Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on apoptosis induced by Oxaliplatin and 5-FU in cervical cancer cells. a SHP-2 expression were detected by western blot in Hela cells and C33A cells. SHP-2 expression were detected by western blot in Hela cells, which were transfected SHP-2 shRNA1 or SHP-2 shRNA2 ( b ), and in C33A cells, which were transfected pCMV-SHP2-HA plasmid ( e ). Induction of apoptosis were measured by Annexin-V/PI double-staining assay in Hela cells, which were transfected SHP-2 shRNA1 or SHP-2 shRNA2 ( c , d ), and C33A cells, which were transfected pCMV-SHP2-HA plasmid ( f , g ) and treated different concentrations of Oxaliplatin ( c , f ) and 5-FU ( d , g ). Data are presented as mean ± SD. *P < 0.05, **P < 0.01

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Double Staining

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on the apoptosis induced by Oxaliplatin and 5-FU through autophagy. Cleaved caspases and cleaved PARP expression were detected by western blot in Hela cells ( a ), and C33A cells ( b ). Cells were transfected SHP-2 shRNA1 or pCMV-SHP2-HA plasmid and treated Oxaliplatin (100 μM) or 5-FU (800 μM). Data are presented as mean ± SD. *P < 0.05, **P < 0.01 compared with Oxaliplatin group. # P < 0.05, ## P < 0.01 compared with 5-FU group. Intracellular ROS levels was measured by flow cytometry in Hela cells ( c ) and C33A cells ( d ). LC3 expression were detected by western blot in Hela cells ( e ) and C33A cells ( f ). Data are presented as mean ± SD. *P < 0.05, **P < 0.01

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on mitochondria damage in cervical cancer cells. Hela cells were transfected SHP-2 shRNA1 and treated CCCP (10 μM). a The change of ΔΨm was detected by confocal laser scanning microscope using JC-1 staining. b The Ca 2+ level in cytoplasm was detected by flow cytometry. c , d Intracellular ROS levels was measured by flow cytometry. Data are presented as mean ± SD. **P < 0.01

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Transfection, Laser-Scanning Microscopy, Staining, Flow Cytometry

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on autophagy activation induced by CCCP in Hela cells. a , b LC3 expression were detected by western blot. Hela cells were transfected SHP-2 shRNA1 and treated CCCP (10 μM) for different time. Data are presented as mean ± SD. **P < 0.01. Induction of GFP + dots in Hela cells expressing GFP-LC3. Cells were transfected SHP-2 shRNA1 and treated CCCP (10 μM). Images of individual cells c and quantified of GFP-LC3 + dots ( d ). Data are presented as mean ± SD. **P < 0.01

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Activation Assay, Expressing, Western Blot, Transfection

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on autophagy activation induced by CCCP in C33A cells. a , b LC3 expression were detected by western blot. C33A cells were transfected pCMV-SHP2-HA plasmid and treated CCCP (10 μM). Data are presented as mean ± SD. **P < 0.01 compared with CCCP treatment group. c Expressing mCherry-GFP-LC3 C33A cells were observed under a confocal microscopy after pCMV-SHP2-HA plasmid transfection or CCCP (10 μM) and Baf A1 (30 nM) treatment. Representative images are shown. d The average numbers of yellow or red puncta were obtained from three countings. Data are presented as mean ± SD. **P < 0.01 compared with CCCP treatment group

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: Effect of SHP-2 on clearance of damaged mitochondria. a Confocal microscopy of Hela cells transfected SHP-2 shRNA1 or treated CCCP (10 μM), immunostained for p62 (green) and Tom20 (red). b Confocal microscopy of GFP-LC3 + Hela cells transfected SHP-2 shRNA1 or treated CCCP (10 μM), immunostained for endogenous Tom20. c, d Subcellular distribution of p62, LC3-II, and Parkin a transfected SHP-2 shRNA1 or treated CCCP (10 μM). Data are presented as mean ± SD. **P < 0.01 compared with CCCP treatment group. Data are representative of at least three experiments

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Confocal Microscopy, Transfection

Journal: Cancer Cell International

Article Title: SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

doi: 10.1186/s12935-018-0505-3

Figure Lengend Snippet: The clearance of damaged mitochondria is Parkin-dependent. a Confocal microscopy of Hela cells transfected SHP-2 shRNA1 or treated CCCP (10 μM), immunostained for Parkin (green) and Tom20 (red). b Confocal microscopy of C33A cells transfected Parkin shRNA or pCMV-SHP2-HA plasmid, immunostained for p62 (green) and Tom20 (red). c Confocal microscopy of C33A cells transfected Parkin shRNA or pCMV-SHP2-HA plasmid, immunostained for Poly-Ub (green) and Tom20 (red). Data are representative of at least three experiments

Article Snippet: GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Techniques: Confocal Microscopy, Transfection, shRNA, Plasmid Preparation