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: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Spermine treatment inhibited NLRP3-mediated inflammatory response in DSS-treated mice. ( A – E ) Genes expressions related to NLRP3-mediated inflammatory response in the colon were measured. ( F – I ) The levels of pro-inflammatory cytokines in the serum. ( J ) Detection of NLRP3 inflammasome protein expression levels in the colon using immunohistochemistry. Data are presented as mean ± SEM (* p < 0.05, *** p < 0.001 vs. control group. ## p < 0.01, and ### p < 0.001 vs. DSS group).

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Immunohistochemistry, Control

Journal: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Potential mechanism of Spermine treatment alleviating DSS-induced colitis. First, spermidine enhances mitochondrial autophagy by upregulating the expression of PINK1/Parkin, thereby eliminating damaged mitochondria and reducing the production of reactive oxygen species. This, in turn, inhibits the activation of the NLRP3 inflammasome and the subsequent release of IL-1β/IL-18, thereby alleviating the inflammatory response. Secondly, spermidine can reverse the intestinal flora imbalance induced by DSS, manifested as a decrease in the ratio of Firmicutes to Bacteroidetes, inhibition of Blautia enrichment, and restoration of the abundance of beneficial Muribaculaceae. These two pathways work together in synergy, jointly forming an integrated mechanism by which spermidine inhibits the NLRP3-mediated inflammatory response, promotes mitochondrial autophagy, and improves intestinal flora imbalance to alleviate DSS-induced colitis.

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A is a more Potent Tumor Suppressor than PTEN-WT. (A) In the PTEN-4A mutant protein, the serine-threonine cluster (S380, T382, T383, S385) in the PTEN C-tail is replaced by alanine residues, converting it into an open/active conformer. (B) Lentiviral transduction, followed by puromycin selection, was used to stably express Flag PTEN-WT and Flag PTEN-4A proteins in PTEN deficient NSCLC cell line H1299. The expression levels of both PTEN-WT and PTEN-4A proteins were similar. (C) PTEN-4A inhibited cell proliferation significantly more than PTEN-WT in a standard cell proliferation assays (i). Even in the presence of a proliferative signal such as leptin, PTEN-4A remained a potent inhibitor of cell proliferation (ii). Data are derived from experiments performed in triplicates ± S.E. (n = 3, *p<0.05). (D) Cell proliferation assays on H1299, PTEN-WT and PTEN-4A cells measured by the ECIS method also revealed that PTEN-4A (black line) significantly suppressed cell proliferation as compared to PTEN-WT (pink line). (E) Likewise, PTEN-4A (blue line) significantly inhibited the migratory potential of H1299 lung cancer cells as compared to PTEN-WT (red line).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Mutagenesis, Transduction, Selection, Stable Transfection, Expressing, Derivative Assay

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Preferentially Localizes to the Nucleus. (A) Total nuclear proteins isolated from H1299 cells stably expressing Flag-tagged PTEN-WT and PTEN-4A revealed that PTEN-4A preferentially localized to the nucleus as compared to PTEN-WT, as examined by immunoblotting with Flag antibodies. (B) Lamin B1, an exclusively nuclear protein, was used as a loading control and to normalize densitometric values obtained for Flag-tagged PTEN-WT and PTEN-4A protein levels in the nucleus. Levels of PTEN-4A was approximately 2.5-fold higher in the nucleus than PTEN-WT. (C) Preferential nuclear localization for PTEN-4A detected by immunofluorescence signals of GFP-tagged PTEN-WT and GFP-tagged PTEN-4A protein expression in 293T cells. Expression of H2B mCherry stained the nucleus red. Data are derived from three independent experiments ± S.E. (n = 3, *p<0.05).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Isolation, Stable Transfection, Expressing, Western Blot, Control, Immunofluorescence, Staining, Derivative Assay

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Preferentially Inhibits E2F1-mediated Transcription. (A) Transcriptional assays on four E2F1-responsive promoter-luciferase reporter plasmids co-transfected with E2F1 expression plasmid in H1299 cancer cells indicate that the four promoters are all activated by E2F1 expression. (B) PTEN-4A preferentially suppresses (∼3 fold) the transcription from an artificial E2F1 reporter (E2F-Luc) compared to PTEN-WT. (C) PTEN-4A suppresses transcription mediated by the native E2F1 promoter 2.5 fold more than PTEN-WT. (D) Both PTEN-WT and PTEN-4A likewise suppress transcription mediated by the cyclin E1 promoter. However, PTEN-4A suppresses transcription by the cyclin E1-promoter approximately 2 fold more than PTEN-WT. (E) Both PTEN-WT and PTEN-4A suppress cyclin D1-transcription. However, PTEN-4A suppresses transcription by the cyclin D1-promoter 1.5 fold more than PTEN-WT. (F) As compared to PTEN-4A (lane 2), PTEN catalytic mutants, PTEN C124S 4A (lane 3) and PTEN G129E 4A (lane 4) cannot suppress E2F1-mediated transcription as detected by E2F1-Luc reporter activity in H1299 cells. (G) A PTEN-4A mutant lacking the nuclear localization sequence of PTEN (PTEN d32-4A) cannot suppress E2F1-mediated transcription (lanes 2 and 3), compared to PTEN-4A. (H and I) PTEN-WT, PTEN-4A, PTEN C124S-4A, PTEN G129E-4A and PTEN d32-4A containing expression plasmids stably express the proteins upon transient transfection. (J) Expression of GFP-tagged PTEN-4A and PTEN d32-4A followed by immunofluorescence detection indicate that PTEN d32-4A is excluded from the nucleus. The nucleus is stained red by using a H2B mCherry construct. Positive co-localization (yellow color) was counted independently by two researchers using ImageJ after merging Texas Red and GFP field images. Ten fields/transfection were recorded by each of the two researchers and the data obtained are represented from three independent experiments carried out in triplicate, represented as mean ± S.E. (*p value ≤0.05).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Mutagenesis, Sequencing, Stable Transfection, Immunofluorescence, Staining, Construct

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Physically Interacts with E2F1 Protein and at E2F1 DNA Binding Sites on Chromatin. (A) Both Flag-PTEN-WT and Flag-PTEN-4A proteins physically associated with the HA-E2F1 containing protein complex in co-immunoprecipitation assays performed on cell extracts derived from 293T cells (indicated by arrows). (B) Endogenous PTEN and E2F1 proteins interact in 293T cell nuclear extracts. (C) Chromatin-immunoprecipitation assays indicate that both PTEN-4A and E2F1 associated with E2F1 DNA-binding sites on the chromatin at the native cyclin D1 and cyclin E1 promoters in the PTEN-4A stable cell line.

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Binding Assay, Immunoprecipitation, Derivative Assay, Chromatin Immunoprecipitation, Stable Transfection