Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 targets histone reader and writer to inhibit histone H3 and H4 acetylation in CRC. (A) Crystal violet staining of indicated cell lines were treated with NEO2734 for 72 hours. (B) MTS analysis of indicated cell lines treated with NEO2734. (C) Average IC 50 of each cell line. Error bars indicate 95% CI. (D) Crystal violet staining of HCT116 and LOVO cells treated with indicated drugs and concentrations for 72 hours. (E) Cellular thermal shift assay performed in HCT116 cells. Graphs shows mean soluble protein remaining after incubation at indicated temperatures, with error bars representing +/- SD (only one direction shown for each point for clarity). Representative western blot of each protein and temperature point shown below the graphs. Quantification derived from densitometric measured of 3 independent experiments, with density of each temperature point normalized to the density of a room temp control for that experiment. (F) Western blot of acetylated histones (H3K9/K14, H4K16), total histones, and c-MYC in indicated cells over an 8-hour treatment course. (G) qRT-PCR of myc mRNA in HCT116 cells treated with NEO2734 10µM for the indicated timepoints, normalized to gapdh mRNA at each timepoint. ***p < 0.0005 compared to 0h.

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: Staining, Thermal Shift Assay, Incubation, Western Blot, Derivative Assay, Control, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 induces apoptosis in CRC cells. (A) Annexin V flow cytometry of HCT116 cells after 24 h of treatment with NEO2734 10µM or DMSO control. (B) Western blot of cleaved caspase 3, total and cleaved caspase 8, and total and cleaved caspase 9, in HCT116 cells treated with NEO2734 10µM for indicated times. Cleaved caspase 3 also shown for LOVO, RKO, and DLD-1 cells. (C) Crystal violet staining of HCT116 cells treated with NEO2734 10µM or control, with or without zVAD-fmk 10µM. Cells treated with zVAD-fmk and NEO2734 were pre-treated with zVAD for 1 hour prior to adding NEO2734. (D) Apoptosis percentage of HCT116 cells treated with NEO2734 10µM or control DMSO, with or without zVAD-fmk 10µM co-treatment, as measured by percent fragmented nuclei. All bar plots represent mean of 3 experiments +/- SD (*p < 0.05; **p < 0.005).

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: Flow Cytometry, Control, Western Blot, Staining

Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 induces apoptosis through p53/PUMA. (A) RT-PCR of PUMA in HCT116 cells treated with NEO2734 10µM or DMSO for 24 h, normalized to GAPDH. (B) Western blot of p53 and PUMA in HCT116 cells treated for up to 48 hours with NEO2734 10µM or DMSO. (C) ChIP analysis of p53 binding to the PUMA promoter in HCT116 cells treated with NEO2734 10uM for 24 h, with rabbit IgG control IP. PUMA promoter was detected by PCR amplification of precipitated DNA fragments using primers flanking the p53 binding site, followed by visualization on agarose gel electrophoresis. (D) Luciferase reporter assay of the p53/ PUMA promoter. HCT116 cells were transfected with pBV-based PUMA firefly luciferase reporter plasmids as previously published , along with CMV-βgal plasmid. 24 hours after transfection, cells were treated with NEO2734 10µM or DMSO. 24 hours after drug treatment, cell lysates were prepared and luminescence was measured. Signals were normalized for cell viability as well as transfection efficiency based on co-transfected βgal. (E) Western blot of PUMA and caspases in parental HCT116 (WT), HCT116 PUMA -KO, and HCT116 p53 -KO cells treated with NEO2734 10µM for 48 hours. (F) Western blot of cytosolic versus mitochondrial fractions of HCT116 WT and PUMA -KO cells treated with NEO2734 10µM for 48 hours, with antibodies against cytochrome C (CytoC) and cytochrome oxygenase IV (Cox IV). (G) Apoptosis percentage as measured by quantification of fragmented nuclei in HCT116 parental and knockout cells treated with NEO2734 10µM for 48 hours. (H) Crystal violet staining of colony formation assay of HCT116 parental and knockout cells treated with NEO2734 10µM for 24 h and then replated at 1:400 dilution. Bar graph represents ratio of colonies from NEO2734 treated cultures versus DMSO cultures treated in parallel, for 24 hours. Mean and S.D of 3 independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.0005).

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Control, Amplification, Agarose Gel Electrophoresis, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Knock-Out, Staining, Colony Assay

Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 induces DR5-dependent apoptosis. (A) RT-PCR of DR5 in HCT116 cells treated with NEO2734 10µM for the indicated times, normalized to GAPDH. (B) Western blotting of DR5 and indicated ER stress response markers in HCT116 cells treated with NEO2734 10µM. (C) Western blotting of indicated cell lines for DR5 after treatment with NEO2734. LOVO cells were treated with NEO2734 1µM, RKO and DLD-1 were treated with NEO2734 10µM. (D) Western blot of DR5 and caspases in parental HCT116 (WT) and HCT116 DR5 -KO cells treated with NEO2734 10µM for 48 hours. (E) Western blot of cytosolic versus mitochondrial fractions of HCT116 WT and DR5 -KO cells treated with NEO2734 10µM for 48 hours, with antibodies against cytochrome C (CytoC) and cytochrome oxygenase IV (Cox IV). (F) Apoptosis percentage as measured by quantification of fragmented nuclei in HCT116 parental and knockout cells treated with NEO2734 10µM for 48 hours. (G) Crystal violet staining of colony formation assay of HCT116 parental and knockout cells treated with NEO2734 10µM for 24 h and then replated at 1:400 dilution. Bar graph represents ratio of colonies from NEO2734-treated cultures versus DMSO cultures treated in parallel, for 24 hours. Mean and S.D of 3 independent experiments are shown (*p<0.05; ***p<0.0005).

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Knock-Out, Staining, Colony Assay

Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 activity depends on both PUMA- and DR5- mediated apoptosis. (A) Western blot of indicated HCT116 cell lines after transfection with either scrambled (scr) or DR5 targeted (DR5-KD) siRNA, followed by treatment with NEO2734 10µM. Cells were transfected 24 hours after plating and then treated with drug 24 hours after transfection. Times are the hours of drug treatment. (B) Apoptosis percentage as measured by quantification of fragmented nuclei in HCT116 parental (WT) and PUMA-KO cells. PUMA-KO cells were transfected with either control (scr) or DR5-targeted siRNA (DR5-KD) at 24 hours after plating, and then treated with drug 24 hours after transfection. All cells were treated with NEO2734 10µM for 48 hours. (C) Crystal violet staining of colony formation assay of HCT116 parental and PUMA-KO cells treated with NEO2734 10µM for 24 h and then replated at 1:400 dilution. Bar graph represents ratio of colonies from NEO2734-treated cultures versus DMSO cultures treated in parallel, for 24 hours. Mean and S.D of 3 independent experiments are shown (*p < 0.05; **p < 0.005; ***p < 0.0005).

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: Activity Assay, Western Blot, Transfection, Control, Staining, Colony Assay

Journal: Frontiers in Oncology

Article Title: Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death

doi: 10.3389/fonc.2022.1018775

Figure Lengend Snippet: NEO2734 induces CRC apoptosis in vivo . (A) Growth curves of HCT116 tumor xenografts implanted in flanks of nude mice. Treatments were started after tumors reached 50-100 mm 3 in size. Tumor volumes were estimated by the formula V=w^2*l/2 (V=volume, w=width or short dimension of tumor, l=length or long dimension of tumor). Results are the mean and S.D. of 6 independent tumors. (B) Representative photographs of NEO2734-treated and control HCT116 xenograft tumors dissected from mice after sacrifice on day 21. (C) Western blot of HCT116 xenograft tumors with indicated antibodies. (D) Representative immunofluorescence staining of indicated markers in control versus NEO2734-treated xenografts. (E) Quantification of c-casp3 and TUNEL positive cells in xenografts. Mean and S.D. of 6 independent tumors are shown (***p < 0.0005).

Article Snippet: Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media.

Techniques: In Vivo, Control, Western Blot, Immunofluorescence, Staining, TUNEL Assay

Journal: Biomolecules

Article Title: Sorbaria kirilowii Ethanol Extract Exerts Anti-Inflammatory Effects In Vitro and In Vivo by Targeting Src/Nuclear Factor (NF)-κB

doi: 10.3390/biom10050741

Figure Lengend Snippet: Effects of Sk-EE on nitric oxide (NO) production and its cytotoxicity analysis in macrophages. ( a , b ) RAW264.7 cells or peritoneal macrophages were pretreated with indicated doses of Sk-EE or l -NAME and induced by LPS (1 µg/mL) for 24 h. NO production was measured by Griess assay. ( c , d ) RAW264.7 cells, peritoneal macrophages, or HEK293T cells were treated with indicated doses of Sk-EE or L-NAME and their cell viability was determined by MTT assay. ( e ) Phytochemical characteristics of Sk-EE were analyzed via HPLC. ( f ) Detected flavonoids and Sk-EE were pretreated to RAW264.7 cells 30 min before LPS induction, and NO production levels were measured through Griess assay. # p < 0.05 and ## p < 0.01 compared to normal group; * p < 0.05 and ** p < 0.01 compared to control group. All data presented ( a – d ) are expressed as mean ± SD of experiments performed with 4 samples. SL: silibinin. GN: genistein. QC: quercetin. KP: kaempherol. +: treatment, −: no treatment.

Article Snippet: RAW264.7 cells (no. TIB-71) and HEK293 cells (no. CRL-1573) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Griess Assay, MTT Assay, Control

Journal: Biomolecules

Article Title: Sorbaria kirilowii Ethanol Extract Exerts Anti-Inflammatory Effects In Vitro and In Vivo by Targeting Src/Nuclear Factor (NF)-κB

doi: 10.3390/biom10050741

Figure Lengend Snippet: Effects of Sk-EE on the mRNA expression of inflammatory genes and nuclear translocation level of inflammatory transcription factors. ( a ) RAW264.7 cells were pretreated with indicated concentrations of Sk-EE and induced by lipopolysaccharide (LPS) (1 µg/mL) for six hours. mRNA expression levels of iNOS, COX-2, IL-1β, and GAPDH (as a control) were measured using reverse-transcription polymerase chain reaction (RT-PCR) and agarose gel electrophoresis. ( b , c ) HEK293T cells were transfected with NF-κB-luc, MyD88 or TRIF, and β-gal (as a control) genes by PEI transfection for 24 h, then treated with indicated doses of Sk-EE for 24 h. The expression of NF-κB was determined by luciferase assay. ( d , e ) RAW264.7 cells were pretreated with Sk-EE (200 µg/mL) for the indicated times and nuclear fractions or whole-cell lysates for detecting p65, p50, p-p50, p-p65, and Lamin A/C were analyzed by Western blotting. # p < 0.05 and ## p < 0.01 compared to normal group; * p < 0.05 and ** p < 0.01 compared to control group. Data presented ( b , c ) are expressed as mean ± SD of experiments performed with 6 samples. NF: nuclear faction. +: treatment, −: no treatment.

Article Snippet: RAW264.7 cells (no. TIB-71) and HEK293 cells (no. CRL-1573) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Translocation Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Luciferase, Western Blot

Journal: Biomolecules

Article Title: Sorbaria kirilowii Ethanol Extract Exerts Anti-Inflammatory Effects In Vitro and In Vivo by Targeting Src/Nuclear Factor (NF)-κB

doi: 10.3390/biom10050741

Figure Lengend Snippet: Effects of Sk-EE on the protein expression of the NF-κB signaling pathway and target proteins. ( a , b ) RAW264.7 cells were pretreated with Sk-EE (200 µg/mL) and induced by LPS (1 µg/mL) for the indicated time. The expression levels of total or phosphorylated forms of IKKα/β, IκBα, Syk, Src, and control protein β-actin were analyzed through western blotting. ( c , d ) HEK293T cells were transfected with Myc-Syc or HA-Src by PEI transfection for 24 h and treated with indicated doses of Sk-EE for 24 h. The protein expression levels of Myc, Syk, p-Syk, HA, Src, p-Src, and the control protein β-actin were analyzed by western blotting. ( e , f ) RAW264.7 cells were transfected with Myc-Syk or HA-Src through PEI transfection for 24 h and treated with Sk-EE (200 µg/mL) for 24 h. Binding affinities of Sk-EE and Syk or Src were determined by cellular thermal shift assay. WCL: whole-cell lysate. +: treatment, −: no treatment.

Article Snippet: RAW264.7 cells (no. TIB-71) and HEK293 cells (no. CRL-1573) were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Control, Western Blot, Transfection, Binding Assay, Thermal Shift Assay