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: Theranostics

Article Title: Targeting the splicing factor NONO inhibits GBM progression through GPX1 intron retention

doi: 10.7150/thno.72248

Figure Lengend Snippet: Expression of GPX1 and CCN1 is regulated by NONO-mediated pre-mRNA splicing. A Global splicing efficiency analysis at splicing sites after transfection of U251 and P3 cells with siNC and siNONO. Splicing efficiency was determined as “transread count/5' and 3' intron end first base coverage”. B The top 100 downregulated genes in the P3 sequencing data based on siNC versus siNONO. The expression data was z-transformed. C Venn plot displaying the significantly downregulated mRNAs in both U251 and P3 sequencing data. The genes with decreased splicing efficiency were selected based on splicing analysis. D Sashimi plot visualization of RNA-seq reads mapping to GPX1 and CCN1 in U251 cells in response to NONO knockdown. E Western blot analysis for GPX1 and CCN1 of U251 and P3 transfected with siNC and siNONO (n = 3). F Western blot analysis for GPX1 and CCN1 of LN229- and P3-NONO-OE or -NC (n = 3). G The schematic representation of primers designed for pre-mRNA and mRNA of GPX1 and CCN1 . H qRT-PCR analysis of pre-mRNA and mRNA for GPX1 in U251 and P3 transfected with siNC, siNONO-1 and siNONO-2 (n = 3). GAPDH was used for normalization. I RNA FISH probes for pre-mRNA or mRNA were used for detection in U251 (n = 3). Scale bar = 20 μm. J The NONO binding motif in the intron of GPX1 pre-mRNA. K RNA pulldown assay with GPX1 pre-mRNA, mRNA and anti-sense pre-mRNA to detect binding with NONO in U251 (n = 3). L The RIP-PCR assay with NONO to detect GPX1 pre-mRNA and mRNA (n = 3). Input was used for normalization and IgG was used for negative control. M Representative images of RNA FISH for GPX1 pre-mRNA (green) and GPX1 mRNA (red), and immunofluorescence for NONO (blue) in U251 (n = 3). The right diagram shows the relative gray value of staining on the X-axis (AB). Scale bar = 25 μm. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following primary antibodies were used: anti-NONO (ab70335; Abcam), anti-N-cadherin (13116S; Cell Signaling Technology), anti-CD44 (3570S; Cell Signaling Technology), anti-BCL2 (4223S; Cell Signaling Technology), anti-BAX (50599-1-Ig; ProteinTech; Wuhan, China), anti-GPX1 (ab22604; Abcam), anti-CCN1 (14479S; Cell Signaling Technology), anti-SFPQ (15585-1-AP; ProteinTech), anti-PSPC1 (ab104238; Abcam), and anti-ZEB1 (3396S; Cell Signaling Technology).

Techniques: Expressing, Transfection, Sequencing, Transformation Assay, RNA Sequencing Assay, Western Blot, Quantitative RT-PCR, Binding Assay, Negative Control, Immunofluorescence, Staining

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