Journal: ACS Synthetic Biology

Article Title: Modular Synthetic Biology Toolkit for Filamentous Fungi

doi: 10.1021/acssynbio.1c00260

Figure Lengend Snippet: Genetic Modules and Other Vectors in the Fungal Toolkit for Modular Cloning (FTK) a

Article Snippet: pFTK054 , 171326 , CDS1 , QS (QA-1S) codon optimized, quinic acid repressor , pICH41308 , pAC-Qsco, (Addgene ID 46106) , ( ) .

Techniques: Cloning, Plasmid Preparation, Selection, Marker, Luciferase, Binding Assay, Sequencing, CRISPR

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: (A) Lung cancer cells were co-transfected with the plasmids of hTERT promoter driven-luciferase and pCDNA3.1-CBP or pCDNA3.1-Lac Z for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (B) Lung cancer cells were co-transfected with hTERT promoter driven-luciferase and CBP siRNA or control siRNA for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (C) Up-regulation of hTERT protein expression by the overexpression of CBP. H1299 cells were treated with pcDNA3.1-CBP for 48 h, and the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot in lung cancer cells. (D, E) Downregulation of hTERT protein expression by the knockdown of CBP expression. H1299 cells were transfected with a CBP-specific siRNA for 48 h, the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot (D), and the telomerase activity was assessed (E). All of the measurements represent the means ± SE of three independent experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Transfection, Luciferase, Control, Expressing, Over Expression, Western Blot, Knockdown, Activity Assay

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: ( A ) H1299 cells were transfected with CBP overexpression vector pcDNA3.1-CBP. At different time points after transfection, cell viability was measured by MTT assay. ( B ) H1299 cells were treated with CBP specific siRNA or inhibitor. At different time points after treatment, cell viability was measured by MTT assay. The mean and SE obtained from three independent experiments are plotted (*, P < 0.05,**, P < 0.01). (C) The nude mice containing xenografts of human lung cancer were intratumorally treated with non-specific control siRNA or CBP-specific siRNA, and the tumor volumes ± SE were calculated at different days after treatment. (N=5; *, P < 0.05). (D) Immunohistochemistry of CBP and hTERT from tumor xenografts in nonspecific control siRNA- and CBP-specific siRNA-treated nude mice (400× magnification).

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Transfection, Over Expression, Plasmid Preparation, MTT Assay, Control, Immunohistochemistry

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Control, Binding Assay, Activity Assay

Journal: ACS chemical biology

Article Title: Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors

doi: 10.1021/acschembio.5b00709

Figure Lengend Snippet: Microfluidic mobility shift analysis enabling real-time monitoring of diverse KAT activities. (a) p300 turnover of FITC-H4 (3–14). (b) Crebbp turnover of FITC-H4 (3–14). (c) Morf turnover of FITC-H4 (3–14). (d) Gcn5 turnover of FITC-H3 (5–20). Model separations for each substrate/product pair are provided as Supporting Information (Figure S2).

Article Snippet: Crebbp (1319–1710) and Morf (431–2073) were obtained from SignalChem.

Techniques: Mobility Shift

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 3 | Overexpression of CITED2 at the animal level is able to alleviate PH. (A) The results of gene enrichment analysis in normoxia and hypoxia showed that CITED2 was downregulated in hypoxia, showing a negative correlation. (B–D) Expression of CITED2 mRNA and protein in the pulmonary arteries of model mice. (E) Schematic diagram of lentivirus model mouse construction. (F) The decreased cardiopulmonary function of model mice was effectively reversed by overexpression of CITED2. (G) Right ventricular systolic blood pressure and right ventricular specific gravity were improved in the CITED2 overexpression group. (H, I) H&E and Masson staining of tissue sections of mouse pulmonary arterioles. (K) The ex- pression of PCNA was downregulated in the pulmonary arteries of mice in the lentivirus-treated group. (J) Tissue immunofluorescence experiments showed that the expression of Ki67 in small pulmonary vessels decreased significantly after CITED2 overexpression. A: artery. (L, M) The expression of cycle-related proteins in the pulmonary arteries of mice in the lentivirus-treated group was downregulated. LV: lentivirus. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Expressing, Staining, Immunofluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 4 | Overexpression of CITED2 can affect the proliferation of smooth muscle cells and hinder cell cycle progression. (A) Western blot re- sults of efficiency detection after overexpression of CITED2. (B) The results of the CCK8 experiment indicated that overexpression of CITED2 could affect the growth and viability of mPASMCs. (C) Overexpression of CITED2 affected the expression of PCNA in cells. (D) After overexpression of CITED2, the proportion of cells in the G2/M and S phase was reduced, which affected cell division. (E, F) The results of the EdU cell fluorescence assay and Ki67 cell fluorescence assay indicated that overexpression of CITED2 could reduce cell proliferation. Red and green represent remark- ably proliferating cells. (G, H) Overexpression of CITED2 at the cellular level can affect the expression levels of multiple proteins in the cell cycle. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Western Blot, Expressing, Fluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 6 | FOXJ3 cooperates with SEs to regulate the transcription of CITED2. (A) Schematic representation of overexpression plasmid con- struction and transfection. (B–D) In the dual-luciferase experiment, plasmids containing three SEs motifs were co-transfected with FOXJ3 plasmids into mPASMCs. When both were present, the luciferase activity was the greatest. (Bar = mean ± S.E.M, *p < 0.05).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 9 | Differential effects of CITED2 on PASMC proliferation under normoxia and hypoxia. Normally, the H3K27ac modification of the SEs and promoter regions of CITED2 is significant. Downstream proteins influenced by CITED2, such as cyclin proteins, CDKs and PCNA, decreased. SMC proliferation is also diminished. However, the phenomenon is reversed under hypoxia.

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Modification

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: STRIP2 motivates non-small cell lung cancer progression by modulating the TMBIM6 stability through IGF2BP3 dependent

doi: 10.1186/s13046-022-02573-1

Figure Lengend Snippet: P300/CBP-mediated H3K27ac activates STRIP2 transcription in NSCLC. a Data from the UCSC genome bioinformatics site ( http://genome.ucsc.edu/ ) showed high enrichment of H3K27ac in the promoter of STRIP2. b RT-qPCR analysis of STRIP2 mRNA level in C646 (5 μM)-treated A549 and PC9 cells at the indicated time point. c STRIP2 protein levels were measured by western blotting after C646 (5 μM)-treated A549 and PC9 cells for 24 hours. d The efficiency of P300 and CBP knockdown was performed using RT-qPCR. e The P300, H3K27ac and STRIP2 levels were detected using western blotting after P300 or CBP knockdown. f CHIP assay were used to measure the level of P300 or CBP binding at the promoter of STRIP2 in P300 or CBP deficiency or control A549 cells. Data represent mean ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01 were determined by one-way ANOVA with Tukey’s post hoc analysis. H3K27ac, H3K27 acetylation; CHIP, chromatin immunoprecipitation

Article Snippet: After purifying, the chromatin was incubated with specific CBP antibody (cat. no. 7425; Cell Signaling Technology) or P300 antibody (cat. no. 54062; Cell Signaling Technology) to immunoprecipitation chromatin overnight at 4 °C with rotation and followed by incubation with protein G magnetic beads for 2 h at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Binding Assay, Control, Chromatin Immunoprecipitation