p66shc  (OriGene)

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: p66shc deficiency attenuates high glucose-induced autophagy dysfunction in Schwann cells

doi: 10.4196/kjpp.24.155

Figure Lengend Snippet: S16 rat Schwann cells were treated with glucose for 72 h. (A) Percentages of apoptotic S16 rat Schwann cells by flow cytometry. (B) p66shc protein expression was measured by Western blotting. (C) Schwann cells transfected with siCON and sip66shc and treated with glucose (25 or 150 mM). ROS was measured by DCF-DA fluorescence and flow cytometry. (D) Percentages of apoptotic S16 rat Schwann cells by flow cytometry. β-actin was used as the loading control. Data represent means ± SEM, n = 3. ROS, reactive oxygen species; PI, propidium iodide; DCF-DA, 2,7- dichlorofluorescein. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, siCON + glucose 150 mM compared with siCON + glucose 25 mM; # p < 0.05, sip66shc + glucose 150 mM compared with siCON + glucose 150 mM.

Article Snippet: In duplicate, cultured cells were treated with 25, 50, 100, and 150 mM glucose for 72 h. S16 rat SCs were transfected with short interfering RNA (siRNA) for 72 h (rat p66shc siRNA: sense-UGAGUCUCUGUCAUCGCUGUU and antisense-CAGCGAUGACAGAGACUCAUU [Bioneer]) and negative control siRNA using the Lipofectamine 2000 reagent (Invitrogen) per the manufacturer’s recommendations.

Techniques: Flow Cytometry, Expressing, Western Blot, Transfection, Fluorescence, Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: p66shc deficiency attenuates high glucose-induced autophagy dysfunction in Schwann cells

doi: 10.4196/kjpp.24.155

Figure Lengend Snippet: Schwann cells transfected with siCON and sip66shc and treated with glucose (25 or 150 mM). (A) Expression of autophagy-related proteins (p62 and LC3-II) and p66shc by Western blotting in S16 rat Schwann cells. (B) Immunofluorescence images showing expression of p62 (red) or LC3 (red), and p66shc (green). (C) p62 and LC3 intensity was quantified per cell. Nuclei were stained with DAPI. Scale bar, 20 μm. β-actin was used as the loading control. Data represent means ± SEM, n = 3. DAPI, 4,6-diamidino-2-phenylindole. **p < 0.01 and ***p < 0.001, siCON + glucose 150 mM compared with siCON + glucose 25 mM; # p < 0.05, ## p < 0.01, and ### p < 0.001, sip66shc + glucose 150 mM compared with siCON + glucose 150 mM.

Article Snippet: In duplicate, cultured cells were treated with 25, 50, 100, and 150 mM glucose for 72 h. S16 rat SCs were transfected with short interfering RNA (siRNA) for 72 h (rat p66shc siRNA: sense-UGAGUCUCUGUCAUCGCUGUU and antisense-CAGCGAUGACAGAGACUCAUU [Bioneer]) and negative control siRNA using the Lipofectamine 2000 reagent (Invitrogen) per the manufacturer’s recommendations.

Techniques: Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: p66shc deficiency attenuates high glucose-induced autophagy dysfunction in Schwann cells

doi: 10.4196/kjpp.24.155

Figure Lengend Snippet: (A, B) Expression of p66shc by Western blotting and immunofluorescence staining, respectively in sciatic nerves from CON and T1DM mice. β-actin was used as the loading control. Nuclei were stained with DAPI. Scale bar, 20 μm. Data represent means ± SEM, n = 15. STZ, streptozotocin; CON, control (saline); T1DM, type 1 diabetes; DAPI, 4,6-diamidino-2-phenylindole. **p < 0.01 and ***p < 0.001.

Article Snippet: In duplicate, cultured cells were treated with 25, 50, 100, and 150 mM glucose for 72 h. S16 rat SCs were transfected with short interfering RNA (siRNA) for 72 h (rat p66shc siRNA: sense-UGAGUCUCUGUCAUCGCUGUU and antisense-CAGCGAUGACAGAGACUCAUU [Bioneer]) and negative control siRNA using the Lipofectamine 2000 reagent (Invitrogen) per the manufacturer’s recommendations.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Control, Saline

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: p66shc deficiency attenuates high glucose-induced autophagy dysfunction in Schwann cells

doi: 10.4196/kjpp.24.155

Figure Lengend Snippet: (A) Expression of autophagy-related proteins (p62, and LC3-II) by Western blotting in sciatic nerves from CON, T1DM, p66shc KO, and p66shc KO + T1DM mice. β-actin was used as the loading control. (B) Immunofluorescence of the expression of the autophagy-related proteins p62 (red), or LC3 (red), and p66shc (green) in sciatic nerves from CON, T1DM, p66shc KO, and p66shc KO + T1DM mice. (C) The fluorescent area of p62 and LC3 was quantified. Nuclei were stained with DAPI. Scale bar, 20 μm. Data represent means ± SEM, n = 15. CON, control (saline); T1DM, type 1 diabetes mice; p66shc KO, p66shc knockout mice; p66shc KO + T1DM, p66shc KO type 1 diabetic mice; DAPI, 4,6-diamidino-2-phenylindole. *p < 0.05 and ***p < 0.001, T1DM compared with CON; # p < 0.05, ## p < 0.01, and ### p < 0.001, p66shc KO + T1DM compared with T1DM.

Article Snippet: In duplicate, cultured cells were treated with 25, 50, 100, and 150 mM glucose for 72 h. S16 rat SCs were transfected with short interfering RNA (siRNA) for 72 h (rat p66shc siRNA: sense-UGAGUCUCUGUCAUCGCUGUU and antisense-CAGCGAUGACAGAGACUCAUU [Bioneer]) and negative control siRNA using the Lipofectamine 2000 reagent (Invitrogen) per the manufacturer’s recommendations.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, Saline, Knock-Out

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Combination therapy induced phosphorylation of p66shc to increase ROS production, leading to cell apoptosis. (A) U251 and U87 cells were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. Then, the cells were incubated with DCFH‐DA for 1 h in a 37°C cell culture incubator. Cellular ROS level was detected by flow cytometry. (B) Quantitative analysis of the ROS‐positive cell ratio in A. (C) Expression of p66shc and its phosphorylated protein in U251 and U87 cells with the same treatment above. β‐Actin was used as a loading control. (D) The efficacy of p66shc silencing was assessed by western blotting. β‐actin was used as a loading control. (E) U251 and U87 cells were pretreated with siRNA3# (20 μM) for 48 h, then the cells were treated as described in A. Cellular ROS level after p66shc silencing was detected by flow cytometry. (F) Quantitative analysis of the ROS‐positive cell ratio in E. (G) U251 and U87 cells with or without p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The expression of p66shc, phosphorylated protein and apoptosis‐related proteins. β‐actin was used as a loading control. (H) U251 and U87 cells with p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The ratio of apoptotic cells is determined by flow cytometry. (I) Quantitative data of U251 in H. (J) Quantitative data of U87 in H. Data were analyzed by independent‐samples t ‐test or one‐way ANOVA with tukey's multiple comparisons test. ** p < 0.01, **** p < 0.0001.

Article Snippet: Cells were transfected with p66shc small interfering RNA (siRNA) or control siRNA (RiboBio, Guangdong, China), following the supplier's protocol.

Techniques: Phospho-proteomics, Infection, Incubation, Cell Culture, Flow Cytometry, Expressing, Control, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Schematic diagram of TS‐2021 and Olaparib synergistically enhancing anti‐tumor efficacy. TS‐2021 and olaparib synergistically promote the distribution and replication of TS‐2021 in GBM cells, synergistically reduce the activation of PARP, enhance the DNA damage of GBM cells, and up‐regulate the apoptosis of tumor cells induced by p66shc phosphorylation. The above diagram was created by BioRender.com .

Article Snippet: Cells were transfected with p66shc small interfering RNA (siRNA) or control siRNA (RiboBio, Guangdong, China), following the supplier's protocol.

Techniques: Activation Assay, Phospho-proteomics

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Combination therapy induced phosphorylation of p66shc to increase ROS production, leading to cell apoptosis. (A) U251 and U87 cells were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. Then, the cells were incubated with DCFH‐DA for 1 h in a 37°C cell culture incubator. Cellular ROS level was detected by flow cytometry. (B) Quantitative analysis of the ROS‐positive cell ratio in A. (C) Expression of p66shc and its phosphorylated protein in U251 and U87 cells with the same treatment above. β‐Actin was used as a loading control. (D) The efficacy of p66shc silencing was assessed by western blotting. β‐actin was used as a loading control. (E) U251 and U87 cells were pretreated with siRNA3# (20 μM) for 48 h, then the cells were treated as described in A. Cellular ROS level after p66shc silencing was detected by flow cytometry. (F) Quantitative analysis of the ROS‐positive cell ratio in E. (G) U251 and U87 cells with or without p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The expression of p66shc, phosphorylated protein and apoptosis‐related proteins. β‐actin was used as a loading control. (H) U251 and U87 cells with p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The ratio of apoptotic cells is determined by flow cytometry. (I) Quantitative data of U251 in H. (J) Quantitative data of U87 in H. Data were analyzed by independent‐samples t ‐test or one‐way ANOVA with tukey's multiple comparisons test. ** p < 0.01, **** p < 0.0001.

Article Snippet: The extracted cell proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes (Millipore, Billerica, MA), which were then blocked in blocking buffer (Beyotime, Shanghai, China) and incubated with the following primary antibodies: anti‐caspase‐3 (1:1000, 9665; CST), anti‐PARP (1:1000, 9532; CST), anti‐γH2AX Ser139 (1:1000, AF5836; Beyotime, Shanghai, China), anti‐PAR (1:1000, 83732; CST), anti‐p66shc (1:1000, A7725; ABclonal, Hubei, China), and anti‐phospho‐Ser36‐p66shc (1:1000, YP1075; Immunoway, USA).

Techniques: Phospho-proteomics, Infection, Incubation, Cell Culture, Flow Cytometry, Expressing, Control, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Schematic diagram of TS‐2021 and Olaparib synergistically enhancing anti‐tumor efficacy. TS‐2021 and olaparib synergistically promote the distribution and replication of TS‐2021 in GBM cells, synergistically reduce the activation of PARP, enhance the DNA damage of GBM cells, and up‐regulate the apoptosis of tumor cells induced by p66shc phosphorylation. The above diagram was created by BioRender.com .

Article Snippet: The extracted cell proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes (Millipore, Billerica, MA), which were then blocked in blocking buffer (Beyotime, Shanghai, China) and incubated with the following primary antibodies: anti‐caspase‐3 (1:1000, 9665; CST), anti‐PARP (1:1000, 9532; CST), anti‐γH2AX Ser139 (1:1000, AF5836; Beyotime, Shanghai, China), anti‐PAR (1:1000, 83732; CST), anti‐p66shc (1:1000, A7725; ABclonal, Hubei, China), and anti‐phospho‐Ser36‐p66shc (1:1000, YP1075; Immunoway, USA).

Techniques: Activation Assay, Phospho-proteomics

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Combination therapy induced phosphorylation of p66shc to increase ROS production, leading to cell apoptosis. (A) U251 and U87 cells were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. Then, the cells were incubated with DCFH‐DA for 1 h in a 37°C cell culture incubator. Cellular ROS level was detected by flow cytometry. (B) Quantitative analysis of the ROS‐positive cell ratio in A. (C) Expression of p66shc and its phosphorylated protein in U251 and U87 cells with the same treatment above. β‐Actin was used as a loading control. (D) The efficacy of p66shc silencing was assessed by western blotting. β‐actin was used as a loading control. (E) U251 and U87 cells were pretreated with siRNA3# (20 μM) for 48 h, then the cells were treated as described in A. Cellular ROS level after p66shc silencing was detected by flow cytometry. (F) Quantitative analysis of the ROS‐positive cell ratio in E. (G) U251 and U87 cells with or without p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The expression of p66shc, phosphorylated protein and apoptosis‐related proteins. β‐actin was used as a loading control. (H) U251 and U87 cells with p66shc silencing were treated with olaparib (1 μM) and infected with TS‐2021 (MOI = 300) for 72 h. The ratio of apoptotic cells is determined by flow cytometry. (I) Quantitative data of U251 in H. (J) Quantitative data of U87 in H. Data were analyzed by independent‐samples t ‐test or one‐way ANOVA with tukey's multiple comparisons test. ** p < 0.01, **** p < 0.0001.

Article Snippet: The extracted cell proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes (Millipore, Billerica, MA), which were then blocked in blocking buffer (Beyotime, Shanghai, China) and incubated with the following primary antibodies: anti‐caspase‐3 (1:1000, 9665; CST), anti‐PARP (1:1000, 9532; CST), anti‐γH2AX Ser139 (1:1000, AF5836; Beyotime, Shanghai, China), anti‐PAR (1:1000, 83732; CST), anti‐p66shc (1:1000, A7725; ABclonal, Hubei, China), and anti‐phospho‐Ser36‐p66shc (1:1000, YP1075; Immunoway, USA).

Techniques: Phospho-proteomics, Infection, Incubation, Cell Culture, Flow Cytometry, Expressing, Control, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Olaparib Enhances the Efficacy of Third‐Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc‐Induced Apoptosis

doi: 10.1111/cns.70124

Figure Lengend Snippet: Schematic diagram of TS‐2021 and Olaparib synergistically enhancing anti‐tumor efficacy. TS‐2021 and olaparib synergistically promote the distribution and replication of TS‐2021 in GBM cells, synergistically reduce the activation of PARP, enhance the DNA damage of GBM cells, and up‐regulate the apoptosis of tumor cells induced by p66shc phosphorylation. The above diagram was created by BioRender.com .

Article Snippet: The extracted cell proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes (Millipore, Billerica, MA), which were then blocked in blocking buffer (Beyotime, Shanghai, China) and incubated with the following primary antibodies: anti‐caspase‐3 (1:1000, 9665; CST), anti‐PARP (1:1000, 9532; CST), anti‐γH2AX Ser139 (1:1000, AF5836; Beyotime, Shanghai, China), anti‐PAR (1:1000, 83732; CST), anti‐p66shc (1:1000, A7725; ABclonal, Hubei, China), and anti‐phospho‐Ser36‐p66shc (1:1000, YP1075; Immunoway, USA).

Techniques: Activation Assay, Phospho-proteomics