shRNA; media and virus were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown. (E) Tov21 cells were plated in Boyden chambers and infected with control or GDF15 shRNA. After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (F) Tov21 cells were infected with lentiviral control shRNA or GDF15 shRNA for 48 h. Western blots were performed at least twice for phosphorylated and total 4EBP1, Erk1/2, p38, and Akt; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein above each blot.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Real-time Polymerase Chain Reaction, Infection, shRNA, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Targeting in vitro vasculogenic mimicry and associated stemness transcriptional signature in human ovarian cancer cell models: new emerging roles of caffeic acid phenethyl ester synthetic analogs

doi: 10.3389/fphar.2026.1787101

Figure Lengend Snippet: Effects of CAPE (1) and its analogs (2–5) on in vitro vasculogenic mimicry in ovarian cancer cell models. Representative images and Wimasis-processed overlays of the impact of the vehicle, CAPE (1), and analogs (2–5) (1 μM) on capillary-like structure formation in (A) ES-2 and (B) SKOV3 ovarian cancer cells. (C) VM parameters for tube length, branching points, and total tubes for both ES-2 and SKOV3 cells under each treatment. VM parameters expressed as percent of control. Data are the mean ± SD of 3 independent experiments, and asterisks (*) indicate statistically significant differences versus the vehicle (DMSO) with P < 0.05.

Article Snippet: The human SKOV3 ovarian adenocarcinoma cell line was purchased from Cell Biolabs (San Diego, CA, United States).

Techniques: In Vitro, Control