Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: MCPyV truncated LT induces paranuclear retention and stabilization of KIT. (A) Immunofluorescence detection of KIT (green) and MCPyV LT (CM2B4, red) or sT (CM8E6, red) in KIT-HEK293 cells transfected with MCPyV T-antigens or vector control ( n = 6). (B) Top : illustration of the expression constructs of LT339 and the VPS39-interaction defective mutant LT339 W209A . Bottom : Representative images showing the effect of LT339 and LT339 W209A mutant on localization of KIT (green). LT was detected by CM2B4 (red). ( n = 3) (C) Immunoblots showing the effect of the LT339 and LT339 W209A on KIT expression. The quantification of KIT level is shown below the immunoblots ( n = 9). (D) Top : immunoblot analysis of the effect LT339 and LT339 W209A on KIT protein stability in the presence of cycloheximide (CHX) up to 4 h. Bottom : quantification of KIT protein stability after normalization to 0 h time point ( n = 4). (E) Immunoblots showing KIT protein stability in KIT-HEK293 cells transfected with LT339, LT339 W209A or plasmid control (CTR) treated with KIT ligand (KITLG, 100 ng/mL) or solvent control (PBS) in the presence of CHX (100 μg/mL). (F) Quantification of KIT protein stability after normalization to 0 h time point ( n = 5). Solid lines represent PBS control (KITLG − ) and dotted lines represent KITLG treatment (KITLG + ). (A and B) Nuclei were stained by DAPI (blue). Scale bar: 10 μm. Numbers below the images refer to the proportion of cells with KIT paranuclear dot-like staining to the total number of cells analyzed. (C, D and F) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant were calculated by one-way ANOVA with post-hoc Tukey’s test (C), two-way ANOVA (D) or two-way ANOVA with post-hoc Bonferroni’s test (F).

Article Snippet: For KITLG induced KIT degradation experiments, KIT-293 cells were transfected with LT339, LT339 W209A or CTR for 48 h, followed by addition of CHX (100 μg/mL) and KITLG (100 ng/mL; Proteintech Group, HZ-1024) or PBS.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Construct, Mutagenesis, Western Blot, Solvent, Staining

Journal: Frontiers in Immunology

Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

doi: 10.3389/fimmu.2017.00147

Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: BMdDC culture supernatants (100 μl/well) and BMdDC lysates (25 μg of cell lysate/well) were tested by mouse SCF ELISA kit (Boster Immunoleader, Pleasanton, CA, USA).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Bioprocessing, Flow Cytometry, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Blood

Article Title: A topological view of human CD34 + cell state trajectories from integrated single-cell output and proteomic data

doi: 10.1182/blood-2018-10-878025

Figure Lengend Snippet: Functionally defined progenitors with NM, L, and/or E potential display partially overlapping molecular profiles. (A-B) Distribution of each progenitor type in t-SNE space. Index sort information was used to map each progenitor to its nearest 10 neighbors in the mass cytometry data based on the scaled intensity of expression of CD45RA, CD71, CD45, CD123, CD34, CD33, CD49f, CD10, CD135, CD38, CD90, HLA-DR, and CD133. The nearest neighbors for all members of a given progenitor type were pooled and used to generate a probability density, indicated by the intensity of the color shown. The lowest level contains 95% of the total probability density, with each higher 10% density levels indicated thereafter. The black contour shows the 75th quantile of the overall density. (A) Mappings for all progenitor types assessed visually in methylcellulose assays as shown in Figure 1B. (B) Mappings for a representative selection of lineage competencies assessed in the STC assays as shown in Figure 1C. (C) A hierarchical clustering of the progenitor types analyzed based on a pairwise assessment of differences in the density distributions between all mappings of functionally and phenotypically defined cell types. Closely related groups are highlighted and given a descriptive name. (D) Multidimensional scaling indicating the relative distances (based on the distribution differences) between all phenotypically and functionally defined progenitor subsets.

Article Snippet: In vitro assays Colony formation in methylcellulose was assessed by single, randomly selected, index-sorted cells deposited directly and individually into the 60 inner wells of a flat bottom Nunc 96-well polystyrene plate (Thermo Fisher Scientific, Waltham, MA) preloaded with 50 µL of methylcellulose medium; the medium was supplemented with 50 ng/mL stem cell factor (SCF), 20 ng/mL granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), interleukin-6, granulocyte-colony stimulating factor (G-CSF), and 3 U/mL erythropoietin (EPO) (STEMCELL Technologies).

Techniques: Mass Cytometry, Expressing, Selection