Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: a Representative western blot analysis of GRP78 and AMH from KGN cells at 36 h and 48 h after injection of vehicle or CTX at doses of 250–1250 µg/mL. b Apoptosis assay of KGN cells 36 h and 48 h after injection of vehicle or CTX at doses of 250–1250 µg/mL. Compared with untreated control cells, the level of apoptosis was significantly higher with ≥750 µg/mL CTX. c , d AMH mRNA levels of KGN cells verified by qPCR. e , f The concentration of AMH in the cell-culture medium declined as the CTX dose increased. g , h One-way ANOVA analysis of apoptotic cells displayed on b. All error bars represent the standard error of the mean. * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Western Blot, Injection, Apoptosis Assay, Concentration Assay, Cell Culture

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: KGN cells were treated with CTX or CTX plus GnRHa for 36 h. a AMH concentration in KGN cells verified by western blot. Compared with CTX-treated cells, the accumulation of AMH was remitted by co-treatment with GnRHa. b AMH level in cell-culture medium verified by ELISA. Significant reductions of secreted AMH were prevented by co-treatment with GnRHa. c AMH mRNA levels of cells verified by qPCR. d Typical immunocytochemistry images demonstrate the accumulation of AMH in the cytoplasm of cells treated with 750 µg/mL CTX, which was released by co-treatment with GnRHa. e Colocalization of AMH and GRP78 in KGN cells. Cells were transfected with a plasmid expressing FLAG-tagged AMH, and were treated with the indicated reagents for 36 h. Yellow in the merged images indicates the colocalization of AMH and GRP78. Nuclei were stained with DAPI (4’,6’-diamidino-2-phenylindole). CTX750, 750 µg/mL CTX. CG750, 750 µg/mL CTX+ Goserelin. CTX1000, 1000 µg/mL CTX. CG1000, 1000 µg/mL CTX + Goserelin. Scale bars represent 100 µm. Error bars show the standard error of the mean. One-way ANOVA was used for b – c , * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Concentration Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Transfection, Plasmid Preparation, Expressing, Staining

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: a Expression of proteins including GRP78, ATF4, XBP1, and CHOP in the UPR pathway was assessed by western blotting. KGN cells were treated with 750 or 1000 µg/mL CTX, with or without GnRHa, for 36 h. b , c KGN cells were treated with CTX or CTX plus GnRHa for 36 h. mRNA levels were assessed by RT-qPCR for genes encoding proteins involved in the UPR ( GRP78 , XBP1 , ATF4, and CHOP ), and for genes encoding secretion-related proteins ( SEC12 and SEC16 ). Those mRNAs were upregulated by CTX and downregulated when co-treated with GnRHa. d Typical immunofluorescence staining indicated the localization of AMH within KGN cells. Yellow in the merged images indicates the colocalization of AMH and GRP78. Nuclei were stained with DAPI. Scale bars represent 100 µm. e TM was used for simulation of ER stress, and expressions of proteins in the UPR pathway were assessed by western blotting, the results were consistent with the CTX-treated cells. f TM-induced upregulation of UPR sensors was confirmed by RT-PCR for mRNA expression. One-way ANOVA was used for ( b , c , and f ), error bars show the standard error of the mean. * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: To explore the effect of CTX and/or GnRHa on mouse ovaries, 8-week-old tumor-bearing nude mice were treated with saline, 200 mg/kg CTX, and 200 mg/kg CTX + GnRHa, and ovarian protein were extracted 1 week after treatment. a Expression of proteins in the ER stress pathway was assessed by western blotting. b Expressions of proteins in the autophagy were assessed by western blotting. c Expressions of proteins in the mTOR pathway were assessed by western blotting. d The GRP78, ATF4, XBP1, and CHOP increased significantly in CTX-treated ovaries than the control or CTX + GnRHa group. e Compared with CTX-treated mice, LC3B and Beclin-1 were increased, and P62 was decreased in coadministration with GnRHa mice. f , g The phosphorylation of mTOR and P70S6K were significantly increased in ovaries of CTX alone treated mice. Data are expressed as mean ± SD of three independent experiments. All error bars represent the standard error of the mean. Two-tailed Student’s t test was used for statistical analysis, * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: a Representative western blot analysis of GRP78 and AMH from KGN cells at 36 h and 48 h after injection of vehicle or CTX at doses of 250–1250 µg/mL. b Apoptosis assay of KGN cells 36 h and 48 h after injection of vehicle or CTX at doses of 250–1250 µg/mL. Compared with untreated control cells, the level of apoptosis was significantly higher with ≥750 µg/mL CTX. c , d AMH mRNA levels of KGN cells verified by qPCR. e , f The concentration of AMH in the cell-culture medium declined as the CTX dose increased. g , h One-way ANOVA analysis of apoptotic cells displayed on b. All error bars represent the standard error of the mean. * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Western Blot, Injection, Apoptosis Assay, Concentration Assay, Cell Culture

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: KGN cells were treated with CTX or CTX plus GnRHa for 36 h. a AMH concentration in KGN cells verified by western blot. Compared with CTX-treated cells, the accumulation of AMH was remitted by co-treatment with GnRHa. b AMH level in cell-culture medium verified by ELISA. Significant reductions of secreted AMH were prevented by co-treatment with GnRHa. c AMH mRNA levels of cells verified by qPCR. d Typical immunocytochemistry images demonstrate the accumulation of AMH in the cytoplasm of cells treated with 750 µg/mL CTX, which was released by co-treatment with GnRHa. e Colocalization of AMH and GRP78 in KGN cells. Cells were transfected with a plasmid expressing FLAG-tagged AMH, and were treated with the indicated reagents for 36 h. Yellow in the merged images indicates the colocalization of AMH and GRP78. Nuclei were stained with DAPI (4’,6’-diamidino-2-phenylindole). CTX750, 750 µg/mL CTX. CG750, 750 µg/mL CTX+ Goserelin. CTX1000, 1000 µg/mL CTX. CG1000, 1000 µg/mL CTX + Goserelin. Scale bars represent 100 µm. Error bars show the standard error of the mean. One-way ANOVA was used for b – c , * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Concentration Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Transfection, Plasmid Preparation, Expressing, Staining

Journal: NPJ Breast Cancer

Article Title: GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

doi: 10.1038/s41523-021-00340-7

Figure Lengend Snippet: a Expression of proteins including GRP78, ATF4, XBP1, and CHOP in the UPR pathway was assessed by western blotting. KGN cells were treated with 750 or 1000 µg/mL CTX, with or without GnRHa, for 36 h. b , c KGN cells were treated with CTX or CTX plus GnRHa for 36 h. mRNA levels were assessed by RT-qPCR for genes encoding proteins involved in the UPR ( GRP78 , XBP1 , ATF4, and CHOP ), and for genes encoding secretion-related proteins ( SEC12 and SEC16 ). Those mRNAs were upregulated by CTX and downregulated when co-treated with GnRHa. d Typical immunofluorescence staining indicated the localization of AMH within KGN cells. Yellow in the merged images indicates the colocalization of AMH and GRP78. Nuclei were stained with DAPI. Scale bars represent 100 µm. e TM was used for simulation of ER stress, and expressions of proteins in the UPR pathway were assessed by western blotting, the results were consistent with the CTX-treated cells. f TM-induced upregulation of UPR sensors was confirmed by RT-PCR for mRNA expression. One-way ANOVA was used for ( b , c , and f ), error bars show the standard error of the mean. * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: The primary antibodies used recognized AMH (ab103233, Abcam, Cambridge, UK, 1:100 dilution), glucose-regulated protein 78 (GRP78; 11587-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), cyclic AMP-dependent transcription factor ATF4 (ATF4; BS6475, Bioworld, Nanjing, China, 1:1000 dilution), X-box-binding protein 1 (XBP1; BS70247, Bioworld, 1:1000 dilution), DNA damage-inducible transcript 3 protein (CHOP; BS1527, Bioworld, Nanjing, China, 1:1000 dilution), autophagy-related protein LC3B (LC3B; ab192890, Abcam, Shanghai, China, 1:2000 dilution), beclin-1 (ab207612, Abcam, Shanghai, China, 1:2000 dilution), P62 (P62/SQSTM1;18420-1-AP, Proteintech, 1:1000 dilution), mTOR (2983 T, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Phospho-mTOR (p-mTOR, 5536 T, Cell Signaling Technology, 1:1000 dilution), p70 S6 Kinase (P70S6K, 2708 T, Cell Signaling Technology, 1:1000 dilution), and Phospho-p70 S6 Kinase (p-P70S6K, 9234 T, Cell Signaling Technology, 1:1000 dilution).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: The neuroprotective effect of suramin on 6-OHDA-induced damage in SH-SY5Y cells. (a) SH-SY5Y cells were pretreated with 0.01, 0.1, 1, and 10 μ M suramin for 1 h and then challenged with 20 μ M 6-OHDA for 16 h in the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups. The 6-OHDA-treated group was normalized as 0%. Data are presented as mean ± SEM, and each value contains three replicates and six samples. ∗ Significantly different from the 6-OHDA group. (b) SH-SY5Y cells were pretreated with 10 μ M suramin and then challenged with 20 μ M 6-OHDA for 8 h in the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups. TUNEL staining was performed, and white arrows represent apoptotic cells (scale bar, 100 μ M). The quantification of apoptotic cells is shown. (c) Western blotting of cleaved caspase-3 protein and quantification of each group. Data are presented as mean ± SEM, and each value contains three replicates and six samples. ∗ Significantly different from the control group; # significantly different from the 6-OHDA group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: TUNEL Assay, Staining, Western Blot

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Effect of suramin on 6-OHDA-induced downregulation of phospho-extracellular signal-regulated kinases (p-ERK), phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in SH-SY5Y cells. (a) SH-SY5Y cells were pretreated with 10 μ M suramin for 1 h and then challenged with 20 μ M 6-OHDA for 1 h; western blotting for p-ERK and p-CREB of the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups is shown. SH-SY5Y cells were pretreated with 10 μ M suramin for 1 h and then challenged with 20 μ M 6-OHDA for 8 h; western blotting for BDNF of the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups is shown. β -Actin was used as an internal control. (b) The quantification results of p-ERK relative density are shown. (c) The quantification results of p-CREB relative density are shown. (d) The quantification results of p-ERK relative density are shown. Data are presented as mean ± SEM, and each value contains three replicates and three samples. ∗ Significantly different from the control group; # significantly different from the 6-OHDA group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Binding Assay, Derivative Assay, Western Blot

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Effect of PTP1B knockdown in SH-SY5Y on PTP1B expression and neuroprotective effect against 6-OHDA damage. (a) SH-SY5Y cells were transfected with siRNAs by LipofectAMINE PlusReagent for 3 h and incubated with new medium overnight. Western blotting for PTP1B of the control (negative control), 6-OHDA (negative control), control (positive control), 6-OHDA (positive control), control (PTP1B siRNA), and 6-OHDA (PTP1B siRNA) groups is shown. Data are presented as mean ± SEM, and each value contains three replicates and three samples. ∗ Significantly different from the control (negative control) group. (b) SH-SY5Y cells were transfected with siRNAs by LipofectAMINE PlusReagent for 3 h and incubated with new medium overnight. Cell viability of the control (negative control), 6-OHDA (negative control), control (positive control), 6-OHDA (positive control), control (PTP1B siRNA), and 6-OHDA (PTP1B siRNA) groups is measured. Data are presented as mean ± SEM, and each value contains three replicates and three samples. ∗ Significantly different from the control (negative control) group. # Significantly different from the 6-OHDA (negative control) group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Expressing, Transfection, Incubation, Western Blot, Negative Control, Positive Control

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Effect of PTP1B overexpression in SH-SY5Y on PTP1B expression and neuroprotective effect against 6-OHDA damage. (a) PTP1B-overexpressed SH-SY5Y was established by the transfection of PTP1B plasmid. Western blotting for PTP1B of the control, #1 PTP1B-overexpressed SH-SY5Y, and #2 PTP1B-overexpressed SH-SY5Y groups are shown. (b) Normal SH-SY5Y cells were pretreated with 0.01, 0.1, 1, and 10 μ M suramin for 1 h and then challenged with 20 μ M 6-OHDA for 16 h in the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups. Data are presented as mean ± SEM, and each value contains three replicates and six samples. ∗ Significantly different from the 6-OHDA group. (c) PTP1B-overexpressed SH-SY5Y was established by the transfection of PTP1B plasmid. Cell viability of the control; 6-OHDA; and 6-OHDA plus 100, 10, 1, and 0.1 μ M suramin groups was measured. Data are presented as mean ± SEM, and each value contains three replicates and three samples. ∗ Significantly different from the control (negative control) group. # Significantly different from the 6-OHDA (negative control) group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Neuroprotective effect of suramin on the 6-OHDA-induced zebrafish PD model. (a) Zebrafish were pretreated with 10 μ M suramin from 9 h after fertilization (hpf) to 3 days after fertilization (dpf) and then challenged with 250 µ M 6-OHDA from 2 to 3 dpf. Quantitative PCR of PTP1B for the control and 6-OHDA groups was performed. (b) Zebrafish were pretreated with 10 μ M suramin from 9 hpf to 3 dpf and then challenged with 250 µ M 6-OHDA from 2 to 3 dpf. Quantitative PCR of BDNF for the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups was performed. (c) Zebrafish were pretreated with 10 μ M suramin from 9 hpf to 3 dpf and then challenged with 250 µ M 6-OHDA from 2 to 3 dpf. Quantitative PCR of iNOS for the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups was performed. (d) Zebrafish were pretreated with 10 μ M suramin from 9 hpf to 5 dpf and then challenged with 250 µ M 6-OHDA from 2 to 5 dpf. Western blotting of p-eIF2 and GRP-78 for the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups was performed. (e) Quantitative results of p-eIF2 protein expression. (f) Quantitative result of GRP-78 protein expression. Data are presented as mean ± SEM, and each value contains three replicates and three samples. ∗ Significantly different from the control; # significantly different from the 6-OHDA group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Effect of suramin on 6-OHDA-induced locomotor deficit and tyrosine hydroxylase (TH) expression in the zebrafish PD model. (a) Zebrafish were pretreated with 0.1, 1, and 10 μ M suramin from 9 hpf to 5 dpf and then challenged with 250 µM 6-OHDA from 2 to 5 dpf. The upper panel demonstrates a representative swimming pattern, and the lower panel shows the average total swimming distance. (b) Zebrafish were pretreated with 10 μ M suramin from 9 hpf to 5 dpf and then challenged with 250 µM 6-OHDA from 2 to 5 dpf. Western blotting of TH for the control, 6-OHDA, 6-OHDA plus suramin, and suramin alone groups was performed. Data are presented as mean ± SEM, and each value represents the mean of 16 samples. ∗ Significantly compared with the control group; # compared with the 6-OHDA group.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Expressing, Western Blot

Journal: Parkinson's Disease

Article Title: Therapeutic Role of Protein Tyrosine Phosphatase 1B in Parkinson's Disease via Antineuroinflammation and Neuroprotection In Vitro and In Vivo

doi: 10.1155/2020/8814236

Figure Lengend Snippet: Schematic diagram of PTP1B in IFN- γ -induced neuroinflammation and 6-OHDA-induced neuronal death. IFN- γ could increase PTP1B expression and further modulate downstream cascade, including the upregulation of proinflammatory cytokines, such as iNOS, COX-2, and NF- κ B. Treatment of 6-OHDA could also affect PTP1B and its downstream neuroprotection-related pathway, including the downregulation of p-CREB, GRP-78, and BDNF and upregulation of p-eIF2. Our study revealed that the PTP1B inhibition by suramin could reverse IFN- γ -induced upregulation of iNOS and COX-2 protein expression. Moreover, suramin could modulate M2 type microglia-related protein and increase arginase-1 and Ym-1 protein expression. PTP1B inhibition also reversed the 6-OHDA-induced downregulation of p-CREB and BDNF protein expression. In the anti-ER stress section, suramin further enhanced 6-OHDA-induced upregulation of p-eIF2 expression and reversed 6-OHDA-induced downregulation of GRP-78 expression. The effect of suramin significantly protected dopamine neurons against damage.

Article Snippet: 6-OHDA (6-hydroxydopamine; Sigma, St. Louis, MO, USA; catalog: H4381) β -Actin (loading control; Sigma, St. Louis, MO, USA; catalog: A5441) Suramin (Sigma, St. Louis, MO, USA; catalog: S2671) iNOS (BD Pharmingen, San Diego, CA, USA; catalog: 610600) COX-2 (Cayman Chemical, Ann Arbor, MI, USA; catalog: 160126) NF- κ B (Merck Millipore, Massachusetts, USA; catalog: MAB3026) Ym-1 (Abcam, Biorbyt, Cambridge, UK; catalog: ab192029) Arg-1 (Proteintech, Chicago, USA; catalog: 16001-1-AP) PTP1B (Proteintech, Chicago, USA; catalog: 11334-1-AP) Phospho-eukaryotic initiation factor 2 (p-eIF2) (antibodies-online; catalog: ABIN2776588) Binding immunoglobulin protein (GRP-78) (Proteintech, Chicago, USA; catalog: 11587-1-AP) Phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling Technology, Danvers, MA, USA, Thr202/204; catalog: 9101) Caspase-3 (IMGENEX, San Diego, CA, USA; catalog: Img-144A) Tyrosine hydroxylase (Merck Millipore, Massachusetts, USA; catalog: MAB3018)

Techniques: Expressing, Inhibition