Journal: Cell reports

Article Title: Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression

doi: 10.1016/j.celrep.2018.10.061

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant mouse IL3 , Gold Biotechnology , 1310-03-2.

Techniques: Recombinant, Chromatin Immunoprecipitation, SYBR Green Assay, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

doi: 10.1371/journal.pone.0043696

Figure Lengend Snippet: FL5.12 (A) and 32D.3 (C) cells were incubated with 10 µM Scn and DFO for 48 h (NT: no treatment; -IL-3: in the absence of added IL-3). Apoptosis was assayed by annexin V-FITC staining and DAPI was used as a vital stain; percentages of cells positive for annexin staining are indicated. Average annexin V-positivity from three independent experiments are shown for FL5.12 (B) and 32D.3 (D) cells; error was calculated as the standard deviation of three replicates. Statistical significance is indicated as * p <0.05; ** p <0.01; *** p <0.001. Note that while the effect of adding Scn was significant, the effect was anti -apoptotic.

Article Snippet: 32D.3 (ATCC CRL-11346) and FL5.12 (the kind gift of L. Devireddy (Case Western Reserve University)) cells were cultured in modified RPMI-1640 (ATCC) containing 5% fetal calf serum and 5 ng/mL murine IL-3 (BD).

Techniques: Incubation, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

doi: 10.1371/journal.pone.0043696

Figure Lengend Snippet: (A) FL5.12 cells were transduced with the pCVL-SFFV-muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene functionality. (B) A Western blot of supernatants, concentrated from 32 µL, from FL5.12 cells shows that the transduced cells constitutively express Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate chelators DFO and Ent at 100 µM produce robust apoptosis, while the bidentate chelators at 300 µM do not affect viability.

Article Snippet: 32D.3 (ATCC CRL-11346) and FL5.12 (the kind gift of L. Devireddy (Case Western Reserve University)) cells were cultured in modified RPMI-1640 (ATCC) containing 5% fetal calf serum and 5 ng/mL murine IL-3 (BD).

Techniques: Transduction, Fluorescence, Cytometry, Western Blot, Incubation

Journal: PLoS ONE

Article Title: Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

doi: 10.1371/journal.pone.0043696

Figure Lengend Snippet: (A) 32D.3 and FL5.12 cells in the presence (NT) or absence (-IL3) of IL-3 were incubated for 48 h with 10 µM of the anti-Scn antibody MAB1857; percent annexin-V positivity is indicated. (B) The structure of Fab MAB1857 with Scn, shown in a ribbon representation (Fab in gray and Scn in orange), reveals the interface that is occluded in the complex. Had Scn had an effect on apoptosis through receptor-mediated uptake, the effect of the antibody on the process would have identified a potential receptor-interacting surface on Scn, the rationale for this approach. However, since Scn does not affect apoptosis, an anti-Scn antibody cannot reveal a receptor-interacting surface by blocking a non-existent effect.

Article Snippet: 32D.3 (ATCC CRL-11346) and FL5.12 (the kind gift of L. Devireddy (Case Western Reserve University)) cells were cultured in modified RPMI-1640 (ATCC) containing 5% fetal calf serum and 5 ng/mL murine IL-3 (BD).

Techniques: Incubation, Blocking Assay

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: Expression of Bcl-xL and Bim in Baf-3 cells undergoing apoptosis induced by various death stimuli. (A to C) Baf-3 cells were cultured in IL-3-free medium (top panel), treated with 50 Gy of ionizing radiation (IR) and cultured with IL-3 (middle panel), or cultured in medium containing IL-3 and etoposide (VP16) at 3 μg/ml (bottom panel) for the indicated times. The expression of Bcl-xL protein (A), Bim mRNA (B), or Bim protein (C) was detected by specific antibodies for each protein (A and C) or by an RNase protection assay with a cDNA probe designed to detect mouse BimEL and BimL mRNA (B). (D) Percent viability, determined by trypan blue dye exclusion of Baf-3 cells treated with the various cell death inducers at the time of sample collection.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Expressing, Cell Culture, Rnase Protection Assay

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: Bim phosphorylation. (A) Immunoblot analysis using the Bim antibody with lysates from parental Baf-3 cells (pt) (lanes 1 and 2) or cells overexpressing BimEL and BimL (lanes 3 and 4) cultured in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of IL-3. To demonstrate an extra band comigrating with BimL, we added five times more cell extract to lane 1 than to the other lanes. (B) Lysates from Baf-3 cells overexpressing BimEL and BimL proteins were immunoprecipitated (IP) with preimmune serum (lane 1) or Bim antibody (lanes 2 to 4). After treatment with CIAP in the absence (lane 3) or presence (lane 4) of phosphatase inhibitor, precipitants were separated with SDS-PAGE and Bim proteins were then detected with Bim antibody. (C) Baf-3 cells overexpressing BimEL protein were cultured in medium containing [32P]orthophosphate in the presence (lanes 1 and 2) or absence (lane 3) of IL-3 for 4 h. Immunoprecipitates obtained by preimmune serum (lane 1) or Bim antibody (lanes 2 and 3) were separated by SDS-PAGE. (D) Lysates from Baf-3 cells overexpressing BimEL protein were immunoprecipitated with preimmune serum (lane 2) or antibody against Bim (lane 3), Bcl-2 (lane 4), or Bcl-x (lane 5). After immunoprecipitation and SDS-PAGE followed by blotting, Bim proteins were detected with Bim antibody. Lane 1 is a positive control to detect BimEL protein in whole-cell lysates.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Western Blot, Cell Culture, Immunoprecipitation, SDS Page, Positive Control

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: Induction of apoptosis by each Bim isoform. (A) Immunoblot analysis of Baf-3 cells engeneered to express BimEL (lanes 1 and 2), BimL (lanes 3 and 4), or BimS (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of zinc (100 μM). Samples were collected 4 h after the addition of Zn. (B) Percent viability of cells expressing BimEL, BimL, or BimS after the addition of Zn (100 μM) in the presence of IL-3 (5 ng/ml). (C) Baf-3 cells inducibly expressing BimEL, pMT(BimELns clone 10), were cultured in IL-3-containing medium (5 ng/ml) with various Zn concentrations for 4 h (lanes 1 to 4). For comparison of expression levels, cell extracts from parental Baf-3 cells cultured in the absence of IL-3 for the indicated times (lanes 5 to 7) were blotted on the same membrane. Bim proteins were detected by immunoblot analysis. (D) Percent viability of pMT(BimELns clone 10) cells cultured in IL-3-containing medium (5 ng/ml) with various concentration of Zn.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Western Blot, Expressing, Cell Culture, Concentration Assay

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: Identification of upstream pathways regulating Bim expression. (A) IL-3 was washed from the culture medium of β544 cells, and the cells were cultured in the presence of human GM-CSF (hGM-CSF) or murine IL-3 (mIL3) or in the absence of cytokines. The number of viable cells was determined by trypan blue dye exclusion. (B) Immunoblot analysis using lysates from β544 cells cultured in human GM-CSF (lanes 1 to 14) or in the absence of cytokines (lanes 15 and 16) for the indicated times. Bim (top panel) and Bcl-xL (bottom panel) proteins were detected by specific antibodies to each protein. (C) Phosphorylation of p42/MAPK. Parental Baf-3 cells (lanes 1 and 2) or cells expressing Ras(S17N) induced by the addition of 10−7 M Dex (lanes 3 and 4) were cultured in IL-3-free medium for 2 h (lanes 1 and 3), after which IL-3 was added at 20 ng/ml for 5 min (lanes 2 and 4). p42/MAPK was detected by immunoblot analysis using a specific monoclonal antibody. Cell extracts from 105 cells were loaded per lane. (D) Parental Baf-3 cells (lane 1) or cells containing Dex-inducible Ras(S17N) in the presence or absence of Dex (lanes 2 and 3) were cultured in IL-3-containing medium. The phosphorylated form of Akt was detected with antibody specifically recognizing phosphorylated Akt at Ser473. (E) Parental Baf-3 cells and cells expressing Ras(S17N) were cultured in the presence or absence of wortmannin (WMN). The number of viable cells (left panel) and the percent viability determined by trypan blue dye exclusion (right panel) are shown. The results are from a representative experiment; similar results were obtained in three other independent experiments. (F) Immunoblot analysis with Bim antibodies. Parental Baf-3 cells treated with wortmannin at 0.5 μM (top panel) or cells expressing Ras(S17N) induced by addition of 10−7 M Dex in the absence (middle panel) or presence (bottom panel) of wortmannin were cultured in IL-3-containing medium (5 ng/ml). Cell extracts were prepared after the indicated times.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Expressing, Cell Culture, Western Blot

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: mTOR participates in the regulation of Bim by PI3-K pathways. (A and B) Immunoblot analysis with Bim antibodies. Cells were cultured in IL-3-free medium for the indicated times. Baf-3 cells containing Dex-inducible Ras(G12V) were cultured with 10−7 M Dex in the absence (upper panel) or presence (lower panel) of wortmannin (WMN) (0.5 μM) (A). Baf-3 cells containing Dex-inducible Ras(G12V/V45E) were cultured with 10−7 M Dex (top panel), in the presence of wortmannin (upper middle panel) or rapamycin (Rapa) (10 ng/ml; lower middle panel); Baf-3 cells containing Dex-inducible Ras(G12V/V45E) were engeneered to express an AU1-tagged rapamycin-resistant form of mTOR (AU1-mTOR-rr) (see Materials and Methods) and were cultured with rapamycin (bottom panel) (B). (C) Immunoblot analysis of AU1-tagged mTOR-rr and an endogenous mTOR protein in cell extracts from Baf-3 cells containing Dex-inducible Ras(G12V/V45E) (V45E; lanes 1 and 3) or expressing AU1-mTOR-rr (rr, lanes 2 and 4), using monoclonal antibodies specific for AU1 (lanes 1 and 2) and mTOR (lanes 3 and 4). (D) Percent viability of cells expressing Ras(G12V/V45E), with or without mTOR-rr (rr), in the presence or absence of rapamycin in IL-3-free medium. Also shown is that of cells expressing Ras(G12V/V45E) and MAA-PKB in IL-3-free medium.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Western Blot, Cell Culture, Expressing

Journal:

Article Title: Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

doi: 10.1128/MCB.21.3.854-864.2001

Figure Lengend Snippet: (A) Phosphorylation of Akt in Baf-3 cells. Parental (Pt) Baf-3 cells were cultured in the presence (lane 1) or absence (lane 2) of IL-3 for 12 h. Baf-3 cells containing Dexinducible Ras(G12V/V45E) were cultured in IL-3-containing medium without (lane 3) or with (lane 4) 10−7 M Dex for 16 h. The Dex-treated cells were then cultured in IL-3-free, Dex-containing medium for 12 h without wortmannin (WMN) or rapamycin (Rap) (lane 5), with 10 ng/ml rapamycin (lane 6), or with 100 nM wortmannin (lane 7). Akt phosphorylated at Ser473 (top panel) was detected using specific antibodies. (B) Expression of total Akt protein in IL-3-starved Baf-3 cells as detected by immunoblot analysis with antibody recognizing both phosphorylated and nonphosphorylated Akt. Baf-3 cells engineered to express myr-Akt upon addition of Zn were cultured in IL-3-containing medium in the absence (lane 1) or presence (lane 2) of 100 μM Zn for 16 h. The cells were then transferred into IL-3-free medium containing Zn for the indicated times. (C) Akt kinase assay. Parental cells (Pt) (lanes 1 and 2) and cells expressing a dominant-negative form of Akt (dn-Δkt) (lanes 3 and 4) were starved of IL-3 for 2 h (lanes 1 and 3) and then cultured in the presence of IL-3 (20 ng/ml) for 5 min (lanes 2 and 4). The kinase activity of Akt was determined by histone H2B phosphorylation in immunoprecipitates using Akt antibody. (D) Baf-3 cells containing Dex-inducible Ras(G12V/V45E) were retrovirally engineered to express a dominant negative form of Akt (MAA-PKB) (dnΔkt) and then were cultured in IL-3-free, Dex-containing medium for the indicated times.

Article Snippet: In some experiments, recombinant mouse IL-3 (Wako Pure Chemical, Osaka, Japan) was used at the concentrations indicated in the figure legends.

Techniques: Cell Culture, Expressing, Western Blot, Kinase Assay, Dominant Negative Mutation, Activity Assay