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: Cell Proliferation

Article Title: FGF6 promotes cardiac repair after myocardial infarction by inhibiting the Hippo pathway

doi: 10.1111/cpr.13221

Figure Lengend Snippet: FGF6 inhibits activation of the Hippo pathway in NRCMs after OGD. (A) Western blot was performed to determine the protein levels of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in NRCMs in different group. n >4 per group. (B) Western blot analysis the subcellular localization of YAP in NRCMs in different group. Quantitative analysis of expressions of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in (A), quantitative analysis of expressions of YAP in nuclear extracts. (C) Representative immunofluorescence (green for YAP, blue for DAPI) analysis subcellular localization of YAP in NRCMs in different group. n = 5 per group. (D) RT‐PCR analysis of the mRNA levels of YAP and CTGF in NRCMs in different group. n = 3 per group. Data represent means ± SEM. Two‐tailed Student's t test. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373,927), YAP (CST, 14074), p‐YAP (CST, 13008), p‐ERK1/2 (CST, 9101), ERK1/2 (CST, 9102), p‐LATS1 (CST, 4668), LATS1 (Abcam, ab243656), p‐MST1 (CST, 49332), MST1 (CST, 14946), Cyclin D1 (CST, 55506), Cyclin E1 (CST, 20808), PCNA (CST, 13110), CTGF (Abcam, ab6992), and CYR61 (CST, 39382, Abcam, ab228592). β‐Actin (CST, 4970) was used as the internal reference to normalized protein expression levels.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Proliferation

Article Title: FGF6 promotes cardiac repair after myocardial infarction by inhibiting the Hippo pathway

doi: 10.1111/cpr.13221

Figure Lengend Snippet: FGF6 inhibits activation of the Hippo pathway in CMs after MI. (A) Western blot was performed to determine the protein levels of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61 in the heart from different group mice. n >3 per group. (B) Western blot analysis the subcellular localization of YAP in the heart from different group mice. n = 3 per group. Quantitative analysis of expressions of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF, and CYR61 in (A), quantitative analysis of expressions of YAP in nuclear extracts. (C) Representative immunofluorescence (green for YAP, blue for DAPI) analysis subcellular localization of YAP in the heart section from different group mice. (D) RT‐PCR analysis of the mRNA levels of YAP and CTGF in the heart from different group. n >3 per group; n = 5 per group. Data represent means ± SEM. Two‐tailed Student's t test. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373,927), YAP (CST, 14074), p‐YAP (CST, 13008), p‐ERK1/2 (CST, 9101), ERK1/2 (CST, 9102), p‐LATS1 (CST, 4668), LATS1 (Abcam, ab243656), p‐MST1 (CST, 49332), MST1 (CST, 14946), Cyclin D1 (CST, 55506), Cyclin E1 (CST, 20808), PCNA (CST, 13110), CTGF (Abcam, ab6992), and CYR61 (CST, 39382, Abcam, ab228592). β‐Actin (CST, 4970) was used as the internal reference to normalized protein expression levels.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Proliferation

Article Title: FGF6 promotes cardiac repair after myocardial infarction by inhibiting the Hippo pathway

doi: 10.1111/cpr.13221

Figure Lengend Snippet: FGF6 protects cardiac function via YAP. (A) Schematic diagram demonstrates the animal experiment design. (B) Western blot was performed and quantitatively analysed to determine the protein levels of CTGF, CYR61 in the hearts of different group mice. n = 4 per group. (C) Representative M‐mode echocardiographic recording obtained from different group mice. n >7 per group. (D) Quantitative analysis of LV ejection fraction (EF), fractional shortening (FS). (E) Masson trichrome staining was performed to detect cardiac fibrosis area of different group mice. Scale bar = 2 mm. n = 5 per group. (F) TTC staining was performed to detect cardiac ischemic area of different group mice. Scale bar = 2 mm. n = 5 per group. (G) Quantitative analysis of cardiac fibrosis area in (E). (H) Quantitative analysis of cardiac ischemic area in (F). Data represent means ± SEM. Two‐tailed Student's t test. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373,927), YAP (CST, 14074), p‐YAP (CST, 13008), p‐ERK1/2 (CST, 9101), ERK1/2 (CST, 9102), p‐LATS1 (CST, 4668), LATS1 (Abcam, ab243656), p‐MST1 (CST, 49332), MST1 (CST, 14946), Cyclin D1 (CST, 55506), Cyclin E1 (CST, 20808), PCNA (CST, 13110), CTGF (Abcam, ab6992), and CYR61 (CST, 39382, Abcam, ab228592). β‐Actin (CST, 4970) was used as the internal reference to normalized protein expression levels.

Techniques: Western Blot, Staining, Two Tailed Test

Journal: Cell Proliferation

Article Title: FGF6 promotes cardiac repair after myocardial infarction by inhibiting the Hippo pathway

doi: 10.1111/cpr.13221

Figure Lengend Snippet: FGF6 inhibits the Hippo pathway via ERK1/2. (A) Western blot was performed to determine the protein levels of p‐ERK1/2, ERK1/2, p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP and YAP in NRCMs in different group. Quantitative analysis of expressions of p‐MST1, MST1, p‐LATS1, LATS1, p‐YAP, YAP, CTGF and CYR61. n >3 per group. (B) Western blot analysis the subcellular localization of YAP in NRCMs in different group. n = 3 per group. Quantitative analysis of expressions of YAP in nuclear extracts. (C) Representative immunofluorescence (green for YAP, blue for DAPI) analysis subcellular localization of YAP in NRCMs in different group. n = 5 per group. Data represent means ± SEM. Two‐tailed Student's t test. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: The primary antibodies used are as follows: FGF6 (Santa Cruz, sc‐373,927), YAP (CST, 14074), p‐YAP (CST, 13008), p‐ERK1/2 (CST, 9101), ERK1/2 (CST, 9102), p‐LATS1 (CST, 4668), LATS1 (Abcam, ab243656), p‐MST1 (CST, 49332), MST1 (CST, 14946), Cyclin D1 (CST, 55506), Cyclin E1 (CST, 20808), PCNA (CST, 13110), CTGF (Abcam, ab6992), and CYR61 (CST, 39382, Abcam, ab228592). β‐Actin (CST, 4970) was used as the internal reference to normalized protein expression levels.

Techniques: Western Blot, Immunofluorescence, Two Tailed Test