Journal: Frontiers in Cellular Neuroscience

Article Title: HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway

doi: 10.3389/fncel.2018.00498

Figure Lengend Snippet: HDAC1-silenced MSCs modulated the protein expression of PI3K/Akt pathway in hippocampus. (A) Representative Western blot bands of PTEN, phosphorylated PTEN (p-PTEN), AKT1, phosphorylated AKT1 (p-AKT1), GSK-3β, and phosphorylated GSK-3β (p-GSK-3β) in each group. (B) Quantitative protein levels were normalized with β-actin. Data are presented as mean ± SEM. * p < 0.05 vs. Vehicle, # p < 0.05 vs. MSCs.

Article Snippet: The membrane was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies respectively directed against Bcl2 (1:500, Proteintech, China), Caspase3 (1:500, Proteintech, China), Cleaved Caspase3 (1:500, Proteintech, China), HDAC1 (1:500; Abcam, Cambridge, England), PTEN (1:500; Sangon Biotech, Shanghai, China), Phospho-PTEN (Ser380/Thr382/Thr383; 1:500; Sangon Biotech, Shanghai, China), AKT-1 (1:500; Sangon Biotech, Shanghai, China), Phospho-AKT1 (Ser473; 1:500; Sangon Biotech, Shanghai, China), GSK-3β (1:500; Proteintech, Wuhan, China), Phospho-GSK-3β (Ser9; 1:500; Cell Signaling Technology, American), or β-actin (1:5,000; Sangon Biotech, Shanghai, China) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Molecules (Basel, Switzerland)

Article Title: Berberine Inhibited Growth and Migration of Human Colon Cancer Cell Lines by Increasing Phosphatase and Tensin and Inhibiting Aquaporins 1, 3 and 5 Expressions.

doi: 10.3390/molecules28093823

Figure Lengend Snippet: Figure 11. Effect of berberine on the expression of PTEN, AKT, p-AKT, mTOR and p-mTOR in CRC

Article Snippet: Membranes were subsequently incubated overnight with the following primary antibodies at 4 ◦C: PTEN (AF847, R&D System; 1:1000), p-AKT-S473 (AF887, R&D System; 1:1000), AKT (MAB2055, R&D System; 1:1000), p-mTOR-S2448 (ab109268, Abcam; 1:1000), mTOR (AHO1232, Thermo Fisher Scientific; 1:1000) and beta actin (2118S; Cell Signaling Technology, Inc.; Danvers, MA, USA; 1:1000).

Techniques: Expressing

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 1. ATM regulated TPT-induced PTEN nuclear translocation in A549 and HeLa cells. (A) Repre- sentative photomicrographs showed endogenous PTEN translocation (100£). A549 and HeLa cells were treated with 3 mg/mL TPT and/or 10 mM KU55933 for 24 h. (B) 3£Flag-PTEN-WT plasmids were transfected into the HeLa cells, and the transfection efficiency was confirmed by western blot analysis. (C) Representative photomicrograph images showed exogenous PTEN translocation (100£). HeLa cells were transiently transfected with 3£Flag-PTEN-WT plasmids, and 24 h after transfection, the cells were treated with 3 mg/mL TPT and/or 10 mM KU55933 and/or ATM siRNA for 24 h. (D) Repre- sentative photomicrograph images showing exogenous PTEN translocation (100£). HeLa cells were transiently transfected with 3£Flag-PTEN-WT or 3£Flag-PTENS113A plasmids, and 24 h after transfec- tion, the cells were treated with 6 mg/mL CDDP. (E) Western blot analysis result of exogenous PTEN nuclear translocation in A549 and HeLa cells when treated with 3 mg/mL TPT and/or 10 mM KU55933 for 24 h. (F) Western blotting result of exogenous PTEN nuclear translocation in A549 and HeLa cells when treated with 3 mg/mL TPT and/or ATM siRNA for 24 h.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Translocation Assay, Transfection, Western Blot

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 2. ATM mediated TPT-induced PTEN phosphorylation. (A) Sequence analysis of PTEN was per- formed by Scansite software. The sequence of PTEN was retrieved from GenBank. (B) A549 cells were treated with 3 mg/mL TPT, l-phosphatase, and PPase inhibitors as indicated. Tagged proteins were analyzed by immunoblotting. (C) HeLa cells were transiently transfected with the 3£Flag-PTEN-WT plasmid，then treated with various concentrations of TPT or CDDP. (D) A549 and HeLa cells were transiently transfected with 3£Flag-PTEN-WT plasmids, and 24 h after transfection, the cells were treated with 3 mg/mL TPT; then, immunoprecipitation was conducted with an antibody against Flag. The precipitates were subjected to SDS-PAGE and probed with antibody against phospho-PTENS113. (E) A549 cells were treated with 3 mg/mL TPT for 24 h in the absence or presence of ATM siRNA- or KU55933, and then immunoblotting analysis was performed. (F) In vitro phosphorylation assays with active ATM kinase and purified PTEN recombinant proteins. Reaction products were subjected to SDS-PAGE and probed with antibody against phospho-PTENS113.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Phospho-proteomics, Sequencing, Software, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, In Vitro, Recombinant

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 3. TPT-induced PTEN nuclear translocation was dependent on PTEN (S113) phosphorylation in A549 and HeLa cells. A549 cells and/or HeLa cells were transiently transfected with the 3£Flag-PTEN-WT or 3£Flag-PTENS113A plasmid, and 24 h after transfection, the cells were treated with or without 3 mg/mL TPT. (A) Representative photomicro- graph images showing PTEN translocation (200£). (B) The expression levels of PTEN and p-PTENS113 were analyzed by immunoblotting. (C) Representative photomicrograph images showing PTEN translocation (200£) after treatment of 10 ng/mL of leptomycin B for 24 h.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Translocation Assay, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 4. The PTENS113A mutant enhanced TPT-induced DNA damage in A549 and HeLa cells. (A) A549 and HeLa cells were transiently transfected with the 3£Flag-PTEN-WT or 3£Flag-PTENS113A plasmid, and 24 h after transfection, the cells were treated with or without 3 mg/mL TPT. Representative photo- micrograph images showed accumulation of g-H2AFX as an indicator of DNA damage (100£). (B) Bar graphs representing the percentage of g-H2AFX-positive cells upon treatment with TPT for 24 h (*P < 0.05, **P <0.001, t test, n D 3 , bars represent SEM, Cells containing more than 5 foci were scored as positive). (C) A549 and HeLa cells were transiently transfected with the 3£Flag-PTEN-WT or 3£Flag- PTENS113A plasmid, and 24 h after transfection, the ells were treated with or without 3 mg/mL TPT. The expression levels of g-H2AFX protein were analyzed by immunoblotting.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 6. Effect of PTEN expression level on TPT-induced autophagy in A549 and HeLa cells. (A) A549 and HeLa cells were stably transfected with PTEN shRNA plasmids. Cells were treated with or without 3 mg/mL TPT for 24 h. The expression levels of PTEN, MAP1LC3/LC3, and SQSTM1/p62 protein were analyzed by immunoblotting. Bar graphs represent the relative MAP1LC3/LC3-II and SQSTM1/p62 protein levels normalized to that of GAPDH of different groups (*P < 0.05, **P < 0.001, t test, n D 3 ). (B) A549 and HeLa cells were transiently transfected with the 3£Flag-PTEN-WT plasmids. Cells were treated with or without 3mg/mL TPT for 24 h. The expression levels of Flag, PTEN, MAP1LC3/LC3, and SQSTM1/p62 protein were analyzed by immunoblotting. Bar graphs represent the relative MAP1LC3/LC3-II and SQSTM1/p62 protein levels normalized to that of GAPDH of different groups. (*P < 0.05, **P < 0.001, t test, n D 3 ). (C) The effect of PTENS113 phosphorylation on autophagy in A549 and HeLa cells. The expression levels of MAP1LC3/LC3 pro- tein were analyzed by immunoblotting. (D) HeLa cells were transiently transfected with the 3£Flag-PTEN-WT or 3£Flag-PTENS113A plasmid and YFP-LC3 plasmids. Twenty four h after transfection, the cells were treated with or without 3 mg/mL TPT. Green YFP autofluorescence (YFP-LC3) was analyzed by confocal microscopy (100£). Bar graphs represent the percentage of YFP- LC3-positive cells in control or TPT-treated group (*P < 0.05, **P < 0.001, t test, n D 3 , bars represent SEM, Cells containing more than 5 foci were scored as positive). (E) A549 and HeLa cells were treated with or without 3 mg/ mL TPT for 24 h, followed by treatment with 100 mM bafilomycin A1 (Baf.A1) as indicated. The expression levels of MAP1LC3/LC3 and SQSTM1/p62 protein were analyzed by immunoblotting.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Western Blot, Phospho-proteomics, Plasmid Preparation, Confocal Microscopy, Control

Journal: Autophagy

Article Title: ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

doi: 10.1080/15548627.2015.1009767

Figure Lengend Snippet: Figure 7. PTEN promoted TPT-induced autophagy via activation of the p-JUN-SESN2-AMPK pathway in A549 and HeLa cells. (A) The cells were treated with or without 3 mg/mL TPT, and the expression levels of JUN, p-JUN, SESN2, AMPK, p-AMPK, RPS6KB, and p-RPS6KB protein were analyzed by immunoblot- ting. (B) The cells were treated with or without 3 mg/mL TPT and/or JUN siRNA for 24 h, and the expres- sion levels of JUN, p-JUN, SESN2, AMPK, p-AMPK, RPS6KB, p-RPS6KB, and LC3 protein were analyzed by immunoblotting. Bar graphs represent the relative MAP1LC3/LC3-II protein levels normalized to that of GAPDH of different groups. (*P < 0.05, **P < 0.001, t test, n D 3 ). (C) The cells were treated with or with- out 3 mg/mL TPT and/or AMPK siRNA for 24 h, and the expression levels of p-AMPK, AMPK, RPS6KB, p-RPS6KB, and MAP1LC3/LC3 protein were analyzed by immunoblotting. Bar graphs represent the rel- ative MAP1LC3/LC3-II protein levels normalized to that of GAPDH of different groups. (*P <0.05, **P < 0.001, t test, n D 3 ). (D) The cells were treated with or without 3 mg/mL TPT, and the expression levels of p-GSK3B, GSK3B, p-AKT, and AKT protein were analyzed by immunoblotting.

Article Snippet: Purified ATM protein (active) (14–933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene.

Techniques: Activation Assay, Expressing, Western Blot

Journal: NPJ Breast Cancer

Article Title: Mcam stabilizes a luminal progenitor-like breast cancer cell state via Ck2 control and Src/Akt/Stat3 attenuation

doi: 10.1038/s41523-024-00687-7

Figure Lengend Snippet: Commercial antibodies used in this study

Article Snippet: anti-phospho-Pten (Ser380/Thr382/383) , 44A7 , Cell Signaling Technology , 9549 , Western Blot , 1:1000 , AB_659891.

Techniques: Western Blot, Immunocytochemistry, Immunoprecipitation

Journal: Human Mutation

Article Title: RNA Panel Sequencing Is an Effective Tool to Help Classify Splice Variants for Clinical Oncogenetic Diagnosis

doi: 10.1155/2024/4830045

Figure Lengend Snippet: List of the 48 genes sequenced in the CJP familial cancer panel.

Article Snippet: PTEN expression was determined by IHC on 3 μ m paraffin sections with the PTEN (D4.3) XP rabbit monoclonal antibody (Cell Signaling Technology, Beverly, MA, United States).

Techniques:

Journal: Human Mutation

Article Title: RNA Panel Sequencing Is an Effective Tool to Help Classify Splice Variants for Clinical Oncogenetic Diagnosis

doi: 10.1155/2024/4830045

Figure Lengend Snippet: Summary of the 53 variants studied by our targeted RNA panel.

Article Snippet: PTEN expression was determined by IHC on 3 μ m paraffin sections with the PTEN (D4.3) XP rabbit monoclonal antibody (Cell Signaling Technology, Beverly, MA, United States).

Techniques: Variant Assay, Modification, Reverse Transcription Polymerase Chain Reaction

Journal: Human Mutation

Article Title: RNA Panel Sequencing Is an Effective Tool to Help Classify Splice Variants for Clinical Oncogenetic Diagnosis

doi: 10.1155/2024/4830045

Figure Lengend Snippet: Study of the PTEN c.206+6T>G variant. (a) RT-PCR analysis on blood sample RNA: peripheral blood of the patient with the PTEN c.206+6T>G variant was collected in PAXgene blood RNA tubes. RT-PCR analysis was performed with primers mapping to exons 2 and 5, and PCR products were separated by bioanalyzer electrophoresis. The 370 bp peak corresponds to the reference PTEN transcript, and the 325 bp peak corresponds to a PTEN transcript lacking exon 3. RT-PCR products were then analyzed by Sanger sequencing. (b) Minigene analysis: HeLa cells were transfected with pCAS2 vectors including wild-type or mutant PTEN sequences. Total RNA was isolated, RT-PCR analysis was performed using pCAS primers, and PCR products were separated by bioanalyzer electrophoresis. The 280 bp band corresponds to the reference PTEN transcript, and the 235 bp band corresponds to a PTEN transcript lacking exon 3. (c) PTEN immunohistochemistry: PTEN expression of the endometrium tumor was determined by immunohistochemistry on 3 μ m paraffin sections with a PTEN rabbit antibody (Cell Signaling Technology). The PTEN IHC control slide was used to validate the technique.

Article Snippet: PTEN expression was determined by IHC on 3 μ m paraffin sections with the PTEN (D4.3) XP rabbit monoclonal antibody (Cell Signaling Technology, Beverly, MA, United States).

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Sequencing, Transfection, Mutagenesis, Isolation, Immunohistochemistry, Expressing, Control