Journal: Leukemia

Article Title: Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL.

doi: 10.1038/leu.2012.177

Figure Lengend Snippet: Figure 1. FLT3 transcript expression in normal and malignant immature T cells. (a) FLT3 transcripts are expressed during thymic T-cell maturation are the highest at the earliest stages and progressively decrease with maturation. T-ALL cases show a continuous spectrum of FLT3 transcript expression but a significant number of cases (22.2%) expressed FLT3 at a level significantly higher than whole thymus (fivefold) or even the most immature thymic subpopulations (P ¼ 8 10 4). ISP, immature single positive (CD4); DP TCR NEG, double positive (CD4/8) TCR negative; DP TCR LOW/HIGH, double positive (CD4/8) TCR positive low or high. (b, c) FLT3 expression levels according to the TCR and genotypic subsets, respectively. ***Po0.0001.

Article Snippet: Western blotting of FLT3 was performed using anti-FLT3 antibody (clone S18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphotyrosine (clone 4G10) as previously described.24 Phosphorylation of KIT was analyzed using anti-phospho-c-Kit (Tyr719) (Cell Signaling Technology, Berverly, MA, USA) as previously reported.25 Flow-cytometry monitoring of phosphoepitopes of FLT3 and downstream signaling molecules was performed using a polyclonal antiphospho-FLT3 (Y591) Alexa Fluor 488 (Cell Signaling Technology), monoclonal anti-phospho-STAT5 (Y694) Alexa Fluor 647 (Clone 47; BD Biosciences) and monoclonal anti-phospho-ERK1/2 (T202/Y204) Alexa Fluor 647 (Clone 20a, BD Biosciences).

Techniques: Expressing

Journal: Leukemia

Article Title: Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL.

doi: 10.1038/leu.2012.177

Figure Lengend Snippet: Figure 2. FLT3 expression and activation in T-ALL. (a) FLT3 expression was confirmed at the protein level by flow cytometry in FLT3High T-ALLs, whereas it was undetectable in FLT3Low T-ALLs. (b) Western blot analysis demonstrates that high transcriptional expression of FLT3 is associated with autophosphorylation of the receptor, both in FLT3-mutated and -unmutated cases. (c) Signaling properties of T-ALL depending on their FLT3 status evaluated by flow cytometry, in the presence or absence of FL stimulation (5 ng/ml). FLT3, STAT5 and ERK signaling molecules were monitored 30 min, 1 h and 1 h after FL exposure, respectively. Changes in phosphorylation of signaling proteins were calculated using the log2 ratio of the median fluorescence intensity of cells stained with anti-phosphoepitope antibody versus isotype control, and represented as a color scale. Results are plotted in heatmaps (left) and exemplified for representative cases in overlay histograms (right). (d) Quantitative reverse transcription PCR shows increased Pim-1 expression in FLT3High T-ALL as compared with FLT3Low cases, favoring oncogenic activation of the FLT3 pathway. *Refers to statistical significance Po0.05.

Article Snippet: Western blotting of FLT3 was performed using anti-FLT3 antibody (clone S18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphotyrosine (clone 4G10) as previously described.24 Phosphorylation of KIT was analyzed using anti-phospho-c-Kit (Tyr719) (Cell Signaling Technology, Berverly, MA, USA) as previously reported.25 Flow-cytometry monitoring of phosphoepitopes of FLT3 and downstream signaling molecules was performed using a polyclonal antiphospho-FLT3 (Y591) Alexa Fluor 488 (Cell Signaling Technology), monoclonal anti-phospho-STAT5 (Y694) Alexa Fluor 647 (Clone 47; BD Biosciences) and monoclonal anti-phospho-ERK1/2 (T202/Y204) Alexa Fluor 647 (Clone 20a, BD Biosciences).

Techniques: Expressing, Activation Assay, Cytometry, Western Blot, Phospho-proteomics, Staining, Control, Reverse Transcription

Journal: Leukemia

Article Title: Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL.

doi: 10.1038/leu.2012.177

Figure Lengend Snippet: Figure 3. (a) Sensitivity experiments of primary T-ALL cells upon RK inhibition. Results are expressed as the relative number of surviving cells as compared with an internal control (dimethyl sulfoxide). (b) Flow diagrams show the sensitivity to CEP701 of a representative case (UPN_221). (c) Sensitivity experiments of primary T-ALL cells to alternative FLT3 inhibitors harboring more potent and selective properties, with regard to FLT3. Cases sensitive to CEP701 remain sensitive to AC220. (d) Exposure of cells to RK inhibitors with or without FL does not result in any significant difference in sensitivity (ns, not significant). (e) Dephosphorylation of FLT3 evaluated by flow cytometry in response to FLT3- inhibitor exposure. (f) Dephosphorylation of FLT3 was not impacted by the presence of FL in FLT3-inhibitor-sensitive and -resistant T-ALLs.

Article Snippet: Western blotting of FLT3 was performed using anti-FLT3 antibody (clone S18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphotyrosine (clone 4G10) as previously described.24 Phosphorylation of KIT was analyzed using anti-phospho-c-Kit (Tyr719) (Cell Signaling Technology, Berverly, MA, USA) as previously reported.25 Flow-cytometry monitoring of phosphoepitopes of FLT3 and downstream signaling molecules was performed using a polyclonal antiphospho-FLT3 (Y591) Alexa Fluor 488 (Cell Signaling Technology), monoclonal anti-phospho-STAT5 (Y694) Alexa Fluor 647 (Clone 47; BD Biosciences) and monoclonal anti-phospho-ERK1/2 (T202/Y204) Alexa Fluor 647 (Clone 20a, BD Biosciences).

Techniques: Inhibition, Control, De-Phosphorylation Assay, Cytometry

Journal: Leukemia

Article Title: Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL.

doi: 10.1038/leu.2012.177

Figure Lengend Snippet: Figure 5. RK coinhibition in T-ALL: (a) KIT protein expression by flow cytometry. KIT was detectable neither in FLT3Low T-ALL cases (left) nor in CEP701-sensitive FLT3High T-ALLs (middle). In contrast, CEP701-resistant FLT3High cases, that also expressed high levels of KIT transcript, demonstrated KIT protein expression (right) (b) Differential transcriptional expression level between classes of T-ALL as compared with normal thymocyte development. (c) In vitro sensitivity experiments of FLT3, KIT and dual FLT3/KIT inhibition to reverse ITK resistance. (d) Median-effect plot (left) and isobologram curve (right) for the effect of AC220 and AB1010 on induction of apoptosis. The latter was measured by annexin V/ propidium iodide-based assay on primary blast cells from UPN_647, and results were calculated using CalcuSyn software (see Materials and Methods). The experimental points fall to the left of the predicted line of additive effect, indicating a synergistic interaction of AC220 and AB1010. This is reflected by combination index (CI) values reported in the table below the curves, reflecting an even more synergistic effect as the CI value falls o1. D, drug dose; Fa, fraction affected; fu, fraction unaffected. (e) Flow-cytometric analysis of FLT3 and KIT protein expression. Results indicate coexpression of both RK receptors by T-ALL blast cells. (f) Western blot analysis of KIT phosphorylation upon drug exposure (AB1010 10 mM and AC220 50 nM) showed significant C-Kit phosphorylation decrease after AB1010 but not AC220 exposure.

Article Snippet: Western blotting of FLT3 was performed using anti-FLT3 antibody (clone S18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphotyrosine (clone 4G10) as previously described.24 Phosphorylation of KIT was analyzed using anti-phospho-c-Kit (Tyr719) (Cell Signaling Technology, Berverly, MA, USA) as previously reported.25 Flow-cytometry monitoring of phosphoepitopes of FLT3 and downstream signaling molecules was performed using a polyclonal antiphospho-FLT3 (Y591) Alexa Fluor 488 (Cell Signaling Technology), monoclonal anti-phospho-STAT5 (Y694) Alexa Fluor 647 (Clone 47; BD Biosciences) and monoclonal anti-phospho-ERK1/2 (T202/Y204) Alexa Fluor 647 (Clone 20a, BD Biosciences).

Techniques: Expressing, Cytometry, In Vitro, Inhibition, Software, Western Blot, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conservation of CD1 intracellular trafficking patterns between mammalian species.

doi: 10.4049/jimmunol.169.12.6951

Figure Lengend Snippet: FIGURE 1. Expression of CD1 on cells in the guinea pig lymph node. A, CD1 staining (red) of sporadic cells with dendritic morphology (arrows) in the paracortical (PC) regions of the lymph node of guinea pigs. Note the large secondary lymphoid follicle with many CD1 B cells with a weakly staining germinal center (GC). Closeup of CD1 stained DC in the lymph node PC demonstrating interdigitating processes (arrows) emanating from the cell body (B and C). This region of the lymph node is dominated by T cells. Tissue was stained with BCD1b3.1 Ab, visualized with an anti-mouse avidin-biotin- peroxidase complex and counterstained with hematoxylin. Magnifications are 100 (A), 400 (B), and 600 (C).

Article Snippet: After incubating the supernatants in the wells for 1 h, the fusion protein was detected with a biotinylated anti-mouse Flt-3L Ab (R&D Systems, Minneapolis, MN), followed by incubations with alkaline phosphatase-conjugated streptavidin and paranitrophenyl phosphate.

Techniques: Expressing, Staining, Avidin-Biotin Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conservation of CD1 intracellular trafficking patterns between mammalian species.

doi: 10.4049/jimmunol.169.12.6951

Figure Lengend Snippet: FIGURE 4. Morphological and functional characterization of guinea pig DC. A and B, Scanning electron microscopy of purified DC from guinea pigs. Note the M. tuberculosis bacteria at the left in A for size comparison. Bars are 2 m (A) and 5 m (B). C, A cytospin preparation of purified DC stained with Diff-Quik demonstrating cell surface projections, 600. D, A cytospin of purified DC stained with the pan gpCD1 Ab 6B5 and visualized with an anti-mouse avidin-biotin-peroxidase complex. Positive staining is red with hematoxylin counterstain. E, Phagocytosis of live GFP-expressing M. tuber- culosis bacteria (green) by cultured guinea pig DC that were counterstained with Alexa-594-conjugated 6B5 Ab (red). Note the long projection (white arrows) on the left emanating from the cell body. F, The IgG1 isotype control Ab (P3) used in flow cytometry of purified guinea pig DC. G, High levels of MHC class II (IVA12) expression by DC. H, CD1 (6B5) expression on purified DC. Note the biphasic population of cells. I, Demonstration that DC from Flt-3LFc-treated strain 2 guinea pigs are able to support an MLR using allogeneic responder cells (2 105) from a Hartley strain guinea pig. The magnitude of the response is larger using DC from Flt-3LFc-treated animals compared with DC from a vehicle control-treated animal or whole splenocytes. f are DC from a Flt-3LFc-treated strain 2 guinea pig; Πare DC from PBS-treated strain 2 guinea pig; are strain 2 guinea pig whole splenocytes; are autologous splenocytes from the responder Hartley strain guinea pig. The x-axis of the graph reflects the total number of cells added to the well from each preparation. The actual DC in each well is dependent upon the purity of the preparation. The Flt-3L-treated guinea pig splenic preparation was 72% DC, while the PBS-treated animal was 31% DC.

Article Snippet: After incubating the supernatants in the wells for 1 h, the fusion protein was detected with a biotinylated anti-mouse Flt-3L Ab (R&D Systems, Minneapolis, MN), followed by incubations with alkaline phosphatase-conjugated streptavidin and paranitrophenyl phosphate.

Techniques: Functional Assay, Electron Microscopy, Bacteria, Comparison, Staining, Diff-Quik, Avidin-Biotin Assay, Expressing, Cell Culture, Control, Cytometry

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Induction of potent TRAIL-mediated tumoricidal activity by hFLEX/Furin/TRAIL recombinant DNA construct.

doi: 10.1016/j.ymthe.2004.02.021

Figure Lengend Snippet: FIG. 2. Western blot analysis. (A) Expression of pFETZ-, pFFT-, and pFFZT-encoded proteins in COS7 cells following transfection. The presence of fusion proteins and cleaved protein products was detected using anti-TRAIL Ab. (B) Expression of pFFT, pFFZT, and pFETZ in vivo. pFFT-, pFFZT-, and pFETZ-encoded proteins could be detected in sera collected from mice injected with pFFT, pFFZT, or pFETZ with (i) anti-TRAIL Ab and (ii) anti-Flt3L Ab. (C) pFFT-, pFFZT-, or pFETZ- encoded proteins were consistently detected in sera collected from groups of mice injected with pFFT, pFFZT, or pFETZ, respectively. (D) Analysis of FFT, FFZT, and FETZ protein by 6% native PAGE. Cleaved form of TRAIL was assembled in stable trimers upon addition of isoleucine zipper sequence (arrow) compared to the monomeric cleaved TRAIL (dotted-line arrow).

Article Snippet: The membrane was then incubated with 5 Ag/ml rabbit anti-TRAIL polyclonal antibody or goat anti-Flt3L polyclonal antibody (Santa Cruz Biotechnology) for 1.5 h at room temperature.

Techniques: Western Blot, Expressing, Transfection, In Vivo, Injection, Clear Native PAGE, Sequencing