Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) p27 mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of p27 mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of p27 protein levels normalized to actin expression. (C) p27 protein expression in CLL cells and in healthy B-cells (controls). (D) A representative immunoblot showing p27 and actin expression. The arrows mark examples of CLL patient samples with low p27 expression. T, tonsils. (E) Immnofluorescence analysis showing nuclear p27 in CLL cells from four patients. Nuclei are stained with DAPI.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot, Staining

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Myc mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of Myc mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of Myc protein levels normalized to actin expression. (C) Myc protein expression in CLL cells and in healthy B-cells (controls). (D) Representative immunoblot showing Myc and actin expression. T, tonsils

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing p27, Myc and actin expression in cells from CLL patients and from tonsil cells (T, tonsils). (B) Expression levels of Myc and p27 in CLL patients. The protein levels of p27 and Myc as determined by film densitometry and normalized to actin, were classified in three categories with respect to the mean of expression in controls. (C) Classification of patients with low and high Myc protein expression according to their p27 levels.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Immunoblot showing p27 and Myc expression in MEC1 cell line transfected with a p27 expression vector (pCEFL-p27) and the corresponding empty vector. Proteins were analyzed 24 h after transfection. Actin levels are shown to asses protein loading. (B) MEC1 cells were transfected with pEYFPp27 vector and 24 h later the cell cycle of transfected cells was analysed by flow cytometry. The empty vector (pEYFP) was used as controls. The percentage of cells in the G0/G1 phase is indicated. (C) p27 expression rescued fludarabine-induced cell death. Cells were transfected with pEYFPp27 and pEYFP vector and 24 h after transfection the cells were treated with 10 μM fludarabine and cell death was determined 24 h later by trypan blue exclusion assay. (D) MEC1 cells were transfected and treated as in C, and the apoptosis was determined by annexin V binding, assessed by flow cytometry. (E) MEC1 cells were transfected and treated as in C and the levels of active caspase 3 were determined by immunoblot. (F) Correlation of high expression of p27 protein with higher Bcl2 levels in CLL cells.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Trypan Blue Exclusion Assay, Binding Assay

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the levels of cyclins A and E in CLL cells with and without Myc/p27. Actin levels were also determined to assess protein loading. (B) Correlation between Myc and cyclin A protein levels in CLL cells. (C) Immunoblot showing Myc and p27 protein levels in four patient's cells selected for the experiments shown in D. (D) Molecular filtration chromatographic separation of CLL protein extracts followed by immunoblot for cyclins A, E and Cdk2. The elution of the 55 and 30 kDa proteins is shown at the top. (E) Immunoblot (IB) showing the levels of Myc and p27 in three CLL samples and MEC1 cells and kinase assay of Cdk2 in the same extracts. Proteins were immunoprecipitated with anti Cdk2 and the presence of both cyclin E, p27 and Cdk2 were determined by immunoblot. Lower panel: kinase assays were performed using HisCK − as kinase substrate. M, mock kinase reaction without extract. (F) Immunoprecipitation of Cdk2 and kinase assay of the immunoprecipitates of MEC1 cells and MEC1 cells incubated with lysates from a CLL sample (#01). (G) Immunoprecipitation of Cdk2 and kinase assay from MEC1 cells and from mixed lysates prepared with MEC1 cells and two CLL samples (#01 and #10).

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Filtration, Kinase Assay, Immunoprecipitation, Incubation

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the protein levels of Myc, p27 and Skp2 in CLL cells. Actin levels are also shown as protein loading control. (B) p27 protein levels in CLL cells with high or low expression of Skp2. (C) Myc protein levels in CLL cells with high or low expression of Skp2.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Control, Expressing

Journal: Journal of immunotoxicology

Article Title: The early growth response factor-1 contributes to interleukin-13 production by mast cells in response to stem cell factor stimulation.

doi: 10.1080/15476910802129612

Figure Lengend Snippet: FIG. 4. Egr-1 deficiency has no effects on c-Kit and IgE receptor expression or mast-cell viability. (A) Bone marrow cells from wild-type mice or Egr-1– deficient mice were cultured in conditioned media in vitro for 4 wk and exam- ined by flow cytometry for c-Kit and IgE receptor expression. For c-Kit anal- ysis, BMMCs were stained with FITC conjugated rat anti-mouse c-Kit mAb or FITC conjugated rat IgG2a isotypic control. For analysis of IgE receptor expression, BMMC were sensitized with IgE overnight and then stained with FITC-conjugated anti-IgE antibody (mouse IgG1). No difference in c-Kit or IgE receptor expression was observed between Egr-1+/+ and Egr-1−/−BMMCs. (B) BMMC from Egr-1-deficient mice or wild-type mice cultured in complete media containing WEHI-3B supernatants (a source of IL-3) were healthy and showed ≥96% viability. To induce mast cell death, WEHI-3B supernatant was removed from culture media for various days. Mast cell viability was examined by trypan blue exclusion assay. Data were expressed as mean ± SD (n = 3 independent experiments).

Article Snippet: Fluorescein isothiocyanate (FITC) anti-mouse CD117 monoclonal antibody (mAb) (CL8936F), FITC rat IgG2a (CLCR2A01) were purchased from Cedarlane Laboratories Limited (Ontario, Canada).

Techniques: Expressing, Cell Culture, In Vitro, Cytometry, Staining, Control, Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: Immunohistochemical stainings for HSP60 A. and p27 Kip1 B. in pancreatic sections from db/db and db/m + mice were shown. Original magnification, ×400, scale bar: 100 μm; x1000, scale bar: 50 μm. The protein expressions of HSP60 C. and p27 Kip1 D. in pancreatic islets isolated from db/db and db/m + were determined by Western blotting. Moreover, the nuclear p27 Kip1 protein expression was also determined E. . Protein levels were quantified by densitometry and normalized by β-actin levels. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus db/m + mice.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Immunohistochemical staining, Isolation, Western Blot, Expressing

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: Effects of AGEs on the cell viability A. , p27 Kip1 protein expression B. , and HSP60 protein expression C. in RINm5f cells were shown. Cells were treated with AGE-BSA (5-100 μg/ml in A or 0.02-1 μg/ml in B and C) for 24 hours. Cell viability was determined by WST-8 assay. The protein expression was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Moreover, effects of AGEs on the cell number (D-a), cell hypertrophy index (D-b), and cell area (D-c) of RINm5f cells were investigated. Cells were treated with AGE-BSA (0.1-1 μg/ml) for 24 hours. The viable cell number was determined by trypan blue exclusion assay. The cell hypertrophy index and cell diameter were measured as described under “Materials and Methods”. Data are presented as means ± SEM (n ≥ 5). * P < 0.05, versus BSA.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: A. The effect of AGEs on RAGE protein expression in RINm5f cells. Cells were treated with AGE-BSA or non-glycated BSA (0.02-0.5 μg/ml) for 24 hours. The protein expression was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Data are presented as means ± SEM (n ≥ 5). * P < 0.05, versus BSA. C: control, B: BSA, A: AGE-BSA. (B-E) After the pretreatment of RAGE neutralizing antibody (10 μg/ml) for 1 hour, RINm5f cells were treated with AGE-BSA or non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expression of HSP60 B. , cell hypertrophy index C. , ATP content D. , and insulin production E. were detected as described under “Materials and Methods”. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus BSA, # P < 0.05, versus AGE-BSA.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: RINm5f cells were transfected with pM51-HSP60 (0-4 μg/ml) for 48 hours. The pM51 empty vector was as a negative control. Transfection of pM51-HSP60 or pM51 vector control (1 μg/ml) for 48 hours, and then cells were treated with AGE-BSA and non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expressions of HSP60 and p27 Kip1 A. , cell hypertrophy index B. , cell diameter C. , insulin secretion D. , and ATP production E. were detected as described under “Materials and Methods”. Data are presented as means ± SEM ( n ≥ 5). * P < 0.01, versus BSA, # P < 0.01, versus pM51/AGE-BSA. NS: non-significant.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Transfection, Plasmid Preparation, Negative Control, Control

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: RINm5f cells were treated with AGE-BSA (0.5-50 μg/ml) and non-glycated BSA (50 μg/ml) for 24 hours. A. The levels of cellular H 2 O 2 were detected by ELISA. Data are presented as means ± SEM ( n ≥ 5). * P < 0.05, versus BSA. B. ROS production was also determined by flow cytometric assay. NAC, N-acetyl-L-cysteine. C. RINm5f cells were treated with AGE-BSA and non-glycated BSA (10 μg/ml) for 24 hours in the presence or absence of RAGE neutralizing antibody. The levels of cellular H 2 O 2 were detected by ELISA. D. Effect of antioxidant N-acetyl-L-cysteine (NAC) on HSP60 protein expression in AGE-BSA-treated RINm5f cells. After pretreatment with NAC (2 mM) for 1 hour, the cells were treated with AGE-BSA or non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expression of HSP60 was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus BSA, # P < 0.05, versus AGE-BSA. In some experiments, the H 2 O 2 productions in islets of db/db and db/m+ mice were measured E. . Data are presented as means ± SEM ( n ≥ 10). **P < 0.01, versus db/m+ mice.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Western Blot

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: The immunohistochemical staining for insulin (A-a), AGEs (A-b), and HSP60 (A-c) were performed on the pancreatic sections (islet areas) of normal subject and diabetic patient. Original magnification, ×400, scale bar: 100 μm; x1000, scale bar: 50 μm. Moreover, the islet area B. and β-cell area C. in islets of normal subject and diabetic patient with 6 random areas per section was determined by ImageJ software. Data are presented as mean ± SEM. * P < 0.05, diabetic patient versus normal subject.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Immunohistochemical staining, Staining, Software

Journal: Frontiers in Oncology

Article Title: STAT3 and mutp53 Engage a Positive Feedback Loop Involving HSP90 and the Mevalonate Pathway

doi: 10.3389/fonc.2020.01102

Figure Lengend Snippet: STAT3 inhibition in wtp53 U87 cells reduces cell survival and increase p53 and p21 expression and inhibits the mevalonate pathway. U87 cells were cultured in the absence or in the presence of 100 μM AG490 for 48 h, and cell survival and STAT3, p53, p21, HSP90, and MVK expression were analyzed, respectively, by trypan blue exclusion assay (A) and by western blot (B–F) . The histograms (A) represent the mean plus S.D. of more than 3 experiments * P < 0.05; in western blot (B–F) β Actin was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of specific band and control of 3 different experiments. In (G) , FACS analysis of ROS production, by U373, Panc1, and U87 treated or not with 100 μM AG490, measured by DCFDA staining. The mean of fluorescence intensity is indicated. Solid gray peaks represent the controls. One representative experiment out of 3 is shown.

Article Snippet: To evaluate the expression of proteins we used the following antibodies: mouse monoclonal anti-STAT3 (1:500) (BD Transduction Laboratories, 610,189), rabbit polyclonal anti-phospho-STAT3 (1:500) (p-Tyr705, clone D3A7, Cell Signaling Technology, 9145), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc., sc-126), mouse monoclonal anti-HSP90 (1:100) (Santa Cruz Biotechnology Inc., sc-69703), mouse monoclonal anti-p21 (1:100) (clone F-8, Santa Cruz Biotechnology Inc., sc-271610), mouse monoclonal anti-SREBP1 (1:100) (clone A-4, Santa Cruz Biotechnology Inc., sc-365513), mouse monoclonal anti-MVK (1:100) (clone D-3, Santa Cruz Biotechnology Inc., sc-390669).

Techniques: Inhibition, Expressing, Cell Culture, Trypan Blue Exclusion Assay, Western Blot, Control, Staining, Fluorescence

Journal: Oncogene

Article Title: Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells.

doi: 10.1038/onc.2011.397

Figure Lengend Snippet: Figure 3 Generation of atf3 knockout mice and loss of p53 or atf3 gene abrogates DR5 induction in MEFs. (a) Schematic diagram of the generation of conditional and constitutive atf3 knockout mice. Positions of selected restriction enzymes and of coding exon 2 (E2) as well as of loxP recombination sites (triangles) are indicated (neo: neomycin resistance cassette; DT-A: diphtheria toxin). The targeting strategy and primer sequences are described in Materials and methods. (b) Southern hybridization of ES cells treated with adenovirus expressing Cre-recombinase. Cellular DNA samples were digested with SpeI and hybridized with the internal probe as indicated in (a). (c) Wt and atf3 deleted genome DNA eluted from MEFs were amplified by PCR with two primer sets (#1 and #3) indicated in (a) (left panel). MEFs were treated with 2.0 mM CPT, and ATF3 expression was assessed by western blotting (right panel). (d, e) Wt and p53 null MEFs (d) or atf3 null MEFs (e) were treated with 2.0 mM CPT for the indicated time, and assayed for atf3 and dr5 mRNAs by qRT–PCR. In (e), the induction of dr5 mRNA was also assayed after the human ATF3 was re-introduced into atf3 null MEFs as in Materials and methods. Relative expression of transcripts was normalized to GAPDH. (f) Wt and p53 null or atf3 null MEFs were treated with 2.0 mM CPT for 12 h, then, DR5, ATF3 and p53 proteins were analyzed by western blotting.

Article Snippet: Antibodies used were as follow: anti-ATF3 (C19), anti-p53 (DO-1 and FL-393) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-b-actin (AC-74) from Sigma-Aldrich (St Louis, MO, USA), anti-DR5 from ProSci (San Diego, CA, USA), anti-PARP from R&D systems (Minneapolis, MN, USA), anticaspase3 from BD Transduction Laboratories (Franklin Lakes, NJ, USA) and anticleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knock-Out, Hybridization, Expressing, Western Blot, Quantitative RT-PCR

Journal: Oncogene

Article Title: Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells.

doi: 10.1038/onc.2011.397

Figure Lengend Snippet: Figure 4 ATF3 sensitizes HCT116 cells to TRAIL-induced apoptosis. (a) HCT116 p53 wt cells were transfected with siControl or siATF3 oligos. After 24 h, cells were treated with 0.2 mM CPT with or without 2.5 ng/ml TRAIL for another 24 h. Then, trypan blue exclusion assay was carried out as detailed in Materials and methods, and relative values of dead cell numbers are shown with s.e. from three independent experiments. *Po0.05. (b) Nuclei of HCT116 cells treated as in (a) were visualized using 6-diamidino-2-phenylindole staining under a fluorescence microscope. Arrowheads show fragmented nuclei. (c) HCT116 cells stably expressing siGFP or siATF3- 363 were treated with 0.2 mM CPT with or without 2.5 ng/ml TRAIL for 24 h, then fluorescence-activated cell sorting analysis was carried out as in Materials and methods. Relative cell number at sub-G1 fraction was quantitated as sub-G1 population (%). (d) Whole-cell extracts of HCT116 cells treated as in (a) were analyzed by western blotting using anti-PARP, anticaspase3, anticleaved caspase3, anti-ATF3 or anti-DR5 antibodies. (e) HCT116 cells stably expressing ATF3 or GFP were treated with 0.2 mM CPT for the indicated time, and whole-cell extracts were analyzed by western blotting using anti-DR5, anti-ATF3 or anti-p53 antibodies (upper panel). In the middle panel, cells were treated with 0.2 mM CPT with or without 2.5 ng/ml TRAIL for 24 h, and trypan blue exclusion assay was carried out as in (a). Data represent means with s.e. of three independent experiments.*Po0.05. Cells treated as in the middle panel were subjected to fluorescence-activated cell sorting analysis (lower panel) as in (c).

Article Snippet: Antibodies used were as follow: anti-ATF3 (C19), anti-p53 (DO-1 and FL-393) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-b-actin (AC-74) from Sigma-Aldrich (St Louis, MO, USA), anti-DR5 from ProSci (San Diego, CA, USA), anti-PARP from R&D systems (Minneapolis, MN, USA), anticaspase3 from BD Transduction Laboratories (Franklin Lakes, NJ, USA) and anticleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Trypan Blue Exclusion Assay, Staining, Microscopy, Stable Transfection, Expressing, FACS, Western Blot

Journal: Oncogene

Article Title: Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells.

doi: 10.1038/onc.2011.397

Figure Lengend Snippet: Figure 6 ATF3 binds to the ATF/CRE motifs of the human DR5 gene promoter and interacts with p53. (a) Nuclear extracts of HCT116 p53 wt cells were prepared after treatment with 0.2 mM CPT for 24 h, and subjected to DNAP assay using biotinylated ATF-BS2, BS3 or BS4 probes as detailed in Materials and methods (upper panel). In the lower panel, ten times molar excess of wt or mutant-type(mt) oligonucleotide was used as competitor. (b) Whole lysates of cells treated as in (a) were incubated with 1 mg each of anti-ATF3 or anti-p53 antibody overnight, and immunoprecipitates were captured and subjected to western blotting. Lysates were also immunoprecipitated with 1 mg normal IgG as a control. Arrows indicate specific bands. Arrowheads denote IgG. (c) Cells treated as in (a) were subjected to Re-ChIP assay. Briefly, the first ChIP was performed by 1 mg each of anti- ATF3 or anti-p53 antibody, and the eluted samples were sequentially immunoprecipitated with the second antibody. Lysates were also immunoprecipitated with 1 mg normal IgG as a control. Precipitated chromatin was analyzed by PCR using primer sequences flanking the ATF3 or p53 binding site as in Materials and methods. Primer set for GAPDH was also used as a negative control.

Article Snippet: Antibodies used were as follow: anti-ATF3 (C19), anti-p53 (DO-1 and FL-393) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-b-actin (AC-74) from Sigma-Aldrich (St Louis, MO, USA), anti-DR5 from ProSci (San Diego, CA, USA), anti-PARP from R&D systems (Minneapolis, MN, USA), anticaspase3 from BD Transduction Laboratories (Franklin Lakes, NJ, USA) and anticleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Mutagenesis, Incubation, Western Blot, Immunoprecipitation, Control, Binding Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: ROS levels are increased in HepG2 cells following co-treatment with ABT-737 and curcumin. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which a 2′,7′-dichlorofluorescin diacetate probe was used to measure the levels of cellular ROS. HepG2 cells were treated with 2 µ M curcumin and 10 µ M ABT-737 for 24 h in the presence or absence of the antioxidant, NAC (10 mM). Subsequently, the levels of HepG2 cell apoptosis were analyzed using (B) Hoechst 33258 staining (white arrows indicate cells undergoing apoptosis with nuclear fragmentation visualized by Hoechst staining), (C) Annexin V-fluorescein isothiocyanate/PI staining and (D) caspase-3 activity detection. Data are expressed as the mean ± standard deviation. * P<0.05. ROS, reactive oxygen species; PI, propidium iodide; NAC, N-acetyl-L-cysteine.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Staining, Activity Assay, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: Increased levels of ROS promote the sustained activation of JNK, which leads to the apoptosis of HepG2 cells. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which the total cellular proteins were extracted. Subsequently, the protein levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (B) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of 10 µ M SP600125 (a JNK inhibitor). Following treatment, apoptosis of the HepG2 cells was assessed using annexin V-fluorescein isothiocyanate/PI staining. (C) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of antioxidant, NAC (10 mM). Subsequently, the levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (D) HepG2 cells were separately transfected with control siRNA or ASK1 siRNA for 24 h, following which the cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h. A Trypan blue exclusion assay was then used to measure the total cell death ratio. * P<0.05 vs. control. ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; p-, phosphorylated; siRNA, small interfering RNA; PI, propidium iodide.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Activation Assay, Western Blot, Staining, Transfection, Control, Trypan Blue Exclusion Assay, Small Interfering RNA