Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Expressing, Western Blot, Stable Transfection, Colony Assay, Wound Healing Assay, Migration, Microscopy

Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. - D. The levels of β-catenin, E-cadherin, Snail, Twist, and actin were determined by Western blotting with extracts from parental MDCK (A-D), MDCK-TAp73-KD A. , MDCK-p21-KD B. , MDCK-PUMA-KD C. , and MDCK-ΔNp73-KD D. cells. E. The levels of E-cadherin, Snail and Twist transcripts were measured by qRT-PCR in parental MDCK cells and MDCK cells with knockdown of TAp73, ΔNp73, p21 or PUMA. The level of Actin was measured as an internal control. F. A model for the role of p73, p21 and PUMA in MDCK cell morphogenesis.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cell reports

Article Title: In vivo RNA-seq and ChIP-seq analyses show an obligatory role for the C terminus of p53 in conferring tissue-specific radiation sensitivity

doi: 10.1016/j.celrep.2023.112216

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal antibody to PUMA , Cell Signaling , 14570S; RRID:AB_2798517.

Techniques: Recombinant, Protein Extraction, Membrane, Staining, Protease Inhibitor, Plasmid Preparation, TUNEL Assay, Purification, ChIP-qPCR, Software

Journal: Cell Death & Disease

Article Title: Therapeutic targeting of ARID1A-deficient cancer cells with RITA (Reactivating p53 and inducing tumor apoptosis)

doi: 10.1038/s41419-024-06751-1

Figure Lengend Snippet: A Interaction between ARID1A and p53. Coimmunoprecipitation assays were performed in HCT116 cell lines as described in “Methods” section. Input (cell lysates without immunoprecipitation) and IgG served as positive and negative controls, respectively. B RT-qPCR analysis of CDKN1A , PUMA , and NOXA mRNA level in HCT116 ARID1A +/+ and ARID1A − / − clones. ** P < 0.01, one-sample t -test. C Upregulation of p53, PUMA, and NOXA levels and downregulation of p21 level in HCT116 ARID1A − / − cell lines. D Downregulation of p53, PUMA, and NOXA levels and upregulation of p21 level in RKO ARID1A OE cell lines. E Upregulation of p53, PUMA, and NOXA levels and downregulation of p21 level by ARID1A silencing. F Downregulation of p53, PUMA, and NOXA levels and upregulation of p21 level by ARID1A overexpression. G Upregulation of p53, p21, PUMA, and NOXA levels with the increasing concentration of RITA treatment for 48 h in HCT116 cells. H Upregulation of p53, p21, PUMA, and NOXA level by 100 nM RITA treatment for 48 h in HCT116 ARID1A +/+ and ARID1A − / − cell lines. I , J Effect of RITA on the cell cycle progression of HCT116 ARID1A − / − cells. Cells were treated with 100 nM RITA for 48 h before flow cytometry. Cell cycle distribution ( I ) and cell population quantification ( J ) are shown. Error bars represent s.d. ( n = 3 independent experiments). ANOVA ** P < 0.01. K , L Effect of RITA on the cell cycle progression of RKO ARID1A − / − cells. Cells were treated with 10 μM RITA for 48 h before flow cytometry. Cell cycle distribution ( K ) and cell population quantification ( L ) are shown. Error bars represent s.d. ( n = 3 independent experiments). ANOVA ** P < 0.01.

Article Snippet: Each aliquot of protein sample was run on an SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Cell Signaling Technology, #12354S, 1:2000 dilution), Cleaved PARP (Cell Signaling Technology, #5625S, 1:2000 dilution), Cleaved caspase3 (Cell Signaling Technology, #9661S, 1:500 dilution), MDM2 (Cell Signaling Technology, #86934S, 1:1000 dilution), p53 (Cell Signaling Technology, #2527S, 1:2000 dilution), p21 (Cell Signaling Technology, #2947S, 1:2000 dilution), PUMA (Cell Signaling Technology, #98672S, 1:2000 dilution), NOXA (Cell Signaling Technology, #14766S, 1:2000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-BRCA1 (Ser1524) (Cell Signaling Technology, #9009T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), Phospho-p53 (Ser15) (Cell Signaling Technology, #9286T, 1:1000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), MDM4 (Proteintech, #28747-1-AP, 1:1000 dilution), FANCD2 (Proteintech, # 28619-1-AP, 1:1000 dilution) and β-actin (Cell Signaling Technology, #3700S, 1:4000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (ZEN-Bioscience, 511203).

Techniques: Immunoprecipitation, Quantitative RT-PCR, Clone Assay, Over Expression, Concentration Assay, Flow Cytometry

Journal: Oncology reports

Article Title: MicroRNA-296 inhibits colorectal cancer cell growth and enhances apoptosis by targeting ARRB1-mediated AKT activation.

doi: 10.3892/or.2018.6806

Figure Lengend Snippet: Figure 2. Effects of miR-296 on cell proliferation, cell apoptosis and the expression of associated molecules in SW480 and HCT-116 cell lines. (A) Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that miR-296 expression was significantly enhanced in SW480 and HCT-116 cells transfected with miR-296 mimics compared to cells transfected with miR-NC. *P<0.05 vs. miR-NC-transfected cells. (B) Effect of miR-296 upregulation on SW480 and HCT-116 cell proliferation as measured by MTT assay. *P<0.05 vs. miR-NC-transfected cells. (C) Effect of miR-296 upregulation on SW480 and HCT-116 cell apoptosis rates. The number of apoptotic cells was significantly higher in SW480 and HCT-116 cells overexpressing miR-296 as measured by propidium iodide staining and flow cytometry. *P<0.05. (D) Morphological analysis of SW480 and HCT-116 cell apoptosis induced by miR-296 mimics. The morphology of colorectal cancer cells were imaged by TEM. Magnification in transmission electron microscopy analysis was x6,000. (E) Altered AKT signaling and apoptosis are associated with overexpression of miR-296. Detection of p-AKT, AKT, p-STAT3, STAT3 and apoptosis-associated proteins in SW480 and HCT-116 cells was performed by western blotting. miR, microRNA; NC, negative control; p, phosphorylated; AKT, RAC-α serine/threonine- protein kinase; STAT3, signal transducer and activator of transcription 3; mTOR, serine/threonine-protein kinase mTOR; Bcl-2, B cell lymphoma 2; Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2 associated X, apoptosis regulator; Noxa, NADPH oxidase activator; Puma, p53 upregulated modulator of apoptosis.

Article Snippet: Primary antibodies used were against ARRB1 (catalog no. sc-377015; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), AKT (catalog no. GTX110613; 1:600; GeneTex, Inc., Irvine, CA, USA), B cell lymphoma (Bcl)-2 (catalog no. sc-20067; 1:500; Santa Cruz Biotechnology, Inc.), signal transducer and activator of transcription 3 (STAT3) (catalog no. ab-11935; 1:500; Abcam, Cambridge, MA, USA), Bcl-2associated X protein (Bax) (catalog no. sc-23959; 1:500; Santa Cruz Biotechnology, Inc.), Bcl-2 homologous antagonist/killer (Bak) (catalog no. sc-7873; 1:500; Santa Cruz Biotechnology, Inc.), NADPH oxidase activator (Noxa) (catalog no. 14766; 1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), p53 upregulated modulator of apoptosis (PUMA) (catalog no. 24633; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (catalog no. ab8227; 1:1,000; Abcam).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, MTT Assay, Staining, Flow Cytometry, Transmission Assay, Electron Microscopy, Over Expression, Western Blot, Negative Control

Journal: Oncology reports

Article Title: MicroRNA-296 inhibits colorectal cancer cell growth and enhances apoptosis by targeting ARRB1-mediated AKT activation.

doi: 10.3892/or.2018.6806

Figure Lengend Snippet: Figure 4. Overexpression of ARRB1 partially rescues miR-296-regulated cell growth and apoptosis in SW480 and HCT-116 cells. (A) mRNA levels of ARRB1 measured in five CRC cell lines by RT-qPCR. *P<0.05 vs. HT-29, SW620 and LoVo cells. (B) Expression of ARRB1 protein in five CRC cell lines measured by western blotting. (C) The mRNA levels of ARRB1 expression were suppressed in SW480 and HCT-116 cells after transfection of ARRB1 siRNA for 72 h, detected by RT-qPCR. *P<0.05 vs. GFP-siRNA-transfected cells. (D) Western blot analysis showing the results of ARRB1 expression in HCT-116 and SW480 cells after ARRB1-siRNA transfection. (E) Effect of ARRB1 underexpression on SW480 and HCT-116 cell proliferation, as measured by MTT assay. *P<0.05 vs. GFP-siRNA-transfected cells. (F) Effect of ARRB1 underexpression on SW480 and HCT-116 cell apoptosis rates. The number of apoptotic cells was significantly higher in SW480 and HCT-116 cells underexpressing ARRB1, as measured by apoptosis assay. *P<0.05. (G) Western blot analysis of CRC cells co-transfected with miR-296 mimics and Lenti-ARRB1. (H) MTT assay of SW480 and HCT-116 cells co-transfected with miR-296 mimics and Lenti-ARRB1 or the control. *P<0.05 vs. SW480 and HCT-116 cells co-transfected with miR-296 mimics and Lenti-ARRB1. (I) Apoptosis assay of SW480 and HCT-116 cells co-transfected with miR-296 mimics and Lenti-ARRB1 or the control. *P<0.05, **P<0.01. (J) ARRB1 overexpression blocks the effect of miR-296 on dephosphorylation of AKT signaling without affecting total AKT and STAT3 expression. Western blot analysis also indicated that miR-296 suppressed Bcl-2 protein expression, via regulation of ARRB1. Western blot analyses were performed in triplicate, and representative results are shown. miR, microRNA; NC, negative control; CRC, colorectal cancer; ARRB1, arrestin β1; si, small interfering; GFP, green fluorescent protein; p, phosphorylated; AKT, RAC-α serine/ threonine-protein kinase; STAT3, signal transducer and activator of transcription 3; Bcl-2, B cell lymphoma-2.

Article Snippet: Primary antibodies used were against ARRB1 (catalog no. sc-377015; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), AKT (catalog no. GTX110613; 1:600; GeneTex, Inc., Irvine, CA, USA), B cell lymphoma (Bcl)-2 (catalog no. sc-20067; 1:500; Santa Cruz Biotechnology, Inc.), signal transducer and activator of transcription 3 (STAT3) (catalog no. ab-11935; 1:500; Abcam, Cambridge, MA, USA), Bcl-2associated X protein (Bax) (catalog no. sc-23959; 1:500; Santa Cruz Biotechnology, Inc.), Bcl-2 homologous antagonist/killer (Bak) (catalog no. sc-7873; 1:500; Santa Cruz Biotechnology, Inc.), NADPH oxidase activator (Noxa) (catalog no. 14766; 1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), p53 upregulated modulator of apoptosis (PUMA) (catalog no. 24633; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (catalog no. ab8227; 1:1,000; Abcam).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Transfection, MTT Assay, Apoptosis Assay, Control, De-Phosphorylation Assay, Negative Control