Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 overexpression antagonizes cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with GFP-HSP70 plasmid or control plasmid. After 24 h, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) Nuclear morphology of apoptotic cells was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P<0.01, with comparisons indicated by lines. HSP70, heat shock protein 70; GFP, green fluorescent protein; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin; CON, control.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Staining, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: Pretreatment with an HSP70 inhibitor enhances cisplatin-induced HGC-27 cells apoptosis. (A) HGC-27 cells were treated with different concentrations of PES for 24 h, and cell viability was measured using a Cell Counting Kit-8 assay. ** P<0.01 compared with untreated cells. (B) HGC-27 cells were pretreated with PES (8 µ M) for 2 h and then stimulated with cisplatin (5 µ g/ml) for 24 h. Expression levels of apoptosis-related proteins PARP, cleaved-caspase-3 and pro-caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P<0.01 with comparisons indicated by lines. HSP70, heat shock protein 70; PES, pifithrin- µ ; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Cell Counting, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 downregulation enhances cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with HSP70 shRNA plasmid and control plasmid, and at 48 h post-transfection, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) The morphology of apoptotic cell nuclei was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Flow cytometry was used to determine apoptosis rates (representative plots and quantification is shown). ** P<0.01 with comparisons indicated by lines. HSP70, heat shock protein 70; sh, short hairpin; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin; CON, control.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

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

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 affects cisplatin-induced MAPK signaling pathway activation. (A) HGC-27 cells were treated with cisplatin (5 µ g/ml) for different amounts of time and the phosphorylation and total protein levels of (A) p38, ERK and JNK, and (B) Src, Akt and IκB, were monitored by western blotting. (C) HSP70-overexpressing and control plasmids were transfected into HGC-27 cells. (D) HGC-27 cells were pretreated with PES for 2 h and then stimulated with cisplatin for 6 or 8 h. (E) HSP70-shRNA and control plasmids were transfected into HGC-27 cells. After transfection, the cells were treated with cisplatin for 6 or 8 h and the phosphorylation and total protein levels of p38, ERK and JNK were monitored by western blotting. * P<0.05 compared with control cells at 6 h; # P<0.05 compared with control cells at 8 h. HSP70, heat shock protein 70; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Src, SRC proto-oncogene non-receptor tyrosine kinase; Akt, AKT serine/threonine kinase 1; IκB, inhibitor of κB; sh, short hairpin; DDP, cisplatin; CON, control; p-, phosphorylated; PES, pifithrin- µ .

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Transfection, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: MAPK pathway inhibition enhances cisplatin-induced HGC-27 cell apoptosis. (A) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 24 h. Expression levels of PARP, cleaved caspase-3 and pro-caspase-3 were detected by western blotting. (B) Apoptotic rate was determined by flow cytometry (representative plots and quantification is shown). (C) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 6 h. Phosphorylation of p38, ERK, JNK and the levels of HSP70 were detected by western blotting. * P<0.05 and ** P<0.01, with comparisons indicated by lines. MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PARP, poly-ADP-ribose-polymerase; HSP70, heat shock protein 70; DDP, cisplatin; p-, phosphorylated.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: Schematic diagram illustrating the signaling pathway involved in the regulation of HSP70 in cisplatin-induced HGC-27 cell apoptosis. HSP70, heat shock protein 70; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: