Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 3. Biologic effect of increased miR-130a expression. Stable transfection of 32Dcl3 cells with the pEGP-miR-130a, pEGP-miR-223, or pEGP-miR-Null vectors was performed, generating 3 pEGP-miR-130a (Cl 3, Cl 6, and Cl 7) clones, 1 pEGP-miR223 (miR-223) clone, and 1 pEGP-miR-null (null) clone. (A) Relative miR-130a expression in Cl 3, Cl 6, Cl 7, null, and miR-223 was determined by real-time PCR and normalized to Sno234 expression. (B) Relative miR-223 expres- sion in clone miR-223 and null was determined by real-time PCR and normalized to Sno234 expression. (C) Immunoblot showing Smad4 protein expression in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones. The graph shows the relative expression of Smad4 protein relative to the expression of -actin. The highest value among those being compared was assigned the value 1, and the remaining values were recalculated accordingly. (D) The relative expression level of Smad4 mRNA in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones was measured by real-time PCR and normalized to -actin expression. Data are shown as the mean SD from triplicate measurements. (E) Cell proliferation assays were performed with the clones Cl 3, Cl 6, Cl 7, null, and miR-223 in the presence or absence of human recombinant TGF-1 (20nM). The graph shows the relative number of cells after 4 days when TGF-1– stimulated cells are compared with unstimulated cells for each clone. The mean reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 8% for Cl 3, 31% for Cl 6, 8% for Cl 7, 43% for null, and 52% for miR-223 (the values are based on 3 independent growth experiments). The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-1–

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, Stable Transfection, Clone Assay, Real-time Polymerase Chain Reaction, Western Blot, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 4. The effect of knocking-down Smad4 expression with a shRNA against Smad4 mRNA. (A) Immunoblot showing Smad4 protein expression in 32Dcl3 stably transfected with a plasmid encoding a short hairpin against Smad4 (sh-Smad4) or a nonsilencing short hairpin (sh-neg), respectively. The graph shows the relative expression of Smad4 normalized to -actin expression. (B) The relative expression level of Smad4 mRNA in the 32Dcl3 clones sh-Smad4 and sh-neg, respectively. Smad4 mRNA expression was measured by real-time PCR and normalized to -actin expression. Data are shown as the mean SD from triplicate measurements. (C) Cell proliferation assays were performed with the 32Dcl3 clones sh-Smad4 and sh-neg in the presence or absence of human recombinant TGF-1 (20nM). The graph shows the relative number of cells after 4 days when TGF-1–stimulated cells are compared with unstimulated cells for each clone. The reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 19% for sh-Smad4 and 47% for sh-neg. The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-1–stimulated cells was recalculated accordingly.

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, shRNA, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Real-time Polymerase Chain Reaction, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 5. TGF-1 sensitivity is restored by antisense RNA against miRNA-130a or expression of a Smad4 mRNA lacking miR-130a–binding sites. (A) 2 32Dcl3 clones stably transfected with pmiRZIP-130a (pZIP-130a–cl 1 and pZIP-130a–cl 2) or pmiRZIP-null (pZIP-null–cl 1and pZIP-null–cl 2) were tested for Smad4 expression by immunoblotting. The graph shows the relative expression of Smad4 normalized to -actin expression. (B) Cell proliferation assays were performed with pZIP-null–cl 1, pZIP-null–cl 2, pZIP-130a–cl 1, and pZIP-130a–cl 2 in the presence or absence of human recombinant TGF-1 (20nM). The mean reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 36% for pZIP-null–cl 1, 39% for

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, Binding Assay, Clone Assay, Stable Transfection, Transfection, Western Blot, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 6. Identification of miR-130a binding sites in the Smad4 mRNA. (A) Schematic drawing showing the cloning of the predicted miR-130a binding sites in the 3-UTR of pMIR-REPORT. (B-C) 32Dcl3 cells were transfected individually with the firefly expression vector pMIR-REPORT containing 1 of the 4 different constructs (wt1/wt2, mut1/wt2, wt1/mut2, and mut1/mut2) along with a renilla luciferase vector (for normalization) and pre–miR-130a/scrambled miRNA (B) or LNA-130a/LNA-neg (C), respectively. Relative firefly luciferase activity is shown after normalizing to the renilla luminescence. Data are shown as the mean SD from triplicate measure- ments. The expression from the transfections with the controls (miR-neg and LNA-neg) was assigned the value 1, and the relative expression measured from the constructs cotransfected with miR-130a or LNA-130a was recalculated accordingly.

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Binding Assay, Cloning, Transfection, Expressing, Plasmid Preparation, Construct, Luciferase, Activity Assay

Journal: Oncogene

Article Title: p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

doi: 10.1038/onc.2009.260

Figure Lengend Snippet: Figure 1 Stromal-derived factor-1a (SDF-1a) differentially activates Cdc42 and Rac1 GTPases in 32D and 32Dp210 cells. (a and b) 32D and 32Dp210 cells deprived of serum for 18 h were treated with or without murine SDF-1a (500 ng/ml) for 5 min for measuring the amounts of Cdc42–GTP (a) and Rac1–GTP (b) by GST–PBD (glutathione S-transferase–Rac/Cdc42 binding do- main of p21 activated kinase 1 protein) pull-down assays. The western blot analysis was performed using Cdc42 and Rac1 antibodies. The top panel shows the western blot analysis for Cdc42 (a) and Rac1 (b) in the GTP-bound form that associates with GST–PBD and 8% of total input. By densitometric scanning, active Rho GTPase was measured and its percentage relative to total Rho GTPase was calculated. Average values from three individual experiments are shown below the western blot data. The intensity ratio of Rho GTPase in GTP-bound form to total Rho GTPase in lysate of 32D cells without SDF-1a treatment was set to 1, and the relative fold of active Rho GTPase for each condition is shown in the lower panel as means±s.d. (n ¼ 3) (‘*’ indicates Po0.05; ‘**’ indicates Po0.01). (c) The expression of chemokine (CXC motif) receptor 4 (CXCR4) in 32D and 32Dp210 cells was detected by western blot using anti- CXCR4 antibody. Actin is shown as a loading control.

Article Snippet: Mouse 32D (32Dcl3) cells were purchased from American Type Culture Collection and maintained in the same medium used for transformed 32Dp210 cell line with 25% WEHI-cultured conditioned medium as a source of interleukin-3.

Techniques: Derivative Assay, Binding Assay, Western Blot, Expressing, Control

Journal: Oncogene

Article Title: p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

doi: 10.1038/onc.2009.260

Figure Lengend Snippet: Figure 2 Inhibition of p210BcrAbl tyrosine kinase inactivates Cdc42 and Rac1 GTPases and restores SDF-1a chemotaxis. (a and b) Murine 32D and 32Dp210 cells were maintained in serum-free medium for 12 h and then treated with or without Gleevec (2 mM) for additional 6 h. Human K562 cells were incubated in serum-free medium in the presence or absence of Gleevec (5 mM) for 18 h. Cells were then collected for GST–PBD pull-down assays. The representative amounts of active and total Cdc42 (a) and Rac1 (b) are shown in the top panel. The intensity ratio of Rho GTPase in GTP-bound form to total Rho GTPase in lysate of 32D/K562 cells without Gleevec treatment was set to 1, and the relative fold of active Rho GTPase for each condition is shown in the lower panel as means±s.d. (n ¼ 3) (‘*’ indicates Po0.05; ‘**’ indicates Po0.01). (c) 32D and 32Dp210 cells treated as described above were then re-suspended in fresh medium and seeded in triplicate into the upper chamber of the transwell. The lower chamber of the transwell was filled with serum-free medium with or without murine SDF-1a (100 ng/ml). After 4 h, transmigrated cells recovered from the lower chamber were counted. The percentage of migration was calculated by dividing the transmigrated number by the total cells in the initial suspension (105). Data are represented as mean±s.d. of six transwell experiments from two independent treatments (n ¼ 6) (‘*’ indicates Po0.05; ‘**’ indicates Po0.01).

Article Snippet: Mouse 32D (32Dcl3) cells were purchased from American Type Culture Collection and maintained in the same medium used for transformed 32Dp210 cell line with 25% WEHI-cultured conditioned medium as a source of interleukin-3.

Techniques: Inhibition, Chemotaxis Assay, Incubation, Migration, Suspension

Journal: Oncogene

Article Title: p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

doi: 10.1038/onc.2009.260

Figure Lengend Snippet: Figure 4 SDF-1a-induced finger-like projection is abolished by p210BcrAbl-mediated deregulation of Cdc42. (a and b) 32D and 32Dp210 cells deprived of serum for 18 h were re-suspended in serum-free medium and plated onto fibronectin-coated glass slide assembled in the POC-R open cultivation chamber. Cells were then treated with murine SDF-1a (25 ng/ml) for time-lapse recording at 15-s intervals and were imaged using 100 DIC objective for 20 min. The morphology of representative 32D cells treated with or without SDF-1a was shown as indicated in (a). The total numbers of finger-like projection from 80 frames were counted by MetaMorph software and the projection numbers are shown as the mean±s.d. of five cells from at least three independent treatments (‘**’ indicates Po0.01) (b). (c) 32D cells were transfected with green fluorescent protein (GFP) or GFP-tagged dominant active or negative Cdc42 and Rac1 plasmids. Transfected cells were re-suspended in serum-free medium for 18 h and SDF-1a-induced finger-like projections were observed and quantified as described above (n ¼ 5). (d) 32Dp210 cells deprived of serum for 15 h were then treated with or without Gleevec (2 mM) for 3 h. Cells were then stimulated with or without murine SDF-1a (500 ng/ml) for 5 min for measuring the amounts of Cdc42–GTP by GST—PBD pull-down assays and the western blot using Cdc42 antibody. The representative western blot in top panel shows the Cdc42 in the GTP-bound form that associates with GST–PBD and 7% of total input. By densitometric scanning, the intensity ratio of Cdc42 in GTP-bound form to total Cdc42 in lysate of 32Dp210 cells with Gleevec treatment was set to 1, and the relative fold of active Cdc42 for each condition is shown in the lower panel as means±s.d. of three individual experiments (‘*’ indicates Po0.05).

Article Snippet: Mouse 32D (32Dcl3) cells were purchased from American Type Culture Collection and maintained in the same medium used for transformed 32Dp210 cell line with 25% WEHI-cultured conditioned medium as a source of interleukin-3.

Techniques: Software, Transfection, Western Blot

Journal: Oncogene

Article Title: p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

doi: 10.1038/onc.2009.260

Figure Lengend Snippet: Figure 6 The involvement of Vav1 in p210BcrAbl -mediated Cdc42/Rac1 GTPase activation. (a) 32D, 32Dp210 and K562 cells treated with or without Gleevec as described in Figure 2 were lysed and subjected to immunoprecipitation for Vav1. Immunoprecipitates were separated by SDS–PAGE, immunoblotted with antibody against P-Tyr (PY), and then re-probed with Vav1 antibody. (b) 32D cells transfected with GFP or GFP–Vav1(D1–186) were maintained in culture medium for 8h. Transfection efficiency was higher than 60% in all experiments examined by Olympus AX-70 fluorescence microscopy by detecting GFP-positive population. Cells were then subjected to GST–PBD pull-down assays. Rho GTPases in GTP-bound form and in total lysates were analyzed by western blot with anti-Cdc42 antibody and re-probing with anti-Rac1 antibody. By densitometric scanning, the relative fold of active to total Rho GTPase in each lysates was determined from three independent experiments (mean±s.d.) shown in the left panel. Expression levels of GFP and GFP–Vav1(D1–186) were confirmed by western blot using anti-GFP and anti-Vav1 antibodies in the right panel. Actin is shown as a loading control. The molecular weight of GFP–Vav1(D1–186) is the same as endogenous Vav1, and is also detected by Vav1 antibody (c) 32Dp210 cells transfected with control LacZ or Vav1-specific shRNA plasmid were maintained in serum-containing culture medium for 48h. Cells were then subjected to GST–PBD pull-down assays for the detection of Cdc42 and Rac1 activities. The left panel shows the representative blot and quantification data of Cdc42 and Rac1 activities from six independent assays. The right panel shows representative Vav1 knockdown efficiency in total lysates using anti-Vav1 antibody. Actin is shown as a loading control. (d) Proposed model of p210BcrAbl in chemotaxis. p210BcrAbl deregulates Cdc42 GTPase through phosphorylation and activation of Vav1, which abrogates SDF-1a-induced Cdc42 activation, thus blocking chemotaxis in chronic myeloid leukemia (CML).

Article Snippet: Mouse 32D (32Dcl3) cells were purchased from American Type Culture Collection and maintained in the same medium used for transformed 32Dp210 cell line with 25% WEHI-cultured conditioned medium as a source of interleukin-3.

Techniques: Activation Assay, Immunoprecipitation, SDS Page, Transfection, Microscopy, Western Blot, Expressing, Control, Molecular Weight, shRNA, Plasmid Preparation, Knockdown, Chemotaxis Assay, Phospho-proteomics, Blocking Assay

Journal: Immunity

Article Title: Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation

doi: 10.1016/j.immuni.2017.08.005

Figure Lengend Snippet:

Article Snippet: 32D-cl3 (32D) myeloblast cells, RRID CVCL_0119, are derived from Mus musculus , were purchased from ATCC and cultured in RPMI 1640 with 10% FCS, 2 mM L-glutamine,100 U/ml Pen-Strep, and 10 ng/ml IL-3 at 37°C, 5% CO 2 .

Techniques: Virus, Recombinant, Giemsa Stain, Labeling, Gene Expression, ATP Bioluminescent Assay, Cell Based Assay, Software

Journal: PLoS ONE

Article Title: Aciculatin Inhibits Granulocyte Colony-Stimulating Factor Production by Human Interleukin 1β-Stimulated Fibroblast-Like Synoviocytes

doi: 10.1371/journal.pone.0042389

Figure Lengend Snippet: (A, C, and D) FLS were incubated with 0, 1, 3, or 10 µM aciculatin (A1, A3, and A10) for 30 min, and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. Whole cell extracts were then prepared for western blot analysis for the indicated proteins (A and C); equal amounts of cell culture media (“conditioned medium”) were collected and concentrated 10-fold (v/v) (lanes 1–4) or PBS only (lane 5), and then immunoprecipitated with 1 µg of anti-G-CSF antibody, followed by immunoblot analysis using anti-G-CSF antibody or anti-β-actin antibody (as an internal control) (D). (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a specific STAT3 DNA probe. (E) Ten-fold concentrated conditioned medium was prepared from FLS incubated with or without aciculatin, and then with IL-1β as in (D), or with IL-1β plus an anti-G-CSF antibody. 32Dcl3 cells were incubated for 10 days with a medium containing 50% of these different conditioned mediums. The cells were then were subjected to Wright-Giemsa staining to detect neutrophils (top row) or washed twice with PBS, incubated at 4°C for 45 min with anti-CD11b FITC-conjugated and anti-CD11a/CD18 PE-conjugated antibodies, and their fluorescence was analyzed by FACScan flow cytometry (bottom row). Magnification = ×100; scale bar = 20 µm. In (A) and (C), the extents of indicated proteins expression were quantitated using a densitometer with the Image-Pro plus software, and the relative levels were calculated as the ratios of proteins to GAPDH or β-actin protein levels. The results are expressed as the mean ± SEM, with n = 3. * p <0.05 and ** p <0.01 compared with the control group; # p <0.05 and ## p <0.01 for the comparisons of the groups indicated.

Article Snippet: Mouse myelomonocytic leukemia WEHI-3 cells and mouse bone marrow 32Dcl3 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Incubation, Western Blot, Cell Culture, Immunoprecipitation, Control, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Staining, Fluorescence, Flow Cytometry, Expressing, Software

Journal: Molecular Medicine Reports

Article Title: Protective effects of ethyl pyruvate on lipopolysaccharide-induced acute lung injury through inhibition of autophagy in neutrophils

doi: 10.3892/mmr.2017.6118

Figure Lengend Snippet: Ethyl pyruvate inhibits autophagy in neutrophils. (A) Murine myeloid cells, 32Dcl, 3 were transiently transfected with siScramble or siATG5 overnight, and the cells were lysed. Lysates were analyzed by immunoblotting with ATG5 antibody. (B) Murine myeloid cells, 32Dcl3, transiently transfected with siScramble or siATG5 overnight, were treated with fMLP (200 nM) or LPS (100 ng/ml) for 14 h, and supernatants from cultured neutrophils were collected and subjected to ELISA for detecting MPO. (C) Murine myeloid cells, 32Dcl3, were treated with fMLP (200 nM) or LPS (100 ng/ml) for 14 h in the absence or presence of EP. Supernatants were collected and the cells were lysed. Lysates were analyzed by immunoblotting with LC3 antibody. Images presented here were representatives of three experiments. Supernatants were subjected to ELISA for (D) TNF-α and (E) MPO. Images presented here were representatives of three experiments. The data are presented as the mean ± standard deviation of three independent experiments (*P<0.05). ATG5, autophagy related 5; fMLP, N-Formyl-Met-Leu-Phe; LPS, lipopolysaccharide; MPO, myeloperoxidase; EP, ethyl pyruvate, LC3, microtubule-associated protein 1A/1B-light chain 3; TNF-α, tumor necrosis factor-α.

Article Snippet: The murine myeloid cell line, 32Dcl3 (CRL-11346), was obtained from American Type Culture Collection (Manassas, VA, USA), and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum.

Techniques: Transfection, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Synergy between Proteasome Inhibitors and Imatinib Mesylate in Chronic Myeloid Leukemia

doi: 10.1371/journal.pone.0006257

Figure Lengend Snippet: The cells are purified using magnetic cell sorting method, and clonogenic assays are carried out using 1×10 3 cells and methylcellulose medium containing IL-3, GM-CSF and SCF. (A through C): Effects of IM/BOR and IM/PSI on colony forming activity of CD34+ cells from 6 CML patients at chronic phase (A, upper panel) and 4 cases at blastic crisis (A, lower panel), 4 from healthy donors (B), or 3 from UCB (C). (D): Effects of IM/BOR and IM/PSI on cell growth of CD34+ cells from CML patients at CP (n = 3), detected by trypan blue exclusion assay. (E): CD34+ cells from 4 CML patients at blastic crisis were cultured for 72 hours at presence or absence of agents indicated, and viable cells were counted by trypan blue exclusion. Treatment protocols: 1, control; 2, IM 0.1 µM; 3, BOR 10 nM; 4, IM in combination with BOR; 5, PSI 15 nM; 6, IM in combination with PSI.

Article Snippet: Clonogenic assays were carried out using methylcellulose medium with recombinant cytokines MethoCult H4434 (containing human IL-3, GM-CSF, SCF and Erythropoietin; for human origin CD34+ cells), M3434 (containing mouse IL-3, SCF, human IL-6 and human Erythropoietin; for 32Dcl3 cells), or H4230 [without cytokines; for BCR-ABL-expressing 32Dc13 (hereafter, 32D/BCR-ABL) cells] at present or absent of IM/BOR/PSI according to manufacturer's technical manual (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Purification, FACS, Activity Assay, Trypan Blue Exclusion Assay, Cell Culture, Control