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: 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: 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: 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: Cell Death & Disease

Article Title: Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

doi: 10.1038/cddis.2015.410

Figure Lengend Snippet: Involvement of mTOR in GPAT1-induced mESCs anti-apoptosis under hypoxia. ( a ) Cells were transfected with gpat1 and NT siRNA for 24 h before glucosamine (10 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Collected samples are lysed, and p-mTOR, GPAT1, and β -actin protein expressions were measured by using western blotting. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with glucosamine. ( b ) Cells were pretreated with rapamycin (10 nM) before glucosamine (10 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, and β -actin. Each result shown is representative of three independent experiments. ( c ) Cells were pretreated with glucosamine and/or rapamycin (10 nM) for 30 min before hypoxia treatment, and cytochrome c, COX IV, and β -tubulin in the cytosolic and mitochondrial fraction were detected by western blot. ( d ) Cells were pretreated with various concentrations of LPA (10 − 6 M–10 − 9 M) before hypoxia treatment for 24 h. Cell viability was measured by trypan blue exclusion assay. Error bars are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( e ) Cells were pretreated with pertussis toxin (100 ng/ml) for 30 min before LPA treatment (0.1 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Total proteins were extracted and blotted with p-mTOR, mTOR, p-S6K1, S6K1, p-4EBP1, 4EBP1, and β -actin. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( f ) p-mTOR was immunostained with p-mTOR antibody, and counter-stained with PI. Fluorescence images were acquired by using confocal microscopy. Fluorescence intensity of p-mTOR was quantified by using ImageJ software. Data are presented as a mean±S.E.M. of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( g ) Cells were pretreated with rapamycin (10 nM) before LPA (0.1 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, and β -actin. Each result shown is representative of three independent experiments. ( h ) Cell viability was measured by using cell counter. Data are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( i ) Viable cells were measured by using annexin V/PI flow cytometry analysis. Annexin V-negative-PI-negative cells (Q3) were considered viable, annexin V-negative-PI-positive cells (Q1) were considered necrotic, annexin V-positive-PI-positive cells (Q2) were considered late apoptotic, and annexin V-positive-PI-negative cells (Q4) were considered early apoptotic. Data are presented as a mean±S.E.M. of two independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. * P <0.05 versus control, # indicates P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. The proposed model for signaling pathways involved in glucosamine-induced mESCs survival under hypoxia ( j )

Article Snippet: Mammalian target of rapamycin (mTOR), p-mTOR (Ser 2448), S6K1, p-S6K1 (Thr 389), 4EBP1, and p-4EBP1 (Thr 37/46) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Western Blot, Control, Trypan Blue Exclusion Assay, Staining, Fluorescence, Confocal Microscopy, Software, Flow Cytometry, Protein-Protein interactions

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: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Reduced DNA damage-mediated apoptosis in cells from the S47 mouse. ( A ) MEFs were generated from Hupki embryos containing either wild-type (WT) p53 or the S47 variant. MEFs were treated with 20 μM etoposide for 24 h, and protein lysates were analyzed by Western blot for the proteins indicated. The data depicted are representative of multiple experiments in multiple independent batches of MEFs. ( B ) Quantification of the relative intensity of cleaved lamin A blots from three independent experiments (as depicted in A ) in primary MEFs from the wild-type and S47 mice untreated or treated with 20 μM etoposide for 24 h. Error bars mark standard deviation. ( C ) Immunohistochemical analysis of the small intestine of wild-type (Wt) and S47 Hupki mice 4 h following exposure to 5 Gy of γ irradiation ( right panels) or untreated ( left panels) for total p53. Bars, 100 μm. ( D ) Quantification of the number of cells staining positively for p53 antisera in equal millimeters of crypts from the small intestines and colons of mice with wild-type p53 or S47. Error bars mark standard error. IR indicates 5 Gy of ionizing radiation. ( E ) Immunohistochemical analysis of the small intestine of wild-type and S47 Hupki mice 4 h following exposure to 5 Gy of γ irradiation ( right panels) or untreated ( left panels) for apoptotic cells (cleaved lamin A). Red arrows mark apoptotic cells. Bars, 100 μm. ( F ) Quantification of apoptosis in the wild-type and S47 small intestines ( left ) and colons ( right ) as cells positive for cleaved lamin A following 5 Gy of radiation. The data depicted are averaged from three fields from three independent experiments in which equal millimeters of crypts were analyzed and quantified. Error bars represent standard deviation. (*) P -value <0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Generated, Variant Assay, Western Blot, Standard Deviation, Immunohistochemical staining, Irradiation, Staining

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Marked impairment of cisplatin-mediated apoptosis in S47 cells and mice. ( A ) Primary MEFs from the wild-type (Wt) or S47 Hupki mouse were treated with 10 μM cisplatin (CDDP) for the time points indicated, and protein lysates were analyzed by Western blot analysis for the proteins indicated. The data depicted are representative of three independent experiments in a minimum of three independent batches of MEFs. ( B ) Primary MEFs from the wild-type and S47 mice as well as the p53 knockout mouse (p53 −/− ) were treated with 10 μM CDDP for 24 h, and protein lysates were analyzed by Western blot analysis for the proteins indicated. ( C ) Flow cytometric analysis of Annexin V-positive cells from primary wild-type and S47 MEFs treated with 10 μM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. ( D ) IC 50 analysis for cisplatin (CDDP) in primary wild-type (WT) and S47 MEFs treated with the indicated concentrations of cisplatin for 72 h and analyzed for viability by the Alamar blue assay. The depicted data represent an average of four independent experiments on independent batches of MEFs. Error bars represent standard deviation. ( E ) Human LCLs homozygous for wild-type p53 and the S47 variant were treated with 10 μM CDDP for 24 h, and protein lysates were analyzed by Western blot for the proteins indicated. ( F ) Flow cytometric analysis of Annexin V-positive cells from wild-type and S47 human LCLs treated with 10 μM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. ( G ) IC 50 analysis for cisplatin (CDDP) in wild-type and S47 LCLs treated with the indicated concentrations of cisplatin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. ( H ) IC 50 analysis for adriamycin in wild-type and S47 LCLs treated with the indicated concentrations of adriamycin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. ( I ) Cisplatin-mediated apoptosis, as assessed by cells positive for cleaved lamin A, in the kidneys of wild-type or S47 mice following injection with 20 mg/kg CDDP and analyzed after 48 h. Data are representative of n = 3 per mice group. Bar, 100 μm. ( J ) Clonogenic survival of shARF immortalized wild-type and S47 MEFs treated with the indicated concentrations of cisplatin (CDDP), plated at equal cell numbers 48 h later, and stained with crystal violet after 7 d. ( K ) Quantification of clonogenic survival of immortalized wild-type and S47 MEFs following cisplatin treatment. All values were normalized to the untreated control averaged from three independent experiments. Error bars represent standard deviation. (*) P -value < 0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Western Blot, Knock-Out, Alamar Blue Assay, Standard Deviation, Variant Assay, Staining, Injection, Control

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: The S47 variant is impaired for transactivation of a subset of p53 target genes, including Gls2 , Noxa ( Pmaip1 ), and Sco2 . ( A ) qRT–PCR analysis of p53 target genes in primary wild-type (Wt) and S47 MEFs treated with 10 μM cisplatin (CDDP) for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. ( B ) qRT–PCR analysis of the p53 target genes indicated in independent batches of primary MEFs from wild-type and S47 mice treated with 10 μM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. ( C ) qRT–PCR analysis of the p53 target genes indicated in human LCLs that are homozygous for wild-type p53 or S47 treated with 10 μM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P ≤0.05. SCO2 was not expressed in LCL cells, so these data are not depicted. ( D ) Western analysis for the proteins indicated in wild-type and S47 MEFs pretreated with 10 μM p38MAPK inhibitor SB203580 for 2 h followed by 10 μM cisplatin (CDDP) for 24 h. GAPDH served as the loading control. ( E ) qRT–PCR analysis of the cells in D for the p53 target genes indicated, normalized to control (cyclophilin A). The depicted data represent the average of three independent experiments. Error bars represent standard deviation. (*) P <0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Variant Assay, Quantitative RT-PCR, Control, Standard Deviation, Western Blot

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Impaired DNA-binding ability of the S47 variant. ( A ) ChIP of primary wild-type (Wt) and S47 MEFs treated with 10 μM CDDP for 24 h analyzed using antisera to p53 (CM5) or IgG. The percentage binding normalized to input from qPCR analysis is shown. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P -value < 0.05. ( B ) ChIP analysis of p53 binding to the consensus elements from the genes indicated in human H1299 (p53-null) cells containing doxycycline-inducible wild-type or S47 forms of p53 in the absence and presence of 100 ng/mL doxycycline plus 10 μM cisplatin for 24 h. ChIP was performed using antisera to p53 (fl393G) or normal rabbit IgG. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P -value < 0.05. The IgG results are depicted for the CDKN1A/p21 p53-binding site but were comparable for all other sites analyzed. NOXA was not expressed in this cell line, so this gene was not analyzed.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Binding Assay, Variant Assay, Standard Deviation

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Impaired ferroptosis in S47 cells. ( A ) Representative phase-contrast images of primary MEFs containing wild-type p53 (WT), heterozygous S47/wild type, or S47 treated with 4 μM ferroptosis inducer erastin or vehicle (DMSO) for 8 h (magnification 10×). Data represent the average of three independent studies. Bar, 20 μm. ( B ) Cell viability (Alamar blue) analysis of wild-type, S47/wild-type, or S47 primary MEFs treated with erastin for 72 h. The data represent the average of four independent experiments. Error bars represent standard error of the mean. ( C ) Western blot analysis for GLS2 in wild-type MEFs, wild-type MEFs infected with a lentiviral short hairpin for Gls2 (shGls2), and S47 MEFs untreated or treated with 4 µM erastin for 24 h. GAPDH served as the loading control. In the bottom panel, the percent viability using the Trypan blue exclusion assay is shown. Error bars represent standard deviation. ( D ) qRT–PCR analysis of slc7a11 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. ( E ) qRT–PCR analysis of Ptgs2 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. ( F ) Immunoblot analysis for GPX4 in wild-type and S47 MEFs following treatment with 10 µM CDDP for 24 h. ( G ) Cell viability analysis of wild-type and S47 human LCLs treated with RSL3 for 48 h. The data represent the average of three independent experiments. Error bars represent standard deviation. ( H ) Trypan blue exclusion analysis of the percent viability in wild-type MEFs or wild-type LCLs exposed to 10 µM CDDP, CDDP plus 2 µM ferrostatin-1 (Fer-1), or CDDP plus 20 µM zVAD-fmk. The data represent the average of three independent experiments. Error bars represent standard deviation. ( I ) Proposed model depicting the relative abilities of wild-type p53 and S47 to induce senescence, apoptosis, and ferroptosis and suppress spontaneous tumor initiation.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Western Blot, Infection, Control, Trypan Blue Exclusion Assay, Standard Deviation, Quantitative RT-PCR