Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Thoracic cavity of control mice and β-cat gain-of-function (GOF) mice at E15.5. Dotted lines show the outline of the thymic primordium. In many cases, blood clots were observed in the central core of the thymic primordium in β-cat GOF mice. Representative data from three independent experiments are shown. TH: thymus, H: heart. Bar: 1 mm. ( B ) Labeling of β5t-iCre-activated cell progenies with tdTomato fluorescence in the thymus of control mice and β-cat GOF mice at E15.5. Mouse sections were nuclear counterstained with TO-PRO3. Bottom panels are magnifications of white-boxed areas in the top panels. Representative data from three independent experiments are shown. Bars: indicated in figures. ( C ) Hematoxylin and eosin staining (top) and immunofluorescence staining for K5 and K8 (middle) and β-catenin and β5t (bottom) on sagittal sections of thymic primordium in control mice and β-cat GOF mice at E15.5. Representative results from three independent experiments are shown. Bar: 100 μm. ( D ) Intracellular staining of β-catenin in CD45 − EpCAM + TECs isolated from control mice and β-cat GOF mice at E15.5. Histograms show β-catenin expression in control TECs (blue line) and β-cat GOF TECs (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat GOF TECs, respectively. Plots on the right show net median fluorescence intensity (MFI) values (means and standard error of the means [SEMs], n = 4–5). The numbers in parentheses indicate percentage of control value. *p < 0.05.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Labeling, Fluorescence, Staining, Immunofluorescence, Isolation, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of thymic primordium in control mice and β-cat GOF mice at E11.5–E15.5. The sections were nuclear counterstained with TO-PRO3. Anterior–posterior (A–P) and dorsal–ventral (D–V) orientations of the images are indicated. Representative data from three independent experiments are shown. Bar: 100 μm. ( B ) Quantitative RT-PCR analysis of mRNA expression levels (means and standard error of the means [SEMs], n = 3–4) of indicated genes relative to Gapdh levels in CD45 − EpCAM + thymic epithelial cells (TECs) isolated from the thymus of control mice and β-cat GOF mice at E15.5. ( C ) The numbers of CD45 + thymocytes were analyzed by flow cytometry. Plots show the numbers (means and SEMs, n = 3–4) of CD45 + thymocytes in the thymus of control mice and β-cat GOF mice at E15.5. ( D ) Flow cytometric analysis of double negative (DN) thymocytes from control mice and β-cat GOF mice at E15.5. Shown are profiles of CD44 and CD25 expression. The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Cell numbers (means and SEMs, n = 3–4) of indicated DN thymocyte subpopulations from control mice and β-cat GOF mice at E15.5 are plotted. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Expressing, Isolation, Flow Cytometry

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of the thymic primordium in control mice and β-cat gain-of-function (GOF) mice at E11.5–E15.5. Shown are merged images with nuclear counterstaining (TO-PRO3) (left) and images obtained in each channel (middle, right) for the thymus from control mice and β-cat GOF mice at the indicated stages. Representative data from three independent experiments are shown. Bar: 100 μm. ( B ) Flow cytometric analysis of thymic epithelial cells (TECs) from indicated mice at E15.5, showing the purity of the isolated TECs for quantitative RT-PCR analysis. Shown are representative profiles of CD45, PI, and EpCAM expression in total cells (left) and in isolated CD45 − EpCAM + TECs (right) from control mice and β-cat loss-of-function (LOF) mice. The numbers indicate the frequency of cells within indicated areas.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Immunofluorescence, Staining, Control, Isolation, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Flow cytometric analysis of splenocytes from control mice and β-cat gain-of-function (GOF) mice at 11 wk. Shown are representative dot plot profiles of CD3 and TCRβ expression (left) and CD3 and TCRδ expression (right) in PI − viable cells. The numbers in dot plots indicate the frequency of cells within indicated area. ( B ) Cell numbers (means and standard error of the means [SEMs], n = 4–6) of indicated subpopulations in the spleen (left) and the iLN (right) from control and β-cat GOF mice at 8 wk are plotted. ( C ) Shown are representative dot plots of TCRδ and Vγ5 expression in CD45 + cells in the thymus from control mice and β-cat GOF mice at E15.5. The frequency of TCRδ + Vγ5 + cells is plotted (mean and SEMs; n = 4–6). ( D ) Flow cytometric analysis of dendritic epidermal T cells (DETCs) in the skin epidermis from control mice and β-cat GOF mice at 8 wk. Shown are representative dot plot profiles of CD3 and Vγ5 expression in epidermal cells. The numbers in dot plots indicate the frequency of cells within indicated area. The frequency of CD3 + Vγ5 + cells in epidermal cells is plotted (means and SEMs; n = 3). ( E ) Histograms for Vγ4 and Vγ1 expression in TCRβ - δ + cells in the iLN from control mice and β-cat GOF mice at 8 wk are shown. The frequency of Vγ4 + and Vγ1 + cells is plotted (means and SEMs; n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Thoracic cavity of control mice and β-cat LOF mice at E15.5. Dotted lines show the outline of the thymic primordium. Representative data from three independent experiments are shown. TH: thymus, H: heart. Bar: 1 mm. ( B ) Immunofluorescence staining for β-catenin and Foxn1 (left) or K5 and K8 (right) on sagittal sections of the thymus from control mice and β-cat LOF mice at E15.5. Shown are merged images with nuclear counterstaining (TO-PRO3) (top) and images obtained in each channel (middle, bottom). Representative data from three independent experiments are shown. Bar: 100 μm. ( C ) Intracellular staining of β-catenin in CD45 − EpCAM + thymic epithelial cells (TECs) from control mice and β-cat LOF mice at E15.5. Histograms show β-catenin expression in control TECs (blue line) and β-cat LOF TECs (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat LOF TECs, respectively. Plots show net median fluorescence intensity (MFI) values for β-catenin (means and standard error of the means [SEMs], n = 4). The numbers in parentheses indicate percentage of control value. ( D ) Flow cytometric analysis of enzyme-digested thymic cells from indicated mice at E15.5. Shown are profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Plots show the number of total thymic cells (left) and the frequency and the number of total TECs (middle) and cortical thymic epithelial cell (cTECs; right) from control mice and β-cat LOF mice at E15.5 (means and SEMs, n = 4). **p < 0.01; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Immunofluorescence, Staining, Expressing, Fluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Intracellular staining of β-catenin in UEA1 − Ly51 + cortical thymic epithelial cells (cTECs; left) and UEA1 + Ly51 − medullary thymic epithelial cells (mTECs; right) from control mice and β-cat LOF mice at 2 wk. Histograms show β-catenin expression in cTECs and mTECs from control mice (blue line) and β-cat LOF mice (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat LOF TECs, respectively. Plots show net MFI values for β-catenin in cTECs and mTECs (means and standard error of the means [SEMs], n = 3). The numbers in parentheses indicate percentage of control value. ( B ) Quantitative RT-PCR analysis of mRNA expression levels (means and SEMs, n = 5) of indicated genes relative to Gapdh levels in UEA1 − Ly51 + cTECs (top) and UEA1 + Ly51 − mTECs (bottom) in the thymus of control mice and β-cat LOF mice at 2 wk. ( C ) Flow cytometric analysis of enzyme-digested thymic cells from control mice and β-cat LOF mice at 2 wk. Shown are representative profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( D ) Plots show the number (means and SEMs, n = 6) of cTECs and mTECs in the thymus from control mice and β-cat LOF mice at 2 wk. ( E ) Immunofluorescence analysis of β5t (green), CCL21 (red), and Aire (cyan) on transverse sections of thymus from control mice and β-cat LOF mice at 2 wk. Representative data from three independent experiments are shown. Bar: 100 μm. Ctrl: Control, LOF: β-cat LOF. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Staining, Control, Expressing, Fluorescence, Quantitative RT-PCR, Immunofluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: Flow cytometric analysis of cTECs and mTECs from indicated mice at 2 wk. Shown are representative profiles of CD45, PI, and EpCAM expression of total cells after magnetic depletion of CD45 + cells, UEA1 reactivity and Ly51 expression in CD45 − EpCAM + cells, and isolated cTECs and isolated mTECs from control mice and β-cat loss-of-function (LOF) mice. The numbers indicate the frequency of cells within indicated areas.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Expressing, Isolation, Control

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Bars show body weight (left) and thymus weight (right) at 6 mo in control female mice and β-cat LOF female mice (means and standard error of the means [SEMs], n = 4). ( B ) Appearance of thymus from control mice and β-cat LOF mice at 6 mo. Representative data from four independent experiments are shown. Bar: 1 mm. ( C ) Plots show the number (means and SEMs, n = 4) of total thymocytes in the thymus from control mice and β-cat LOF mice at 6 mo. ( D ) Flow cytometric analysis of enzyme-digested thymic cells from control mice and β-cat LOF mice at 6 mo. Shown are representative profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Plots show the number (means and SEMs, n = 4) of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) in the thymus from control mice and β-cat LOF mice at 6 mo. ( F ) Immunofluorescence analysis of K5 (green) and K8 (magenta) on transverse sections of thymus from control mice and β-cat LOF mice at 6 mo. Representative data from three independent experiments are shown. Bar: 100 μm. Ctrl: Control, LOF: β-cat LOF. *p < 0.05; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Expressing, Immunofluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet:

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Transduction, Plasmid Preparation, Recombinant, Sequencing, DNA Library Preparation, Software

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: dG Treatment Kills Samhd1 −/− Cells by Apoptosis (A) BMDMs were treated with 0.5 mM of each dN for 24 h and stained with Annexin V and 7AAD. AnnexinV + 7AAD − and AnnexinV + 7AAD + cells were quantified by flow cytometry. Data from triplicate measurements are shown with mean ± SD. p values determined with two-way ANOVA are indicated. (B) Caspase activity was assessed in BMDMs 6 h after treatment with the indicated doses of dG, or Staurosporine as control, using the Caspase 3/7 Glo assay. For each genotype, values from untreated control cells were set to 100. Data from triplicate measurements are shown with mean ± SD. The p value determined with an unpaired t test is indicated. (C and D) Live-cell imaging of Samhd1 −/− BMDMs treated with 0.5 mM dG. Alexa 488-labeled Annexin V and propidium iodide (PI) were added to the culture medium to visualize early apoptotic cells and cells that lost membrane integrity, respectively. (C) Representative images of a Samhd1 −/− cell treated with dG. Numbers show the time after dG exposure (h:min). (D) Enumeration of AnnexinV + PI + cells after 24 h of treatment with or without 0.5 mM dG. Six images per condition were analyzed, and means ± SEM are shown. The p value determined with an unpaired t test is indicated. (E and F) BMDMs were treated with 0.5 mM dG or 1 μg/mL cycloheximide (CHX, added to WT cells in F) for 8 hours. (E) Levels of the indicated proteins in total cell extracts were determined by western blot. (F) Cells were fractionated into cytosol and a pellet containing organelles. Levels of the indicated proteins were determined by western blot. β-Actin served as a loading control. (G) WT and Samhd1 −/− BMDMs were co-cultured at the indicated ratios. Cell viability was determined as in Figure 1 A 24 h after treatment with 0.5 mM dG. Data from triplicate measurements are shown with mean ± SD. (A)–(G) are representative of at least three independent experiments. ns, p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S1 .

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Staining, Flow Cytometry, Activity Assay, Control, Glo Assay, Live Cell Imaging, Labeling, Membrane, Western Blot, Cell Culture

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: dG Induces the Death of Cancer Cell Lines (A) HeLa cells were infected with VLPs containing Vpx (VLP vpx ) or not (VLP ctrl ). After 24 h, cells were treated with 0.5 mM of each dN for an additional 24 h. Cell viability was assessed as in Figure 1 A. (B and C) HeLa (B) and MDA-MB231 (C) cells were left uninfected (NI) or were infected with VLPs containing Vpx (VLP vpx ) or not (VLP ctrl ). After 24 h, cells were treated with 0.5 mM dG, and brightfield images were acquired after an additional 10–12 h. Scale bars represent 300 μm. (D) MDA-MB231 cells were treated as in (C), and confluency was monitored after dG addition using a live-cell imaging system in the incubator (Incucyte). The mean of 9 measurements ± SD is shown. (E–G) Wild-type and Samhd1 −/− B16F10 cells were treated with dG as indicated for 20 h. (E and F) Cells were then stained with Annexin V and 7AAD and analyzed by flow cytometry. Representative fluorescence-activated cell sorting (FACS) plots are shown in (E), and Annexin V + 7AAD − and Annexin V + 7AAD + cells were quantified in (F). (G) Confluency was determined as in (D). (H) Jurkat cells were treated for 20 h with dG as indicated or with 25 μM etoposide. Cell viability was determined as in Figure 1 A. (I) Jurkat cells were treated with dG as indicated for 20 h and then seeded in semi-solid medium containing dG. After 13 days, cell colonies were counted, and the number colonies per field of view are shown. (J and K) Jurkat cells were reconstituted with hemagglutinin (HA)-tagged wild-type or K11A mutant SAMHD1 using a lentivector. Uninfected cells (NI) served as control. (J) Cells were then treated with dG for 48 h. Cell viability was determined as in Figure 1 A. (K) SAMHD1 levels in total cell extracts were determined by western blot. β-Actin served as a loading control. (A), (D)–(H), and (J)–(K) are representative of three independent experiments and (B) and (C) of two experiments. In (A), (F)–(H), and (J), dots represent technical triplicates and means ± SD are shown. In (I), data from two independent experiments were pooled, and dots represent the mean of technical duplicates per experiment. The p values determined with two-way ANOVA are indicated. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Infection, Live Cell Imaging, Staining, Flow Cytometry, Fluorescence, FACS, Mutagenesis, Control, Western Blot

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: PNP Inhibitors and dG Synergistically Induce Cell Death in Cells Lacking SAMHD1 (A–C) BMDMs were treated with the indicated doses of dG and forodesine. Viability was tested as in Figure 1 A after 24 or 48 h. (D) BMDMs treated for 24 h with dG and forodesine were fixed and stained with crystal violet. After washing, cell-associated dye was solubilized and quantified by absorbance at 570 nm. For each genotype, values from untreated control cells were set to 100%. (E and F) BMDMs were treated for 8 h with dG and forodesine. Levels of PARP and cleaved PARP (E) or cleaved CASPASE 3 and SAMHD1 (F) in total cell extracts were determined by western blot. β-Actin served as a loading control. cld, cleaved. (G–I) Jurkat cells were reconstituted with SAMHD1 as described in J and 3K. Uninfected cells (NI) served as control. (G and H) Cells were treated for 18 h with 10 μM dG and 1 μM forodesine. Cells were then stained with Annexin V and 7AAD and analyzed by flow cytometry. Representative FACS plots are shown in (G) and Annexin V + 7AAD − cells are quantified in (H). (I) SAMDH1 levels in total cell extracts were determined by western blot. β-Actin served as a loading control. (J) HeLa cells were infected with VLPs containing Vpx (VLP vpx ) or not (VLP ctrl ). After 6 h, cells were treated with 20 μM dG and 2 μM forodesine, and brightfield images were acquired after an additional 48 h. Scale bar represents 300 μm. (K) BMDMs were treated with the indicated doses of dG and forodesine. Viability was tested as in Figure 1 A after 24 h. Means from three biological replicates are shown ± SEM. (L) Samhd1 −/− BMDMs were treated with the indicated doses of dG in the presence or absence of 1 μM forodesine. Cell viability was determined by CellTiter-Glo assay after 24 h. Data were normalized by setting the values for the lowest and highest dG concentrations to 100 and 0, respectively. Means from three biological replicates are shown ± SEM. Half maximal inhibitory concentration (IC 50 ) values were calculated from the non-linear regression curves shown on the graph. (M and N) BMDMs were treated with the indicated doses of dG and homo-DFPP-DG (M) or 6C-DFPP-DG (N). Viability was tested as in Figure 1 A after 24 h. Data are representative of three independent experiments. In (A)–(D), (M), (N), and (H), dots represent BMDMs from individual mice and technical replicates, respectively. Mean ± SD is shown. The p values determined with two-way ANOVA are indicated. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Staining, Control, Western Blot, Flow Cytometry, Infection, Glo Assay, Concentration Assay

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet:

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Virus, Recombinant, Staining, Blocking Assay, Cell Viability Assay, Plasmid Preparation, Software

Journal: Biomacromolecules

Article Title: Combining Biologically Active β-Lactams Integrin Agonists with Poly( l -lactic acid) Nanofibers: Enhancement of Human Mesenchymal Stem Cell Adhesion

doi: 10.1021/acs.biomac.9b01550

Figure Lengend Snippet: Qualitative and quantitative analysis of specific proteins involved in the cell adhesive process to plain PLLA and SR610-PLLA (6.18 wt %), GM18-PLLA (7.48 wt %), and LT25-PLLA (6.31 wt %) after 2 h from seeding. (A) Western blotting analysis. Bar graphs show β 1 integrin and vinculin expression level obtained normalizing to the β-actin housekeeping protein signal. The activation level of FAK was presented as a ratio between the phosphorylated and total FAK protein after normalization to β-actin. Bars represent the mean values ± SD of results from three experiments ( n = 3). Statistical significance values are indicated as *** p < 0.001 and * p < 0.05. (B) CLSM images, showing the expression of focal adhesion β 1 integrin (green, 488 Alexa Fluor), vinculin (red, 633 Alexa Fluor), and p-FAK (green, 488 Alexa Fluor) on different PLLA scaffolds, were acquired at 40× magnification. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 50 μm. Yellow arrows indicated protein distribution at cellular level. The insets display a protein staining with false coloring from dark purple to bright yellow by use of the fire lookup table (LUT) scheme to highlight differences in the intensities of the signals obtained with ImageJ software. Graphs show the correct total cell fluorescence intensity (CTCF) measured in each sample ( n = 3, *** p < 0.001 and * p < 0.05).

Article Snippet: For focal adhesion detection, cells were incubated with primary mouse anti-α-vinculin antibody (1:500 in 1% bovine serum albumin, BSA, BosterBio, Pleasanton, CA, USA), anti-β 1 -integrin (1:100 in 1% BSA, NSJ Bioreagents, San Diego, CA, USA), or anti-p-FAK (pY397, 1:250, Santa Cruz, USA).

Techniques: Adhesive, Western Blot, Expressing, Activation Assay, Staining, Software, Fluorescence

Journal: Journal of Medicinal Chemistry

Article Title: New Water-Soluble Copper(II) Complexes with Morpholine–Thiosemicarbazone Hybrids: Insights into the Anticancer and Antibacterial Mode of Action

doi: 10.1021/acs.jmedchem.8b01031

Figure Lengend Snippet: Bar graphs showing the percentage of cell death due to apoptosis (left) and necrosis (right) in A2780 cells treated with increasing concentrations of HL 2 , 2 (2IC 50 and 6IC 50 ) and CuCl 2 and detected by Annexin V/PI apoptosis assay. Statistical analysis was performed by two-tailed T -test using GraphPad Prism software (GraphPad Software Inc., CA) with p < 0.05 considered as significant (* p < 0.05, ** p < 0.01).

Article Snippet: Cycloheximide, oligomycin, and annexin V-FITC apoptosis detection reagent (500×) were purchased from Abcam (Cambridge, UK).

Techniques: Apoptosis Assay, Two Tailed Test, Software

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment

doi: 10.1002/ijc.28432

Figure Lengend Snippet: PPARγ protein expression in untreated mammary glands from PPARγ-WT and PPARγ-MSE KO strains. Representative immunofluorescence images illustrates ( a and b ) PPARγ (FITC; green) and ( c and d ) β-casein (Alexa Fluor 594; red) expression in lactating glands from both PPARγ-WT and PPARγ-MSE KO mice. An accompanying composite image ( e and f ) shows PPARγ and β-casein expression together with DAPI-stained nuclei. All photos were taken at ×600. ( g ) PPARγ expression was analyzed by Western blot in untreated mammary glands (MG) from both strains of virgin mice and mice three days after initiation of involution (Invol). White adipose tissue (WAT) from untreated PPARγ-WT mice was included as a positive control for PPARγ. α-actinin served as a loading control. ( h ) Densitometry was performed using ImageJ software.

Article Snippet: Slides were rinsed with TBS and then incubated in secondary antibodies for FITC (Santa Cruz, 1:500) and Alexa Fluor 594 (Invitrogen, 1:500) in 5% BSA/TBS for 15 min at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Positive Control, Control, Software

Journal: Cell stem cell

Article Title: Chronic inflammation directs an olfactory stem cell functional switch from neuroregeneration to immune defense

doi: 10.1016/j.stem.2019.08.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: Rabbit anti-Krt5 (1:800, PRB-160P; Covance), Rabbit anti-Ki67 (1:500, Ab16667; Abcam), Rat anti-BrdU (1:400, Ab6326; Abcam), Mouse anti-Krt14 (1:800, MA5–11599; Thermo Fisher), Mouse anti-β-Tubulin III (1:200, MAB1637; Millipore), Rabbit anti-∆NP63 (1:1,000, 619001; BioLegend), Mouse anti-P63 (1:200, sc-8431; Santa Cruz), Rabbit anti-RelA (1:200, Sc-372; Santa Cruz), Rat anti-CD45 (1:200, 14–0451-81; Ebioscience), Rat anti-F4/80(1:500, MCA497GA; Bio-Rad), Rat anti-Ly6G (1:500, 127601; Biolegend), Rat anti-CD3 (1:200, 14–0032-81; Ebioscience), Goat anti-OMP (1:1000, 544–10001; Wako), Goat anti Sox2 (1:200, sc-17320; Santa Cruz), Goat anti-CCL19 (1:50, AF880; R&D), Goat anti-CXCL10 (1:100, AF-466-NA; R&D), Mouse anti Human CCL2 (1:100, MAB2791; R&D), Rabbit anti Human-CD45 (1:400, Ab40763; Abcam), Mouse anti-CD45 (1:500, 304002; Biolegend), Mouse anti-human CD3 (1:300, 300413; Biolegend), Alexa Fluor® 488 anti-human CD3 (1:200, 300454; Biolegend), Mouse anti-Human CD4 (1: 200, 555344, BD Pharmingen), and Alexa Fluor® 488 anti-β-Tubulin III (1:1000, 801203; Biolegend).

Techniques: Immunohistochemistry, Flow Cytometry, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Software

Journal: Cell reports

Article Title: Mutations in an Innate Immunity Pathway Are Associated with Poor Overall Survival Outcomes and Hypoxic Signaling in Cancer

doi: 10.1016/j.celrep.2018.11.093

Figure Lengend Snippet: See also . (A) mRNA expression fold changes (FC) and p values for complement regulators CD35, CD46, CD55, and CD59 in different colorectal cancer/adenocarcinoma versus normal tissue pairs. Data were acquired from https://www.oncomine.org/resource/login.html (GSE20916) . (B) Relative expression of CD55 (log 10 conversion) in COADREAD patients is shown against hypoxia signature expression (log 10 conversion) . Two-tailed p value is shown for the Pearson r (correlation coefficient). (C) KM curve for colorectal cancer patients with high (red) or low (blue) CD55 mRNA expression levels is shown. This analysis was based on the PrognoScan database ( http://dna00.bio.kyutech.ac.jp/PrognoScan/ ) using the publicly available Gene Expression Omnibus data ( https://www.ncbi.nlm.nih.gov/geo ) with the accession number GSE14333 ( ; ). p = 0.011327. (D) mRNA expression of CD55/18S is shown. qPCR was carried out following exposure of HCT116 cells to 0, 6, or 24 hr of hypoxia (1% O 2 ) or 24 hr of hypoxia followed by 1 hr reoxygenation (21% O 2 ). ** = p value < 0.01, 1-way ANOVA with Tukey’s multiple comparison test. Error bars represent the SEM for a representative experiment. n = 3. (E) HCT116 cells were treated with 0, 6, or 24 hr of hypoxia (1% O 2 ) or 24 hr of hypoxia followed by 1 hr reoxygenation (21% O 2 ). Western blotting (WB) was carried with the antibodies indicated. β-actin, loading control. n = 3. (F) CT26 cells were treated with 0 or 24 hr of hypoxia (1% O 2 ). WB was carried with the antibodies indicated. β-actin, loading control. n = 3. (G) Graph shows the % CMC/total lysis in HCT116 cells treated in 21% O 2 (normoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis (and total cell number) following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3. (H) Graph shows the % CMC/total lysis in HCT116 cells treated with 24 hr of 1% O 2 (hypoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis (and total cell number) following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3 (I) Graph shows the % CMC/total lysis in CT26 cells treated in 21% O 2 (normoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis following treatment with either normal human serum or heat-inactivated normal human serum. * = p value < 0.05, unpaired t test. Error bars represent the SEM for a representative experiment. n = 3. (J) Graph shows the % CMC/total lysis in CT26 cells treated with 24 hr of 1% O 2 (hypoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3.

Article Snippet: Antibodies used were anti-human mouse CD55 (BioRad, concentration # MCA914: 1:500), anti-mouse rat CD55 (R&D # MAB5376, concentration: 1:500) β-actin (Sigma # A5441, concentration: 1:5000), H3 (Abcam, #ab1791, concentration: 1:1000), HIF1α (BD-Biosciences, #610959, concentration: 1:500), HIF2α (Novus, # NB100–122, concentration: 1:500), ARNT (Novus, # NB100–124, concentration: 1:500), CD59 (Santa Cruz, concentration # sc-133171, 1:500).

Techniques: Expressing, Two Tailed Test, Western Blot, Lysis, Blocking Assay

Journal: Cell reports

Article Title: Mutations in an Innate Immunity Pathway Are Associated with Poor Overall Survival Outcomes and Hypoxic Signaling in Cancer

doi: 10.1016/j.celrep.2018.11.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used were anti-human mouse CD55 (BioRad, concentration # MCA914: 1:500), anti-mouse rat CD55 (R&D # MAB5376, concentration: 1:500) β-actin (Sigma # A5441, concentration: 1:5000), H3 (Abcam, #ab1791, concentration: 1:1000), HIF1α (BD-Biosciences, #610959, concentration: 1:500), HIF2α (Novus, # NB100–122, concentration: 1:500), ARNT (Novus, # NB100–124, concentration: 1:500), CD59 (Santa Cruz, concentration # sc-133171, 1:500).

Techniques: Blocking Assay, Viability Assay, Recombinant, shRNA, Software

Journal: International Journal of Biological Sciences

Article Title: YTHDF2 Suppresses Notch Signaling through Post-transcriptional Regulation on Notch1

doi: 10.7150/ijbs.61573

Figure Lengend Snippet: Effect of YTHDF2 Nuclear Translocation by Heat Shock Stress on Notch Signal and Cell Survival. A & B. Subcellular localization of YTHDF2 in HeLa cells before and after heat shock stress. HeLa cells stood 2h After heat shock (42°C, 1h) and were fixed and incubated with NICD antibody and YTHDF2 antibody. Cells were washed and incubated with FITC (for YTHDF2 Ab) or TRITC-conjugated secondary antibodies (for NICD Ab). After staining with DAPI, each fluorescence image merged to show the location of proteins in the cytoplasm and the nucleus. C. Immunoblotting of HeLa cells at the indicated time after heat shock stress (42°C, 1h). N, no heat shock stress. The intensity of the protein bands was analyzed using ImageJ software (NIH, Bethesda, NY, USA). D. HeLa cells were treated after heat shock stress (42°C, 1h). Cells were fractionated and subjected to western blot analyses. Fractionation was verified by using nuclear lamin A/C antibody (nuclear fraction) or GAPDH antibody (cytosolic fraction). The intensity of each fractional protein band was analyzed using ImageJ software (NIH, Bethesda, NY, USA). Relative amounts of nuclear and cytoplasmic NICD and YTHDF2 proteins in four replicates of fractionation experiments were graphed and normalized to nuclear lamin A/C and GAPDH, respectively. E. HeLa cells in Fig. C (42°C, 1h) were used for RNA extraction and qRT-PCR. Relative levels of indicated transcripts are normalized to GAPDH . The results represent the means ± S.D. of three independent experiments performed in triplicate. *, P<0.05. F. HeLa cells were transfected with NICD for 48 h and then treated with heat shock stress (42°C, 1h). Cells were assayed with MTT at the indicated time. The results represent the means ± S.D. of three independent experiments performed in triplicate. *, P<0.05. G. Relative values of Subcellular localization of YTHDF2 or YTHDF2 mutants in HeLa cells after heat shock stress (42 °C, 1 h). HeLa cells were transfected with 0.5 μg of GFP fused YTHDF2 WT or Mutants expression vectors. Standing for 2 h after heat shock (42 °C, 1 h), cells were fixed and stained with DAPI. Measurements were expressed relative to the number of fluorescent cells. Among cells with fluorescence in a unit area, the value when fluorescence exists only in the cytoplasm was set to the basic value, and the value when it exists in the nucleus was calculated. The results represent the means ± S.D. of three independent experiments performed in triplicate. *, P<0.05. H. A proposed model for YTHDF2 regulating Notch signal in response to heat shock stress. The illustration shows that YTHDF2-mediated Notch1 mRNA decay suppresses the expression of Notch target genes through YTH domain binding of m 6 A RNA. Under heat stress, YTHDF2 migrates to the nucleus and can restore the expression of Notch target genes required for cell survival and proliferation. CSL, CBF1/suppressor hairless/Lag-1; NEXT, Notch1 extracellular truncation; ADAM, a disintegrin and metalloproteinase; CoA, coactivators; CoR, corepressors; PM, plasma membrane; NM, nuclear membrane

Article Snippet: FITC (1:500, # 209-095-082) and TRITC (1:500, # 209-025-082) antibodies were purchased from Jackson Immuno Research Laboratories (Jackson Immuno Research Laboratories, West Grove, PA, USA). m 6 A antibody (#A-1801) was purchased from EpiGentek (EpiGentek, Farmingdale, NY, USA).

Techniques: Translocation Assay, Incubation, Staining, Fluorescence, Western Blot, Software, Fractionation, RNA Extraction, Quantitative RT-PCR, Transfection, Expressing, Binding Assay, Clinical Proteomics, Membrane