Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: DCP-Bio1 labeling reveals sulfenic acid modification of FLT3ITD cysteine residues. (A, B) MV4-11 cells (A) or RS4-11 cells (B), expressing endogenously FLT3ITD or wild-type FLT3, respectively, were serum-starved and treated as indicated with diphenyleneiodonium (DPI, 10 μM, 4 h), H 2 O 2 (100 μM, 30 min), or FL (100 ng/ml, 10 min). Cells were lysed in presence (or for control absence) of 1 mM DCP-Bio1, and subjected to pulldown with streptavidine (SA) or corresponding control (ctrl) beads. Pulldowns and corresponding lysate aliquots were analyzed for FLT3 (pan-FLT3 S18) by immunoblotting. pFLT3 was also assessed (pY591 phosphorylation) to detect FL stimulation. (C) HEK293 cells were transiently transfected with the indicated plasmids (EV, empty vector). Two days after transfection cells were processed as in A, and B. Note that all parts of the blot in C are from the same membrane and exposure, but have been rearranged for better clarity. Shown results are representative of three experiments with consistent results. (D) DCP-Bio1 labeling of FLT3ITD in HEK293 cells. Cells were pre-treated with DPI at the indicated concentrations or 10 mM N -acetylcysteine (NAC) for 4 h and then processed for DCP-Bio1 labeling as in (A) and (B). Lower panel shows the quantification of 4 independent experiments. *p < 0.05 for comparing with control by t -test.

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Labeling, Modification, Expressing, Control, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Membrane

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Positions and indirect assessment of oxidation of cysteine residues in the FLT3 cytoplasmic domain. (A) Alignment of class III RTKs reveals conserved (highlighted yellow) and non-conserved unpaired cysteines in the FLT3 cytoplasmic domain. (B) Position of cysteine residues in the cytoplasmic domain of FLT3. The structure of FLT3 (PDB 1RJB ) is shown in ribbon representation. The main chain is colored in blue and the Cys residues are highlighted in yellow and their positions in the full-length protein are indicated by black numbers. Several functional motifs of the protein are also indicated: Juxtamembrane domain Tyr572-Trp603 in dark grey, catalytic loop His809-Asn816 in pink, activation loop Asp829-Ala856 in light green. (C) HEK293 cells were transiently transfected with the indicated plasmids expressing FLT3ITD or corresponding Cys-Ser mutants. Two days after transfection, cells were processed as described in and pulldowns with streptavidine (SA) beads were assessed for presence of FLT3 (S18 antibody). For FLT3ITD, two forms are detectable: The larger (ca. 170 kDa), mature, complex glycosylated (CG) form, and the smaller (ca. 150 kDa) major immature, high-mannose (HM) form. (D) Multiple experiments as in (C) were quantified and the ratio of FLT3 signals in pulldowns and whole cell lysate (WCL) aliquots was calculated. Data are depicted as medians within boxes from first quartile (25th percentile) to the third quartile (75th percentile) and whiskers ranging from the 10th to the 90th percentiles of independent experiments. (n = 6, *p < 0.05 by one-way ANOVA for comparison with control). Results for Cys828Ser, Cys925Ser, and Cys945Ser were very variable and not included in the statistical analysis.

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Functional Assay, Activation Assay, Transfection, Expressing, Comparison, Control

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Differential role of FLT3ITD cysteine residues for signal transduction. (A, B) HEK293 cells were transiently transfected with the FLT3ITD constructs as indicated or empty vector (EV). Whole cell lysates (WCL) were subjected to SDS-PAGE and immunoblotting analysis of the indicated signaling proteins using the same antibodies as in and . The complex glycosylated (CG) form and the high-mannose (HM) form are indicated. Blots were probed for phosphoproteins first, then stripped and reprobed for total proteins. (A) Representative experiment. (B) Quantification of multiple experiments (n = 4). Values were calculated as arbitrary units (a.u.) relative to not mutated FLT3ITD and are given as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001 by one-way ANOVA compared to control. (C,D) 32D cells stably transduced with the indicated FLT3ITD expression constructs, or parental 32D cells were washed and starved 4 h from serum and IL-3, then subjected to lysis and immunoblotting analysis as in A, B. (C) Representative experiments. Note that for cysteines 807, 828, and 925, also Cys-Ala mutants were generated and analyzed (left panel). (D) Quantification of multiple experiments (n = 4–7). Only Cys-Ser mutants are included. Values were calculated as arbitrary units (a.u.) relative to not mutated FLT3ITD and are given as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001 by one-way ANOVA compared to control.

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Transduction, Transfection, Construct, Plasmid Preparation, SDS Page, Western Blot, Control, Stable Transfection, Expressing, Lysis, Generated

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Signaling phenotypes of FLT3ITD cysteine mutants are not caused by altered dimerization. HEK293 cells were transiently transfected with the indicated FLT3 expression constructs. Dimers were stabilized by crosslinking with BS3, controls were performed in absence of crosslinker (−). Samples were analyzed by SDS-PAGE with 5.4% gels and immunoblotting. (A) Effect of FLT3 ligand (FL) on dimerization of wild-type (WT) FLT3. Cells were starved from serum 4 h before ligand stimulation. FLT3 was enriched by wheat-germ agglutinin (WGA) pulldown before analysis. The positions of FLT3 monomers (Mon), which exist as immature high-mannose form (HM), or complex glycosylated (CG) mature form, and of FLT3 dimers (Dim) are indicated (detection with pan-FLT3 S18). (B) Spontaneous dimer formation of FLT3ITD, and of the efficiently dimerizing FLT3ITD Cys694S mutant. Analysis of whole cell lysates (WCL) (C) Comparison of dimerization of FLT3ITD and different Cys-Ser mutants as indicated. The blot was also probed with anti-pFLT3 (pY591) to detect the autophosphorylation activity of dimers and monomers.

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Transfection, Expressing, Construct, SDS Page, Western Blot, Mutagenesis, Comparison, Activity Assay

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: FLT3ITD cysteine residues are essential for transforming activity. 32D cell pools stably transduced with the indicated FLT3ITD versions were subjected to viability/proliferation assays with 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT). Assays were performed in absence (A) or presence (B) of IL-3 A. Mean values ± SEM of 3–5 experiments performed with 8 technical replicas are shown. *p < 0.05, **p < 0.01, ***p < 0.001 for comparison with control (FLT3ITD) by one-way ANOVA. Colony assays in methylcellulose (in absence of IL-3) with 32D cell pools stably transduced with the indicated FLT3ITD versions. (C) Representative images. (D) Quantification of independent assays (means ± SEM, n = 8–13, ***p < 0.001 for comparison with control (FLT3ITD) by one-way ANOVA).

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Activity Assay, Stable Transfection, Transduction, Comparison, Control

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Summary of effects of individual Cys-Ser mutations in different assays. Significant up-or downregulations are indicated by arrows, two arrows symbolize effects of more than 50%. Arrows in parentheses indicate trends or qualitative observations. A lack of significant effects is symbolized with a dash. For DCP-Bio1 labeling, ‘uncertain’ indicates large variation of signals in independent assays, which precluded statistical analysis. For detailed description of the different assays and corresponding conclusions, see the text.

Article Snippet: A lentiviral expression construct for FLT3ITD containing an EGFP/blasticidine selection cassette (pLV EGFP/Bsd) and a corresponding control vector (pLV mCherry/Bsd) were obtained from VectorBuilder/Cyagen (Santa Clara, CA, USA).

Techniques: Labeling