Journal: OncoTargets and therapy

Article Title: YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells

doi: 10.2147/OTT.S102837

Figure Lengend Snippet: YAP confers resistance to cisplatin in ovarian cancer cells. Notes: ( A ) Cells were treated with different concentrations (0–50 μmol/L) of cisplatin for 48 hours. Cell viability was assessed using the CCK-8 assay. Data represent the mean and standard deviation values from three independent experiments (** P <0.01). ( B ) The 50% maximal inhibitory concentration (IC 50 ) values of cisplatin in the cisplatin-sensitive parent cell line OV2008 and its resistant clone C13K were calculated. Each value represents the mean ± standard deviation values from three independent experiments (** P <0.01). ( C ) The messenger RNA levels of YAP, Cyr61, CTGF, and CCND1 in the cells were examined using a real-time polymerase chain-reaction kit. Data represent the mean and standard deviation values from three independent experiments (** P <0.01). ( D ) Cell lysates were collected for Western blot analyses of the protein levels of YAP, Cyr61, CTGF, and CCND1.

Article Snippet: The primary antibodies used were as follows: YAP (sc-15407; Santa Cruz Biotechnology), Cyr61 (sc-13100; Santa Cruz Biotechnology), CTGF (sc-101586; Santa Cruz Biotechnology), CCND1 (sc-20044; Santa Cruz Biotechnology), LC3B and Beclin1 (3868S and 3738, respectively; Cell Signaling Technology), cleaved PARP and caspase 3 (9541 and 9661, respectively; Cell Signaling Technology), and Atg-3 and -5 (3415 and 2630, respectively; Cell Signaling Technology).

Techniques: CCK-8 Assay, Standard Deviation, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: OncoTargets and therapy

Article Title: YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells

doi: 10.2147/OTT.S102837

Figure Lengend Snippet: Effects of YAP knockdown on cisplatin sensitivity in resistant C13K cells via impairment of autophagy. Notes: After C13K cells were transfected with YAP small interfering RNA (siRNA) and control siRNA (37.5 nM) for 48 hours, they were treated with 50 μmol/L cisplatin for 48 hours. ( A ) Cell lysates were collected for Western blot analysis of the protein expression of YAP, Cyr61, CTGF, and CCND1. ( B ) Cell viability was determined using a CCK-8 assay. Data represent the mean and standard deviation values from three independent experiments (** P <0.01). ( C ) The fluorescence intensity of rhodamine 123 in the cells was measured by fluorescence-activated cell sorting. Data shown represent the mean ± standard error values from three independent experiments (** P <0.01 vs the control siRNA group). ( D ) Cells were subjected to annexin V–propidium iodide staining. The apoptosis ratio represents the ratio of the number of cells in early stage apoptosis to that in advanced-stage apoptosis. Quantification of apoptotic cells: mean ± standard deviation values represent those from three independent experiments (** P <0.01). ( E ) Western blot analysis of the expression of cleaved caspase 3 and PARP and the autophagy-related proteins Atg-3 and Atg-5 in the control siRNA and YAP siRNA C13K cells. ( F ) Electron transmission microscopy for the detection of autophagosomes in the control siRNA and YAP siRNA C13K cells; the black arrows indicate the autophagic vacuoles (magnification 10,000×). Scale bars 2 μm.

Article Snippet: The primary antibodies used were as follows: YAP (sc-15407; Santa Cruz Biotechnology), Cyr61 (sc-13100; Santa Cruz Biotechnology), CTGF (sc-101586; Santa Cruz Biotechnology), CCND1 (sc-20044; Santa Cruz Biotechnology), LC3B and Beclin1 (3868S and 3738, respectively; Cell Signaling Technology), cleaved PARP and caspase 3 (9541 and 9661, respectively; Cell Signaling Technology), and Atg-3 and -5 (3415 and 2630, respectively; Cell Signaling Technology).

Techniques: Transfection, Small Interfering RNA, Western Blot, Expressing, CCK-8 Assay, Standard Deviation, Fluorescence, FACS, Staining, Transmission Assay, Microscopy

Journal: Journal of Cancer

Article Title: Cyr61 mediates oxaliplatin resistance in colorectal cancer cells by regulating Bcl-xL expression

doi: 10.7150/jca.48891

Figure Lengend Snippet: The level of Cyr61 in the HCT-8/L-OHP and HCT116/L-OHP cell lines. (A) and (D) The mRNA level of Cyr61 was determined by RT-PCR. (B) and (E) The protein level of Cyr61 was determined by western blotting. (C) and (F) The concentration of Cyr61 in the cell supernatant was measured by ELISA. The data are expressed as the mean ± SEM values (n=3). *P < 0.05, **P < 0.01.

Article Snippet: Anti-human Cyr61 monoclonal antibody (093G9) (A gift of professor Ningli Li in Shanghai Jiao Tong University School of Medicine, Shanghai, China), anti-GAPDH and anti-Bcl-xL (Cell Signaling Technology, Inc., Beverly, MA) were used in this study.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cancer

Article Title: Cyr61 mediates oxaliplatin resistance in colorectal cancer cells by regulating Bcl-xL expression

doi: 10.7150/jca.48891

Figure Lengend Snippet: Cyr61 knockdown reduces the resistance of L-OHP-resistant CRC cells. (A) and (D) Lentiviral vectors expressing shCyr61 were transduced into HCT-8/L-OHP cells and HCT116/L-OHP cells to knock down Cyr61 expression, and the concentration of Cyr61 in the cell supernatant was detected by ELISA. (B) and (E) shCyr61 and shNC cells from HCT-8/L-OHP cells and HCT116/L-OHP cells were treated with increasing doses of L-OHP for 72 h and subjected to a CCK8 assay; the IC50 of L-OHP in the cells was then calculated using GraphPad Prism 5.0. (C) and (F) HCT-8 and HCT116 cells were preincubated with exogenous Cyr61 (1 µg/ml) for 24 h and were then treated with increasing doses of L-OHP. After incubation for 72 hours and analysis with a CCK8 assay, the IC50 of L-OHP in the cells was calculated using GraphPad Prism 5.0. The data are expressed as the mean ± SEM values (n=3). *P < 0.05, **P < 0.01.

Article Snippet: Anti-human Cyr61 monoclonal antibody (093G9) (A gift of professor Ningli Li in Shanghai Jiao Tong University School of Medicine, Shanghai, China), anti-GAPDH and anti-Bcl-xL (Cell Signaling Technology, Inc., Beverly, MA) were used in this study.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Incubation

Journal: Journal of Cancer

Article Title: Cyr61 mediates oxaliplatin resistance in colorectal cancer cells by regulating Bcl-xL expression

doi: 10.7150/jca.48891

Figure Lengend Snippet: Cyr61 knockdown enhances L-OHP-induced apoptosis in L-OHP-resistant CRC cells . (A) HCT-8/L-OHP-shCyr61 and HCT-8/L-OHP-shNC cells were treated with L-OHP (50 µg/ml) for 72 h, and apoptosis was assessed with a BD FACSCanto II cytometer. (B) HCT116/L-OHP-shCyr61 and HCT116/L-OHP-shNC cells were treated with L-OHP (30 µg/ml) for 72 h, and the percentages of apoptotic cells were determined with a BD FACSCanto II cytometer. The data are expressed as the mean ± SEM values (n=3). *P < 0.05, **P < 0.01.

Article Snippet: Anti-human Cyr61 monoclonal antibody (093G9) (A gift of professor Ningli Li in Shanghai Jiao Tong University School of Medicine, Shanghai, China), anti-GAPDH and anti-Bcl-xL (Cell Signaling Technology, Inc., Beverly, MA) were used in this study.

Techniques: Cytometry

Journal: Journal of Cancer

Article Title: Cyr61 mediates oxaliplatin resistance in colorectal cancer cells by regulating Bcl-xL expression

doi: 10.7150/jca.48891

Figure Lengend Snippet: Bcl-xL is involved in the effect of Cyr61 on drug resistance. (A) Left panel: Bcl-2, Bcl-xL, XIAP and Survivin mRNA levels in HCT-8/L-OHP and parental HCT-8 cells were determined by real-time PCR; Right panel: Bcl-xL protein levels in HCT-8/L-OHP and parental HCT-8 cells were determined by western blotting. (B) HCT-8/L-OHP-shCyr61 and HCT-8/L-OHP-shNC cells were treated with L-OHP (50 µg/ml) for 72 h, and the protein level of Bcl-xL was detected by western blotting. (C) HCT-8-Cyr61 and HCT-8-NC cells were cultured for 72 h, and the concentration of Cyr61 in the cell supernatant was determined by ELISA. (D) HCT8-Cyr61 and HCT8-NC cells were treated with L-OHP (10 µg/ml) for 72 h, and the protein level of Bcl-xL was determined by western blotting. (E) HCT-8-Cyr61 and HCT-8-NC cells were treated with L-OHP (10 µg/ml), A1155463 (5 µM) (a specific Bcl-xL inhibitor), or L-OHP + A1155463 for 72 h, and apoptosis was analyzed by flow cytometry. (F) Left panel: HCT-8-Cyr61 and HCT-8-NC cells were treated with increasing doses of L-OHP or increasing doses of L-OHP+ A1155463 (5 µM) for 72 h and were then analyzed with a CCK8 assay The IC50 of L-OHP in HCT-8-Cyr61 and HCT-8-NC cells was calculated using GraphPad Prism 5.0. (G) HCT116/L-OHP-shCyr61 and HCT116/L-OHP-shNC cells were treated with L-OHP (30 µg/ml) for 72 h, and the protein level of Bcl-xL was determined by western blotting. (H) HCT116-Cyr61 and HCT116-NC cells were cultured for 72 h, and the concentration of Cyr61 in the cell supernatant was determined by ELISA. (I) HCT116-Cyr61 and HCT116-NC cells were treated with L-OHP (10 µg/ml) for 72 h, and the protein level of Bcl-xL was determined by western blotting. (J) HCT116-Cyr61 and HCT116-NC cells were treated with increasing doses of L-OHP or increasing doses of L-OHP+ A1155463 (5 µM) for 72 h and were then analyzed with a CCK8 assay. Then, the IC50 of L-OHP in HCT116-Cyr61 and HCT116-NC cells was calculated using GraphPad Prism 5.0. The data are expressed as the mean ± SEM values (n=3). *P < 0.05, **P < 0.01.

Article Snippet: Anti-human Cyr61 monoclonal antibody (093G9) (A gift of professor Ningli Li in Shanghai Jiao Tong University School of Medicine, Shanghai, China), anti-GAPDH and anti-Bcl-xL (Cell Signaling Technology, Inc., Beverly, MA) were used in this study.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, CCK-8 Assay

Journal: Journal of Gynecologic Oncology

Article Title: Downregulation of LINC01508 contributes to cisplatin resistance in ovarian cancer via the regulation of the Hippo-YAP pathway

doi: 10.3802/jgo.2021.32.e77

Figure Lengend Snippet: The cell function analysis are all chemically synthesized interference lncRNA and eukaryotic plasmid-mediated lncRNA transient transfection. These have limited gene silencing and overexpression efficiency and show that the correlation between LNC01508 and OC cisplatin resistance is not highly significant. Furthermore, the effect of eukaryotic plasmid-mediated lncRNA overexpression is better than the chemical synthesis of lncRNA. This suggests a stable transfection in vitro to provide knockout and overexpression effects. (A) qPCR detects a lower LINC01508 expression, and the CCK-8 assay measures the cell viability. IC 50 values in LINC01508 inhibition OV2008 cells. (B) qPCR inspects a higher LINC01508 expression, and the CCK-8 assay measures the cell viability. IC 50 values in LINC01508 overexpression C13K cells. (C and D) Colony formation assays were conducted to determine the cell proliferation ability for transfected OC cells. (E and F) GAPDH, Cyr61, ERCC1, and p-AKT protein levels were determined by Western blot. All experiments are repeated in triplicated, and data are shown as mean±SE. lncRNA, long non-coding RNAs; OC, ovarian cancer; qPCR, quantitative real-time polymerase chain reaction; CCK-8, cell counting kit-8; IC 50 , half maximal inhibitory concentration; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Cyr61, cysteine-rich angiogenic inducer 61; ERCC1, excision repair cross complementing‐group 1; p-AKT, phospho-AKT; SE, standard error. * p<0.05; † p<0.01.

Article Snippet: The primary antibodies used were as follows: yes-associated protein (YAP) and cysteine-rich angiogenic inducer 61 (Cyr61) (sc-15407 and sc-13100, respectively; Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH and phospho-AKT (p-AKT) (sc-166574 and sc-135650, respectively; Santa Cruz Biotechnology), phospho-YAP (p-YAP) and excision repair cross complementing‐group 1 (ERCC1) (4911 and 3885s, respectively; Cell Signaling Technology).

Techniques: Cell Function Assay, Synthesized, Plasmid Preparation, Transfection, Over Expression, Stable Transfection, In Vitro, Knock-Out, Expressing, CCK-8 Assay, Inhibition, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Concentration Assay

Journal: Journal of Gynecologic Oncology

Article Title: Downregulation of LINC01508 contributes to cisplatin resistance in ovarian cancer via the regulation of the Hippo-YAP pathway

doi: 10.3802/jgo.2021.32.e77

Figure Lengend Snippet: (A) Western blot analyses of YAP and p-YAP protein expression in OC cells. (B and C) Cell survival rate and colony formation pictures of C13K cells after treatment of verteporfin and different doses of cisplatin. Data were shown as mean±SE of 3 experiments. (D) Cyr61 protein is marked by green fluorescence. Cyr61 protein was mainly localized in the cytoplasm. Nuclear staining with DAPI is shown in blue (magnification ×400). Representative images from 3 independent experiments are shown. OC, ovarian cancer; YAP, yes-associated protein 1; p-YAP, phospho-YAP; SE, standard error; Cyr61, cysteine-rich angiogenic inducer 61; DAPI, diamidino-2-phenylindole; DMSO, dimethyl sulfoxide. * p<0.05; † p<0.01, compared with DMSO group.

Article Snippet: The primary antibodies used were as follows: yes-associated protein (YAP) and cysteine-rich angiogenic inducer 61 (Cyr61) (sc-15407 and sc-13100, respectively; Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH and phospho-AKT (p-AKT) (sc-166574 and sc-135650, respectively; Santa Cruz Biotechnology), phospho-YAP (p-YAP) and excision repair cross complementing‐group 1 (ERCC1) (4911 and 3885s, respectively; Cell Signaling Technology).

Techniques: Western Blot, Expressing, Fluorescence, Staining