Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: Phosphorylated STAT1 modulates AIM2 expression and RTEC PANoptosis. A ) Venn diagram of differentially expressed genes (DEGs) among the GSE9493 and GSE76882 datasets, murine renal allograft RNA-seq (Syn vs Allo), and HK-2 cell RNA-seq (control vs TNF-α). B ) Venn diagram of transcription factors whose putative binding sites within the AIM2 promoter were predicted by hTFtarget, KnockTF, JASPAR, and GTRD. C ) Overlap between DEGs and predicted transcription factors. D-E ) Western blot analysis of the protein levels of STAT1 and p-STAT1 in renal tissue from normal, CAD, Syn, and Allo groups. F ) Representative IHC images of p-STAT1 expression in renal tissue from normal, CAD, Syn, and Allo groups. G-H ) Western blot and quantitative analysis of the protein levels of STAT1, p-STAT1 (Tyr701), and p-STAT1 (Ser727) in HK-2 cells treated with TNF-α (100 ng/mL) for 0, 0.25, 0.5, 1, 3, or 6 h. I-J ) Western blot analysis of the protein levels of ( I ) STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), AIM2, and Fibronectin; ( J ) full-length/cleaved GSDMD, full-length/cleaved CASP1, full-length/cleaved CASP3, and p-MLKL in HK-2 cells treated with TNF-α supplemented with or without Fludarabine (10 μM). K-L ) Western blot analysis of the protein levels of ( K ) STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), AIM2, and Fibronectin; ( L ) full-length/cleaved GSDMD, full-length/cleaved CASP1, full-length/cleaved CASP3, and p-MLKL from the control groups (NC) and STAT1 knockdown groups (Ri-STAT1) treated with TNF-α. M ) Representative IF images of STAT1 and p-STAT1 expression in HK-2 cells from the control and TNF-α-treated groups. N ) Western blot analysis of the protein levels of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in cytoplasmic and nuclear extracts of HK-2 cells treated with TNF-α. O ) Western blot analysis of the protein levels of STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727) from the control groups (WT) and STAT1 phospho-site mutant (STAT1-Y701F, and STAT1-S727A) groups treated with TNF-α. P ) Western blot analysis of the protein levels of STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 from the control groups (WT) and STAT1 phospho-site mutant (STAT1-Y701F, and/or STAT1-S727A) treated with TNF-α.

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Expressing, RNA Sequencing, Control, Binding Assay, Western Blot, Knockdown, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: TNF-α induces STAT1 phosphorylation at Tyr701 to directly bind the AIM2 promoter and enhance transcription, and phosphorylation at Ser727 via the p38/MAPK pathway further augments AIM2 transcription. A ) STAT1 DNA-binding motif obtained from JASPAR. B ) Predicted STAT1-binding sites within the AIM2 promoter (JASPAR). C-E ) HK-2 cells were treated with TNF-α (100 ng/mL) or vehicle for 24 h, then subjected to ChIP using anti-STAT1 antibody; IgG served as the negative control. ( C , D ) qPCR quantification of STAT1 enrichment at site 1 and site 2. ( E ) Agarose-gel verification of site-1 amplification. F ) Relative luciferase activity of cells transfected with pGL3-AIM2-WT reporter plasmid and mutant plasmids (pGL3-AIM2-MUT1 or pGL3-AIM2-MUT2) after treatment ± TNF-α. G-K ) Western blot analysis of the protein levels of JNK, p-JNK, ERK, p-ERK, p38MAPK, p-p38MAPK, p65, p-p65, AKT, and p-AKT in HK-2 cells treated with TNF-α (100 ng/mL) for 0, 0.25, 0.5, 1, 3, and 6 h. L ) Western blot analysis of AIM2 in HK-2 cells treated with TNF-α supplemented with or without (MK-2206 [AKT inhibitor], QNZ [NF-κB inhibitor], SB203580 [p38MAPK inhibitor], SP600125 [JNK inhibitor], or U0126 [MEK/ERK inhibitor]) for 24 h. M ) Western blot and quantitative analysis of Fibronectin, STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 in HK-2 cells treated with TNF-α supplemented with or without SB203580. N ) Western blot analysis of Fibronectin, STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 in HK-2 cells treated with TNF-α supplemented with or without R7050 (TNF-α Antagonist).

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Phospho-proteomics, Binding Assay, Negative Control, Agarose Gel Electrophoresis, Amplification, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: Schematic description of the molecular mechanism. TNF-α drives tubular epithelial injury via the direct induction of STAT1 Y701 phosphorylation and p38 MAPK-mediated STAT1 phosphorylation at S727, cooperatively upregulating AIM2 to execute PANoptosis. PANoptotic cells secrete IL-6 and TGF-β, inducing EMT in adjacent tubular cells, thereby collectively promoting renal allograft interstitial fibrosis.

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-mediated cytotoxicity via TNFR1 was independent of DD and NSD. HEK 293T cells were transfected with empty vector, wild-type TNFR1, ΔDD-TNFR1 or ΔNSD-TNFR1 containing plasmids. (a and c) The cell surface expression of TNFR1 and its mutants (a), and TNFR2 (c) detected by flow cytometry. (b) The transfected HEK 293T cells were stimulated for 24 h with 100 ng/ml sTNF-α or tmTNF-α on fixed NIH3T3 cells at an effector/target ratio of 10:1. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The cytotoxicity was detected by MTT assays. (d) HEK 293T cells were co-transfected with control or TNFR2 siRNA and expression vectors for TNFR1 or its mutants as indicated. After 48 h, the cells were stimulated with tmTNF-α on fixed NIH3T3 for 24 h. The cytotoxicity of tmTNF-α was analyzed by MTT assays. (e) The 24 h-cytotoxicity of R32W-tmTNF-α expressed on COS-7 cells towards HEK 293T cells expressing TNFR1 or mutants thereof. (f) The 24 h-cytotoxicity of sTNF-α to HEK 293T cells co-treated with indicated concentrations of S1P. (g) The 24 h-cytotoxicity of tmTNF-α to HEK 293T cells expressing TNFR1 or its mutants in the presence of 10 μM S1P. All the quantitative data represent means±S.D. of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Neutralization, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α induced formation of DISC via TNFR1 at the plasma membrane. U937 cells were pre-treated with 100 μM MDC for 1 h and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α on fixed COS-7 cells at an E/T ratio of 10:1 for 30 min. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a), cytoplasmic (c) and membrane (d and f) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting with the indicated antibodies. (e) Confocal images of the cellular distribution of FADD or caspase 8 (green) in U937 cells in response to sTNF-α or R32W-tmTNF-α (magnification, × 400). (g) Degradation of Iκ-B was analyzed by western blotting. All immunoprecipitation (IP) or/and western data are representative of three independent experiments. (b and h) The 24 h-cytotoxicity of tmTNF-α (E/T: 10:1) or sTNF-α (100 ng/ml) towards U937 cells transfected with control or FADD siRNA (b) or pre-treated with PDTC (100 μM) for 1 h (h). The data represent means±S.D. of at least three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Clinical Proteomics, Membrane, Neutralization, Immunoprecipitation, Western Blot, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: STAT1 was necessary for tmTNF-induced DISC formation. (a and b) HEK 293T cells were transfected to express wild-type TNFR1, or ΔDD-TNFR1 and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α for 30 min. For neutralization, R32W-tmTNF-α-expressing cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a) and membrane (b) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting. R1: TNFR1; ΔDD: ΔDD-TNFR1. U3A (c–e) or HT1080 (f) cells were transfected by STAT1-containing plasmid or siRNA against STAT1, respectively, followed by stimulation with R32W-tmTNF-α for 30 min. Immunoprecipitation (IP)/western blotting was performed in total (c and f), membrane (d) and cytoplasmic (e) protein with an antibody to TNFR1. Ab, anti-TNF-α antibody; C, Control; T, tmTNF-α. (g) U3A cells were transfected with His-tagged empty vector, His-TNFR1 or His-ΔDD-TNFR1 containing plasmids and stimulated with R32W-tmTNF-α for 30 min. DISC formation was analyzed by IP/western blotting using an anti-His antibody. All the IP/western data are representative of three independent experiments. (h) The 24 h-cytotoxicity of R32W-tmTNF-α to U3A cells pre-treated for 30 min with caspase 8 inhibitor Z-IETD-FMK (4 μM). The data represent means±S.D. of three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Neutralization, Expressing, Membrane, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-mediated cytotoxicity via TNFR1 was independent of DD and NSD. HEK 293T cells were transfected with empty vector, wild-type TNFR1, ΔDD-TNFR1 or ΔNSD-TNFR1 containing plasmids. (a and c) The cell surface expression of TNFR1 and its mutants (a), and TNFR2 (c) detected by flow cytometry. (b) The transfected HEK 293T cells were stimulated for 24 h with 100 ng/ml sTNF-α or tmTNF-α on fixed NIH3T3 cells at an effector/target ratio of 10:1. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The cytotoxicity was detected by MTT assays. (d) HEK 293T cells were co-transfected with control or TNFR2 siRNA and expression vectors for TNFR1 or its mutants as indicated. After 48 h, the cells were stimulated with tmTNF-α on fixed NIH3T3 for 24 h. The cytotoxicity of tmTNF-α was analyzed by MTT assays. (e) The 24 h-cytotoxicity of R32W-tmTNF-α expressed on COS-7 cells towards HEK 293T cells expressing TNFR1 or mutants thereof. (f) The 24 h-cytotoxicity of sTNF-α to HEK 293T cells co-treated with indicated concentrations of S1P. (g) The 24 h-cytotoxicity of tmTNF-α to HEK 293T cells expressing TNFR1 or its mutants in the presence of 10 μM S1P. All the quantitative data represent means±S.D. of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Neutralization, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α induced formation of DISC via TNFR1 at the plasma membrane. U937 cells were pre-treated with 100 μM MDC for 1 h and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α on fixed COS-7 cells at an E/T ratio of 10:1 for 30 min. For neutralization of tmTNF-α, effector cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a), cytoplasmic (c) and membrane (d and f) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting with the indicated antibodies. (e) Confocal images of the cellular distribution of FADD or caspase 8 (green) in U937 cells in response to sTNF-α or R32W-tmTNF-α (magnification, × 400). (g) Degradation of Iκ-B was analyzed by western blotting. All immunoprecipitation (IP) or/and western data are representative of three independent experiments. (b and h) The 24 h-cytotoxicity of tmTNF-α (E/T: 10:1) or sTNF-α (100 ng/ml) towards U937 cells transfected with control or FADD siRNA (b) or pre-treated with PDTC (100 μM) for 1 h (h). The data represent means±S.D. of at least three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Clinical Proteomics, Membrane, Neutralization, Immunoprecipitation, Western Blot, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: STAT1 was necessary for tmTNF-induced DISC formation. (a and b) HEK 293T cells were transfected to express wild-type TNFR1, or ΔDD-TNFR1 and then stimulated with 100 ng/ml sTNF-α or R32W-tmTNF-α for 30 min. For neutralization, R32W-tmTNF-α-expressing cells were treated with anti-TNF-α (Ab) for 30 min prior to the addition to the target cells. The total (a) and membrane (b) protein was immunoprecipitated with an anti-TNFR1 antibody and analyzed by immunoblotting. R1: TNFR1; ΔDD: ΔDD-TNFR1. U3A (c–e) or HT1080 (f) cells were transfected by STAT1-containing plasmid or siRNA against STAT1, respectively, followed by stimulation with R32W-tmTNF-α for 30 min. Immunoprecipitation (IP)/western blotting was performed in total (c and f), membrane (d) and cytoplasmic (e) protein with an antibody to TNFR1. Ab, anti-TNF-α antibody; C, Control; T, tmTNF-α. (g) U3A cells were transfected with His-tagged empty vector, His-TNFR1 or His-ΔDD-TNFR1 containing plasmids and stimulated with R32W-tmTNF-α for 30 min. DISC formation was analyzed by IP/western blotting using an anti-His antibody. All the IP/western data are representative of three independent experiments. (h) The 24 h-cytotoxicity of R32W-tmTNF-α to U3A cells pre-treated for 30 min with caspase 8 inhibitor Z-IETD-FMK (4 μM). The data represent means±S.D. of three independent experiments. ***P<0.001 versus corresponding treatment in the control group

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Transfection, Neutralization, Expressing, Membrane, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1

doi: 10.1038/cdd.2016.162

Figure Lengend Snippet: tmTNF-α-induced STAT1-dependent apoptotic signaling via TNFR1. sTNF-α induces binding of TRADD to the DD of TNFR1. TNFR1-bound TRADD further recruits TRAF2 and cIAP1/2 at the plasma membrane to activate NF-κB pathway. However, after TNFR1 internalization, STAT1 binds to TRADD and is phosphorylated at tyrosine 701, which excludes the binding of TRAF2 and cIAP1 to TRADD and attenuates NF-κB activation. Meanwhile, TRADD recruits FADD and caspase 8 to form DISC in the cytoplasm to mediate apoptosis. In contrast, tmTNF-α induces binding of STAT1 to the SD of TNFR1 and STAT1 serine phosphorylation at 727. The S727 phosphorylated STAT1 further recruits TRADD to form DISC at the plasma membrane to trigger apoptosis, but not NF-κB activation

Article Snippet: The plasmids pcDNA3.1 containing wild-type STAT1, Y701I-STAT1, S727A-STAT1 or DM (Y701I and S727A double mutations)-STAT1 were kindly gifted by Dr. Guanxin Shen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology). siRNA against FADD, STAT1 and TNFR2, and a scrambled control siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Binding Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics

Journal: Cancer research

Article Title: Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress.

doi: 10.1158/0008-5472.can-03-2664

Figure Lengend Snippet: Fig. 1. A, steady-state endogenous levels of the clusterin (CLU) protein (top panel) in OS cell lines, as revealed by immunoblotting analysis of whole cell lysates under nonreducing conditions. The Sa OS and KH OS cells express significantly lower levels of CLU protein as compared with U-2 OS cells. The OS cell lines used are distinct, as far as the status of several oncogenes or tumor suppressor proteins (e.g., p53, middle panel) is concerned. B, CLU protein kinetics in Sa OS (top panel), KH OS (middle panel), and U-2 OS cells (bottom panel) after cell treatment with 0.35 M doxorubicin (DXR) for 24 h. Although CLU protein is induced in all three cell lines after drug treatment, its up- regulation is more intense in the KH OS and Sa OS cells as compared with U-2 OS cells. Secreted (s)-CLU, the mature secreted protein form of 75 kDa; cytoplasmic (c)-CLU, the intracellular precursor protein form of 60 kDa. Protein loading was verified by actin (bottom panels). Molecular weight markers are indicated on the right of each blot. 1836

Article Snippet: Antibodies against actin, Stat1, phosphorylated double-stranded RNAdependent protein kinase (binds a phosphorylated-activated form of PKR), poly(ADP-ribose) polymerase, bcl-2, pan-p53 (detects both wild type and mutant p53), p21, bax, and Ku70 were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Molecular Weight

Journal: Cancer research

Article Title: Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress.

doi: 10.1158/0008-5472.can-03-2664

Figure Lengend Snippet: Fig. 6. A and B, effect of clusterin (CLU) knock down [small interfering RNA (siRNA) treatment for 60 h] in the steady-state levels of proteins related to the cellular anti- or proapoptotic machinery in KH OS (A) and U-2 OS (B) cells. The CLU-specific siRNA oligos used do not affect the levels of the putative nuclear (n)-CLU protein form of 55 kDa (n-CLU55); a minor effect can be seen at the cytoplasmic protein form of 49 kDa (c-CLU49). CLU knockdown induces down-regulation of the bcl-2 protein in both cell lines that, in p53WT U-2 OS cells, is accompanied with p53, Bax, and p21 accumulation. C, U-2 OS cells treated with Sc-I or Cl-I, Cl-I/II oligonucleotides for 70 h were exposed to 0.35 M doxorubicin (DXR) for 24 h, and cell lysates were analyzed for p53 accumulation. CLU knock down cells show a more intense and robust p53 accumulation after DNA damage by DXR, as compared with controls. 1839

Article Snippet: Antibodies against actin, Stat1, phosphorylated double-stranded RNAdependent protein kinase (binds a phosphorylated-activated form of PKR), poly(ADP-ribose) polymerase, bcl-2, pan-p53 (detects both wild type and mutant p53), p21, bax, and Ku70 were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Small Interfering RNA