Journal: Scientific Reports

Article Title: Contribution of reversible histone acetylation to freeze tolerance and recovery in wood frog kidneys

doi: 10.1038/s41598-025-09521-x

Figure Lengend Snippet: Relative protein levels of 7 lysine acetyl transferases (KATs) in R. sylvatica kidneys as determined by Western immunoblotting. ( A ) Histogram showing mean (± SEM, n=4) standardized expression levels of KAT1, KAT2A, KAT2B, KAT3A, KAT5, KAT7, KAT8 under control, 24 h freezing and 8 h thawed conditions. Data are mean ± SEM ( n = 4 independent trials). Statistical significance for freezing and thawing values, relative to the standardized control, was determined using one-way analysis of variance (ANOVA) with Tukey’s post hoc test where a, b, c that share the same letters were not statistically different from one another (*— p < 0.05). ( B ) Representative western blots for individual KMTs under each experimental condition. Original full blots are presented in Supplementary Figure .

Article Snippet: ABclonal Technology supplied antibodies against KAT3A (A1334), KAT5 (A1678); KAT7 (A5823); HDAC8 (A5829); HDAC9 (A1516), p-HDAC4 (AP0280), p-HDAC8 (AP0360), H3K56ac2 (A7256), H2BK5ac (A15621), H3K14ac (A7254), H2AK5ac (A15620), H3K27ac (A7253) and H3K18ac (A7257 antibodies.

Techniques: Western Blot, Expressing, Control

Journal: Cancer Discovery

Article Title: Targeting the p300/CBP Axis in Lethal Prostate Cancer

doi: 10.1158/2159-8290.cd-20-0751

Figure Lengend Snippet: Figure 2. CBP and p300 protein knockdown affects AR-V7 expression and AR signaling independent of C-MYC expression in CRPC models. A–F, 22Rv1 (A–C) and LNCaP95 (D–F) were transfected with a total of 100 nmol/L siRNA with 50 nmol/L of either CBP, p300, p300 plus CBP, or C-MYC siRNA (made up to total with Control siRNA) for 72 hours. The effect of each condition on AR-FL, AR-V7, KLK2, KLK3, FKBP5, TMPRSS2, C-MYC, CBP, and p300 mRNA expression (22Rv1, A; LNCaP95, D) and AR-FL, AR-V7, C-MYC, KLK3, CBP, p300, and GAPDH protein expression was determined (22Rv1, B; LNCaP95, E). Mean mRNA expression (normalized to an average of B2M/GAPDH/HPRT1 and control siRNA; defined as 1.0) with standard error of mean from three individual experiments is shown. Growth assays by CellTiter-Glo were carried out (22Rv1, C; LNCaP95, F; normalized to control siRNA; defined as 1.0) with standard deviation of an individual experiment with six replicates is shown. P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) were calculated for each condition compared with control siRNA (at equivalent concentration) using unpaired Student t test. Western blots are shown in triplicate. G–I, 22Rv1 cells with doxycycline-inducible shRNA to Control (shCON), CBP (shCBP), or p300 (shp300) were treated with 10 ng/μg doxycycline for 48 hours prior to transfection with empty vector plasmid (EV), CBP plasmid (CBP rescue), or p300 plasmid (p300 rescue). The effect of each condition on CBP, p300, AR-FL, AR-V7, KLK3, FKBP5, TMPRSS2, C-MYC, ODC, and CAD mRNA expression (G) and CBP, p300, AR-FL, AR-V7, C-MYC, and GAPDH protein expres- sion (H) was determined after 48 hours. Mean mRNA expression (normalized to 18S and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) were calculated for shCBP:EV compared with shCBP:CBP rescue, and shp300:EV compared with shp300:p300 rescue, using unpaired Student t test. Western blots demonstrate single experiment. The effect of each con- dition on relative growth was determined after 5 days using the PicoGreen dsDNA assay (I). Mean relative growth (normalized to baseline and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) were calculated for shCBP:EV compared with shCBP:CBP rescue, and shp300:EV compared with shp300:p300 rescue, using unpaired Student t test.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein reexpression experiments in doxycycline-inducible cell lines developed.

Techniques: Knockdown, Expressing, Transfection, Control, Standard Deviation, Concentration Assay, Western Blot, shRNA, Plasmid Preparation, Picogreen Assay

Journal: Cancer Discovery

Article Title: Targeting the p300/CBP Axis in Lethal Prostate Cancer

doi: 10.1158/2159-8290.cd-20-0751

Figure Lengend Snippet: Figure 3. CCS1477, a selective p300/CBP inhibitor, downregulates AR and C-MYC signaling and inhibits the growth of prostate cancer models. A, Chemical structure of CCS1477. B, A summary of the derived kinetic parameters from analysis of CCS1477 in an SPR assay (Kon is the rate constant for the association of the protein–ligand reaction; SE, standard error of the mean; Koff is the rate constant for the dissociation of the protein–ligand reaction; Kd is the equilibrium constant for the dissociation equilibrium; T1/2 is the half-life of the protein–ligand reaction). C, Inhibition of cell proliferation by CCS1477 in prostate cancer cell lines representative of hormone dependent, hormone independent, or CRPC with the IC50 determined by either CellTiter- Glo or CyQuant. D, Schematic of RNA-sequencing experimental setup. Briefly, 22Rv1 cells were grown in hormone-deficient media for 72 hours prior to treatment with CCS1477 (96 nmol/L) or vehicle control. RNA sequencing was performed in biological triplicate. E, Gene set enrichment analysis using the hallmarks pathway from the Molecular Signatures Database [false discovery rate (FDR) < 0.25]. Briefly, normalized RNA-sequencing counts were queried for overrepresented pathways with enrichment (left) and de-enrichment (right) after CCS1477 treatment are shown. Leading-edge plots for the top two de-enriched pathways after CCS1477 treatment are shown. Normalized enrichment score (NES) and FDR are shown.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein reexpression experiments in doxycycline-inducible cell lines developed.

Techniques: Derivative Assay, SPR Assay, Inhibition, CyQUANT Assay, RNA Sequencing, Control

Journal: Cancer Discovery

Article Title: Targeting the p300/CBP Axis in Lethal Prostate Cancer

doi: 10.1158/2159-8290.cd-20-0751

Figure Lengend Snippet: Figure 4. CCS1477 inhibits AR and C-MYC signaling in cell line models of CRPC. A–D, 22Rv1 (A and B) and LNCaP95 (C and D) were treated with vehi- cle (0 nmol/L = DMSO 0.1%) or various concentrations of CCS1477 (1, 10, 30, 100, 300, 1,000, 3,000 or 5,000 nmol/L) for 48 hours (72 hours for B). The effect of each condition on AR-FL, AR-V7, KLK2, KLK3, FKBP5, TMPRSS2, C-MYC, CBP, and p300 mRNA expression (22Rv1, A; LNCaP95, C) and AR-FL, AR-V7, C-MYC, and GAPDH protein expression was determined (22Rv1, B; LNCaP95, D). Mean mRNA expression (normalized to an average of B2M/ GAPDH/HPRT1 and control siRNA; defined as 1.0) with standard error of mean from three individual experiments is shown. P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) were calculated for each treatment condition compared with vehicle using unpaired Student t test. Single representative Western blot shown from three separate experiments. (continued on next page)

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein reexpression experiments in doxycycline-inducible cell lines developed.

Techniques: Expressing, Control, Western Blot

Journal: Cancer Discovery

Article Title: Targeting the p300/CBP Axis in Lethal Prostate Cancer

doi: 10.1158/2159-8290.cd-20-0751

Figure Lengend Snippet: Figure 7. CCS1477 regulates key prostate cancer therapeutic targets and modulates blood KLK3 in patients with CRPC. A–F, Patient 1 (A–C) and patient 2 (D–F) were treated with CCS1477 within an ongoing phase I clinical trial (NCT03568656). Patient 1 (A–C) was treated orally with 50 mg twice daily (BD) on a 3-day-on and 4-day-off schedule. Blood KLK3 levels and timing of predose (after 4-day off treatment) and postdose (4-hour post- CCS1477 dose) biopsies are shown (A). The patient remains on trial. Quantification (B) and representative micrographs for hematoxylin and eosin (H&E), AR-FL, AR-V7, C-MYC, KLK3, CBP, p300, and Ki-67 IHC predose and postdose are shown (C). Scale bar, 200 μm. Patient 2 (D–F) was treated orally with 25 mg BD continuously. Blood KLK3 levels and timing of predose (prior to starting treatment) and postdose (3 hours after CCS1477 dose) biopsies are shown (D). The patient has stopped treatment. Quantification (E) and representative micrographs for H&E, AR-FL, AR-V7, C-MYC, KLK3, CBP, p300, and Ki-67 IHC predose and postdose are shown (F). Scale bar, 200 μm.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein reexpression experiments in doxycycline-inducible cell lines developed.

Techniques: Biomarker Discovery

Journal: Oncotarget

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

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 2: The effect of CBP on hTERT promoter activity, hTERT protein expression and telomerase activity. (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: Activity Assay, Expressing, Transfection, Luciferase, Control, Over Expression, Western Blot, Knockdown

Journal: Oncotarget

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

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 3: The effect of CBP on lung tumor growth in vitro and in vivo. (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 (400x 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: In Vitro, In Vivo, 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: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 6: The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells. (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: Acta Histochemica et Cytochemica

Article Title: Expression of Glucocorticoid Receptor and Coactivators in Ependymal Cells of Male Rats

doi: 10.1267/ahc.14021

Figure Lengend Snippet: Expression of coactivators on ependymal cells. ( A ) Expression of p300 (red), which is a glucocorticoid receptor (GR) coactivator, vimentin (green) and DAPI (blue) around the third ventricle (3V) in controls and adrenalectomized (ADX) rats. Bars=20 μm. Arrowheads indicate p300 expression in ependymal cells. ( B ) Percentage of p300-ir cells to vimentin-ir cells around the lateral ventricle (LV), 3V, the cerebral aqueduct (aq) and the fourth ventricle (4V) in control and ADX rats. Statistical differences were determined by unpaired Student’s t -test. Number in each column represents the number of animals used. Values are means±SEM. ( C ) Expression of steroid receptor coactivator 1 (SRC-1, red), which is also a GR coactivator, vimentin (green) and DAPI (blue) around 3V in controls and ADX rats. Bars=20 μm. Arrowheads indicate SRC-1 expression in ependymal cells. ( D ) Percentage of SRC-1-ir cells to vimentin-ir cells. Statistical differences were determined by unpaired Student’s t -test. Values are expressed as means±SEM.

Article Snippet: Briefly, the sections were incubated with anti-CBP/p300 antibody (mouse monoclonal, 1:500, Acris Antibodies GmbH, Herford, Germany) at 4°C for 2 days and then with anti-vimentin antibody (goat, 1:1,000).

Techniques: Expressing, Control

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