β mercaptoethanol  (Millipore)

Journal: The Journal of Experimental Medicine

Article Title: A20 and ABIN-1 synergistically preserve intestinal epithelial cell survival

doi: 10.1084/jem.20180198

Figure Lengend Snippet: A20 and ABIN-1 double-deficient enteroids die primarily via caspase-dependent, RIPK1 kinase-dependent, RIPK3-independent apoptosis. (A) Kaplan-Meier survival curves of the indicated genotypes of mice treated with tamoxifen for 7 d. The number of mice is indicated in the graph legend. (B) Representative confocal microscopy images of PI-stained enteroids with the indicated genotypes treated for 48 h with 4-OHT with or without emricasan or Nec1s, versus mock, as indicated. Bars, 75 or 100 µm, as indicated. (C) Quantitation of cell viability of enteroid cultures treated as indicated (mean ± SD). Viability was measured by using the Cell-Titer Glo 3D assay, and data were normalized to unstimulated cells (%Mock). Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing each stimulation to 4-OHT alone. *, P

Article Snippet: Deletion of A20 or ABIN-1 was performed via treatment with 4-OHT (250 nM; Sigma Aldrich).

Techniques: Mouse Assay, Confocal Microscopy, Staining, Quantitation Assay

Journal: The Journal of Experimental Medicine

Article Title: A20 and ABIN-1 synergistically preserve intestinal epithelial cell survival

doi: 10.1084/jem.20180198

Figure Lengend Snippet: A20 and ABIN-1 double-deficient enteroids are sensitized to TNF-induced apoptosis. (A) Immunoblotting analyses of enteroid cultures treated with 4-OHT for 24 h followed by 2.5 ng/ml TNF for 0, 1.5, and 3 h. Cell lysates were immunoblotted with the antibodies indicated on the right. Solid arrow indicates full-length protein; open arrow indicates cleaved protein. (B) qPCR analyses of mRNA from enteroid cultures of the indicated genotypes after 24 h of 4-OHT treatment followed by stimulation for 0 or 90 min with 2.5 ng/ml TNF. Relative gene abundance was normalized to the mean expression of the housekeeping gene actb (mean ± SD). Statistical significance for individual transcripts was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing each genotype to villin-ER/Cre − enteroids. *, P

Article Snippet: Deletion of A20 or ABIN-1 was performed via treatment with 4-OHT (250 nM; Sigma Aldrich).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A20 and ABIN-1 synergistically preserve intestinal epithelial cell survival

doi: 10.1084/jem.20180198

Figure Lengend Snippet: Combined deletion of A20 and ABIN-1 sensitizes enteroids to TNF-induced cell death. (A) Immunoblot analyses of A20 and ABIN-1 expression in enteroids isolated from villin-ER/Cre + mice of the indicated genotypes and treated with 4-OHT for 24 h followed by stimulation with 2.5 ng/ml TNF for the indicated time points. (B) Representative confocal microscopy images of PI-stained enteroids from indicated genotypes of mice treated with 4-OHT for 24 h followed by 24 h of 2.5 ng/ml TNF versus mock treatment as indicated. Bars, 75 or 100 µm, as indicated. (C) Quantitation of cell viability of enteroid cultures described in B (mean ± SD). (D) qPCR analysis of TNF mRNA from enteroid cultures with the indicated genotypes after 24 h of 4-OHT treatment (mean ± SD). (E) Dose–response curve of TNF-induced cytotoxicity (mean ± SD). Cell viability of enteroids with the indicated genotypes treated for 24 h with 4-OHT followed by 24 h of TNF at the indicated concentrations versus mock treatment. Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect of each concentration to villin-ER/Cre − or villin-ER/Cre + enteroids as indicated. (F) Cell viability of organoids of the indicated genotypes treated with 4-OHT for 24 h followed by stimulation with TWEAK, FasL, or TRAIL for 24 h (mean ± SD). For C, E, and F, viability was measured by using the Cell-Titer Glo 3D assay, and data were normalized to mock-treated cells (%Mock). (C–E) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect to villin-ER/Cre − or villin-ER/Cre + enteroids as indicated. (F) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing each stimulation to 4-OHT alone. *, P

Article Snippet: Deletion of A20 or ABIN-1 was performed via treatment with 4-OHT (250 nM; Sigma Aldrich).

Techniques: Expressing, Isolation, Mouse Assay, Confocal Microscopy, Staining, Quantitation Assay, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Aging (Albany NY)

Article Title: S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α

doi: 10.18632/aging.101760

Figure Lengend Snippet: S100A13 modulates NF-κB activity and SASP expression during cell senescence. ( A ) HCT116 cells were transfected with the control vector, S100A13 wild type or mutant type, and treated with Dox (100 nM) for 3 days. Then cell lysates were subjected to western blot analysis using the indicated antibodies. ( B ) ER:Ras IMR90 cells stably transduced with PHBLV, S100A13 wild type or mutant type were given 4-OHT for 6 days. Cell lysates were then subjected to Western blot analysis for the indicated proteins. ( C ) HCT116 cells were transfected with control (siNC) or two independent siRNAs against S100A13, and treated with Dox (100 nM) for 4 days. Then cell lysates were subjected to western blot analysis using the indicated antibodies. ( D ) ER:Ras IMR90 cells stably transduced with control (siNC) or two independent shRNAs against S100A13, were given 4-OHT for 6 days. Cell lysates were then subjected to Western blot analysis for the indicated proteins. ( E and F ) HCT116 cells were transfected with vector, S100A13 wild type or mutant type, or transfected with control (siNC) or two independent siRNAs against S100A13, and treated with Dox (100 nM) for 4 days. Then mRNA levels of some SASP genes were analyzed by real-time qPCR (n=3). ( G ) HCT116 cells were transfected with wild type S100A13, and treated with Dox (100 nM) for 3 days. Control IgG (0.6 ug/ml) or neutralizing antibody IL-1a (0.6 ug/ml) were added for the last 2 days. Then cell surface-bound IL-1α were analyzed by FACS (n=3). ( H ) HCT116 cells were transfected with siRNA#2 against S100A13, and treated with Dox (100 nM) for 3 days. Solvent or recombinant IL-1a protein (300 ng/ml) were added for the last 2 days. Then cell surface-bound IL-1α were analyzed by FACS (n=3). Three independent experiments were performed. Error bars represent means ± SD (n = 3) *P

Article Snippet: 4-OHT (Sigma) was dissolved in methanol.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Stable Transfection, Transduction, Real-time Polymerase Chain Reaction, FACS, Recombinant

Journal: Aging (Albany NY)

Article Title: S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α

doi: 10.18632/aging.101760

Figure Lengend Snippet: Inhibition the binding of IL-1α to S100A13 or to Cu 2+ suppresses IL-1α secretion, NF-κB activity, and SASP expression. ( A – C ) HCT116 cells were treated with Dox (100 nM) for 4 days in the presence of the indicated doses of Amlexanox or TTM. Then, ( A ) Cell surface-bound IL-1α were analyzed by FACS (n=3). ( B ) Cell lysates were subjected to western blot analysis for the indicated proteins. ( C ) mRNA levels of some SASP genes were analyzed by real-time qPCR (n=3). ( D ) ER:Ras IMR90 cells were given 4-OHT for toal 6 days in the presence of the indicated doses of Amlexanox; fresh medium with 4-OHT and Amlexanox was changed every other day. Cell lysates were then subjected to Western blot analysis for the indicated proteins. ( E–G ) HCT116 cells were treated without or with Dox (100 nM) and the indicated doses of TTM for 4 days. Then, cells were collected, washed with PBS for twice, and, ( E ) Analyzed the intracellular Cu 2+ concentration by ICP-MS (n=3). ( F ) Cell lysates were subjected to western blot analysis for the indicated proteins. ( G ) mRNA levels of some SASP genes were analyzed by real-time qPCR (n=3). ( H ) ER:Ras IMR90 cells were treated with TTM as in (D), then the indicated proteins were analyzed by Western blot. Three independent experiments were performed and analyzed. Error bars represent means ± SD (n = 3) *P

Article Snippet: 4-OHT (Sigma) was dissolved in methanol.

Techniques: Inhibition, Binding Assay, Activity Assay, Expressing, FACS, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay, Mass Spectrometry

Journal: Aging (Albany NY)

Article Title: S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α

doi: 10.18632/aging.101760

Figure Lengend Snippet: S100A13 modulates multiple SASP and cellular senescence regulators. ( A – D ) HCT116 cells were transfected with the control, S100A13 wild type or mutant type ( A and C ), or transfected with the control (siNC) or two independent siRNAs against S100A13 ( B and D ), and then treated with Dox (100 nM) for 3 or 4 days. Then cell lysates were subjected to western blot analysis for the indicated proteins. ( E ) HCT116 cells were transfected with wild type S100A13, and treated with Dox (100 nM) for 3 days. Control IgG (0.6 ug/ml) or neutralizing antibody IL-1a (0.6 ug/ml) were added for the last 2 days. Then the indicated proteins were analyzed by Western blot. ( F ) HCT116 cells were transfected with siRNA#2 against S100A13, and treated with Dox (100 nM) for 3 days. Solvent or recombinant IL-1a protein (300 ng/ml) were added for the last 2 days. Then the indicated proteins were analyzed by Western blot. ( G and H ) ER:Ras IMR90 cells were given 4-OHT for toal 6 days in the presence of the indicated doses of Amlexanox ( G ); or TTM ( H ); then the indicated proteins were analyzed by Western blot. Three independent experiments were performed.

Article Snippet: 4-OHT (Sigma) was dissolved in methanol.

Techniques: Transfection, Mutagenesis, Western Blot, Recombinant

Journal: Aging (Albany NY)

Article Title: S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α

doi: 10.18632/aging.101760

Figure Lengend Snippet: S100A13 is up-regulated and modulates cell surface-bound IL-1α level during cellular senescence. ( A – D ) Normal HCT116 cells either untreated or treated with 100 nM Dox for 4 days. ( A ) Cell lysates were subjected to western blot analysis using the indicated antibodies. ( B ) The mRNA levels of IL-6/IL-8 were analyzed by real-time qPCR. ( C ) The cells were collected, washed, incubated in PBS with FITC-labeled monoclonal antibodies against IL-1α, and processed by FACS analysis to determine the amount of cell surface-bound IL-1α. The histograms were the percent of total fluorescence signal subtracting the background fluorescence for unlabeled cells (n=3). ( D ) The mRNA levels of S100A13 were analyzed by real-time qPCR (n=3). ( E ) ER:Ras IMR90 cells were given 100 nM 4-OHT for the indicated days to induce Ras expression, fresh medium with 4-OHT was changed every other day. Cell lysates were then analyzed for expression of the indicated proteins. ( F ) ER:Ras IMR90 cells were given 4-OHT for 6 days to induce senescence. Cell lysates were then subjected to Western blot analysis for the indicated proteins. ( G and H ) 20PD, 55PD and 43PD 2BS cells were collected, and total proteins and/or total RNAs were extracted, respectively. ( G ) Cell lysates were analyzed by Western blot for the indicated proteins. ( H ) The mRNA levels of S100A13 were analyzed by real-time qPCR (n=3). ( I ) Cells transfected with the siRNA#2 against S100A13 first, then followed by transfection with control vector, the same sense mutation plasmids of S100A13 wild type or mutant type which were insensitive to the siRNA#2 , and then treated with Dox (100 nM) for 3 days, and then cell surface-bound IL-1α were analyzed by FACS (n=3). ( J ) Cells transfected with the control (siNC) or two independent siRNAs against S100A13, then treated with Dox (100 nM) for 4 days, and then cell surface-bound IL-1α were analyzed by FACS (n=3). Three independent experiments were analyzed. Error bars represent means ± SD (n = 3) *P

Article Snippet: 4-OHT (Sigma) was dissolved in methanol.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Incubation, Labeling, FACS, Fluorescence, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Oncogenesis

Article Title: FOXO1 inhibits osteosarcoma oncogenesis via Wnt/β-catenin pathway suppression

doi: 10.1038/oncsis.2015.25

Figure Lengend Snippet: FOXO1 induces growth arrest and apoptosis in osteosarcoma cell lines. OS cell lines stably expressing the empty vector or FOXO1ER were seeded in six-well plates at a density of 2.5 × 10 5 cells per well and treated with 4-OHT (100 n M ) or vehicle. At different time points as indicated, cells were collected for cell cycle analysis ( a ) and apoptosis assay ( b , c ). ( a ) FOXO1 activation inhibits cell-cycle transition. After 24 h, cells were harvested and cell-cycle distribution was analyzed by propidium iodide staining. Bars represent the mean of three measurements±s.d. ( b ) FOXO1 induces cell apoptosis. Apoptosis was measured by annexin V-PE/7-AAD staining. The data are representative of at least three independent experiments that gave similar results. ( c ) The results of ( b ) are represented as specific apoptosis (SA): SA (%)=100(AE−AC)/(1−AC), where AE equals percentage of apoptotic cells in the experimental (4-OHT) group and AC equals percentage of apoptotic cells in the control (vehicle) group. Data are mean±s.d. of at least independent experiments.

Article Snippet: Briefly, cells were seeded in 96-well plates at a density of 3.0 × 103 cells per well and treated with 4-OHT (100 nM ) or vehicle for 2 or 24 h, then fixed with 4% paraformaldehyde solution for 20 min at room temperature and permeabilized with 0.2% Triton X-100 solution for 5 min.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Cell Cycle Assay, Apoptosis Assay, Activation Assay, Staining

Journal: Oncogenesis

Article Title: FOXO1 inhibits osteosarcoma oncogenesis via Wnt/β-catenin pathway suppression

doi: 10.1038/oncsis.2015.25

Figure Lengend Snippet: FOXO1 regulates target gene expression. MG-63 and U2-OS cells expressing FOXO1ER or empty vector were treated with 100 n M 4-OHT or vehicle. One day later, cells were harvested, mRNA and protein was prepared. ( a ) CDKN1B, CyclinD1, TP53INP1, BIM, NOXA and TRAIL mRNA expression was assessed. EV indicates empty vector. The data represent mean±s.d. of least three experiments. ( b ) The expression of BAX, BCL-2, NOXA and BIM protein was detected by immunoblotting. Anti-ACTB antibody was used as a loading control.

Article Snippet: Briefly, cells were seeded in 96-well plates at a density of 3.0 × 103 cells per well and treated with 4-OHT (100 nM ) or vehicle for 2 or 24 h, then fixed with 4% paraformaldehyde solution for 20 min at room temperature and permeabilized with 0.2% Triton X-100 solution for 5 min.

Techniques: Expressing, Plasmid Preparation

Journal: Oncogenesis

Article Title: FOXO1 inhibits osteosarcoma oncogenesis via Wnt/β-catenin pathway suppression

doi: 10.1038/oncsis.2015.25

Figure Lengend Snippet: FOXO1 represses Wnt/β-catenin signaling. MG-63 and U2-OS cells expressing FOXO1ER or empty vector were treated with 100 n M 4-OHT or vehicle. ( a ) One day later, cells were co-transfected with TOPflash luciferase construct and Renilla luciferase vector; the luciferase activity was measured 24 h after transfecting. All experiments were carried out in triplicate. The data represent mean±s.d. of least three experiments. ( b , c ) Two hours (data not shown) or 24 h later, cells were fixed and immunostained with FOXO1 (red) and β-catenin (green) antibodies and visualized under a microscope. 4-OHT induced FOXO1 translocation from the cytoplasm to the nucleus in MG-63 FOXO1ER cells ( b ) and U2-OS FOXOER cells ( c ), and decreased the expression of β-catenin with no influence on the intracellular localization of β-catenin. The experiment was repeated three times with similar observations.

Article Snippet: Briefly, cells were seeded in 96-well plates at a density of 3.0 × 103 cells per well and treated with 4-OHT (100 nM ) or vehicle for 2 or 24 h, then fixed with 4% paraformaldehyde solution for 20 min at room temperature and permeabilized with 0.2% Triton X-100 solution for 5 min.

Techniques: Expressing, Plasmid Preparation, Transfection, Luciferase, Construct, Activity Assay, Microscopy, Translocation Assay

Journal: Oncogenesis

Article Title: FOXO1 inhibits osteosarcoma oncogenesis via Wnt/β-catenin pathway suppression

doi: 10.1038/oncsis.2015.25

Figure Lengend Snippet: FOXO1 inhibits growth of osteosarcoma cell lines and suppresses colony formation capacity. ( a ) Expression of FOXO1ER protein in stably infected MG-63 and U2OS cells was validated by immunoblotting using anti-FOXO1 antibody. Anti-ACTB antibody was used as a loading control. ( b ) Induction of nuclear translocation of FOXO1ER by 4-OHT was verified by immunofluorescence analysis. ( c ) Cell lines were plated in six-well plates at a density of 1 × 10 5 cells per well (day 0). 4-OHT was added every two days at a concentration of 100 n M . Cells were counted with a hemocytometer at days 2, 4 and 6. Cell viability was verified by trypan blue staining. ( d ) Representative photographs of colony formation of MG-63-FOXO1ER and U2OS-FOXO1ER cells 12 days after plating; the number of cells plated in each well is also indicated. All experiments were repeated at least three times. Colony formation was quantified and presented as mean±s.d.

Article Snippet: Briefly, cells were seeded in 96-well plates at a density of 3.0 × 103 cells per well and treated with 4-OHT (100 nM ) or vehicle for 2 or 24 h, then fixed with 4% paraformaldehyde solution for 20 min at room temperature and permeabilized with 0.2% Triton X-100 solution for 5 min.

Techniques: Expressing, Stable Transfection, Infection, Translocation Assay, Immunofluorescence, Concentration Assay, Staining

Journal: The Journal of Cell Biology

Article Title: Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

doi: 10.1083/jcb.200506057

Figure Lengend Snippet: Comparison of the effects of MycER and MycV394DER activation on keratinocyte spreading and motility. 200 nM 4-OHT was added to all cultures 5 d before harvesting. (A and B) Spreading of cells transduced with pBabe (control), MycER, or MycV394DER in the presence or absence of EGF. (A) Quantitation of spreading area per cell. (B) Representative fields of cells labeled with rhodamine-conjugated phalloidin. Pictures were taken using a microscope with a 20× lens with a numerical aperture of 0.50. Bar, 100 μm. (C) Representative detailed fields of a frame from the time-lapse recording (Videos 1–3, available at http://www.jcb.org/content/full/jcb.200506057/DC1 ). Pictures were taken using IMT1 or IMT2 inverted microscopes (Olympus) equipped with monochrome charge-coupled device cameras and a 10× lens with a numerical aperture of 0.25. (D and E) Clonogenic assays of human keratinocytes infected with pBabe (control), MycER, or MycV394DER. Cells were seeded at clonal density and colonies were stained 2 wk later. (D) The absolute number of growing and abortive colonies after plating of 10 4 cells per 35-mm dish. (E) Representative dishes from the experiment shown in D.

Article Snippet: Activation of the steroid-inducible constructs was performed by adding 200 nM 4-OHT (Sigma-Aldrich) to the culture medium.

Techniques: Activation Assay, Transduction, Quantitation Assay, Labeling, Microscopy, Infection, Staining

Journal: The Journal of Cell Biology

Article Title: Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

doi: 10.1083/jcb.200506057

Figure Lengend Snippet: Comparison of the effects of MycER and MycV394DER activation on expression of proteins involved in keratinocyte adhesion. In all experiments, 200 nM 4-OHT was added to all cultures 5 d before harvesting. (A and B) Flow cytometry of cells labeled with antibodies to α6 (A) or β1 (B) integrin subunits. Note that the analysis was performed on cell pools. The heterogeneity in α6 expression most likely results from somewhat varying levels of Myc expression in individual cells. (C) Immunoblots probed with antibodies to the indicated proteins. (D) ChIPs demonstrating binding of Myc but not of MycV394D to the start sites of the integrin β1 and α6 genes in vivo. Chromatin from human keratinocytes infected with the indicated viruses was precipitated with control or α-Myc antibodies. For integrins β1 and α6, primers surrounding the transcription start site were used and, for nucleolin, primers surrounding the Myc-bound E-box sequence were used for the analysis.

Article Snippet: Activation of the steroid-inducible constructs was performed by adding 200 nM 4-OHT (Sigma-Aldrich) to the culture medium.

Techniques: Activation Assay, Expressing, Flow Cytometry, Cytometry, Labeling, Western Blot, Binding Assay, In Vivo, Infection, Sequencing

Journal: The Journal of Cell Biology

Article Title: Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

doi: 10.1083/jcb.200506057

Figure Lengend Snippet: Effects of MycER and MycV394DER on epidermal differentiation in DED cultures. Human keratinocytes were transduced with pBabe (A, D, G, J, M, and P), MycER (B, E, H, K, N, and Q), or MycV394DER (C, F, I, L, O, and R). Cultures were grown in the presence of 200 nM 4-OHT. (A–F) Hematoxylin and eosin staining. (G–I) Immunostaining for involucrin (red). (J–L) Immunostaining for E-cadherin (green). (G–L) DAPI counterstaining (blue). (M–O) Immunostaining for Myc. (P–R) Control staining with secondary reagents only. Pictures A–L were taken using a confocal microscope (Carl Zeiss MicroImaging, Inc.) with a 25× lens with a numerical aperture of 0.80. Pictures M–R were taken using an Axiovert microscope equipped with a MC100 SPOT camera and a 32× lens with a numerical aperture of 0.4. Bars: (A–C) 100 μm; (D–R) 50 μm.

Article Snippet: Activation of the steroid-inducible constructs was performed by adding 200 nM 4-OHT (Sigma-Aldrich) to the culture medium.

Techniques: Transduction, Staining, Immunostaining, Microscopy