Journal: Nature Communications

Article Title: Epigenetic silencing of DNA sensing pathway by FOXM1 blocks stress ligand-dependent antitumor immunity and immune memory

doi: 10.1038/s41467-025-59186-3

Figure Lengend Snippet: a (top) Immunofluorescent staining of FOXM1, CD3 + CD8 + T cells, and CD56 + NK cells in tumor tissues from breast cancer patients based on levels of high versus low FOXM1 expression. Top images: FOXM1 indicated by red; CD56 indicated by green. Bottom images: CD3 is indicated by red; CD8 is green (bottom image). DAPI is stained as blue. The circle indicates a cluster of NK cells in a FOXM1 low patient. (bottom) ULBP1 staining in tissues from breast cancer patients based on levels of high versus low FOXM1 expression. Scale bar indicates 50μm. The experiment was performed with n = 3 patients for each group (FOXM1 high and FOXM1 low). Representative pictures from two patients per group are shown. The number shown on the top of the figure indicates de-identified breast cancer patients. b – d Spearman correlations of FOXM1 RNA levels with tumor purity and immune cell infiltration in breast cancer patients from the TIMER2.0 database. The populations of cells examined include cytotoxic cells (NK cells, CD8 + T cells) and immunosuppressive cells (regulatory T cells [Tregs] and myeloid-derived suppressive cells [MDSC]. p -values for panels b-d were calculated by the TIMER2.0 database using the Spearman correlation coefficient.

Article Snippet: Slides were incubated with either FOXM1(1:150, Cell Signaling Technologies, Cat #5436) and CD56(1:100, Bio-Rad, Cat #MCA2693GA) or CD3(1:100, Bio-Rad), CD8 (1:150, Biolegend), and ULBP1 antibodies (1:100, Booster Biological Technologies, Cat # A04323-1).

Techniques: Staining, Expressing, Derivative Assay

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were stimulated with or without Ebola VLPs (final concentration: 10 μg/mL of EBOV GP) for 6, 24, 48, 72, and 96 hours. Surface markers and intracellular cytokine expression were assessed by flow cytometry. Strategy for gating single and live CD3 – CD56 + NK cells is shown in . ( A and C ) Representative dot blots showing CD3 – CD56 + NK cells plotted for IFN-γ and TNF-α, respectively. ( B and D ) Summary data from 11 independent experiments performed with PBMCs from 11 (24 and 48 hours) or 5 (6, 72, and 96 hours) independent donors are shown for IFN-γ and TNF-α, respectively. Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, as calculated by 2-tailed paired Student’s t test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Concentration Assay, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were stimulated with or without Ebola VLPs as in Figure 2. Cells were stained for the surface and cytotoxicity markers and data acquired and analyzed as described in Methods. Only single and live CD3 – CD56 + NK cells were included in the analysis. Representative dot blots ( A ) and the corresponding summary data from 11 independent experiments performed with PBMCs from 11 (24 and 48 hours) or 5 (6, 72, and 96 hours) independent donors ( B ) showing NK cells’ degranulation (CD107a surface expression) are shown. ( C – F ) Representative histograms for intracellular granzyme B ( C ) and perforin ( E ) expression in gated CD3 – CD56 + NK cells from PBMC cultures stimulated with Ebola VLPs (black) for 48 hours are shown. NK cells from PBMCs left unstimulated are shown in gray. Dotted histograms represent corresponding isotype controls. D (granzyme B) and F (perforin) show corresponding summary data from 8 independent experiments performed with PBMCs from 8 independent donors. ( G ) ADCC killing of target cells mediated by either unexposed (gray circles) or Ebola VLP exposed (black circles) PBMCs. PBMCs exposed to Ebola VLPs for 48 hours were analyzed for their cytotoxic potential by mixing them with the target cells expressing EBOV GP and in the presence of anti-EBOV GP plasma. Combined data from 5 independent experiments with PBMCs from 5 donors are shown. Results are shown as mean ± SEM. ** P < 0.01, *** P < 0.001, as calculated by 2-tailed paired Student’s t test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Staining, Expressing

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were stimulated with or without Ebola VLPs and stained as in and 3. Single and live CD3 – CD56 + NK cells were divided into CD56 bright and CD56 dim NK cells as shown in . Representative dot blots and the corresponding summary data for IFN-γ ( A – D ), TNF-α ( E – H ), and CD107a ( I – L ) from 8 independent experiments with PBMCs from 8 donors are shown. Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 as calculated by 2-tailed paired Student’s t test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Staining

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were left unstimulated or stimulated with Ebola VLPs (10 μg/mL), EBOV GP (10 μg/mL), or EBOV VP40 (5 μg/mL) for 24 hours (IFN-γ and TNF-α) or 48 hours (NK cell degranulation). Cells were stained for the surface and intracellular markers and data acquired as described earlier. Representative dot blots and the corresponding summary data showing gated CD3 – CD56 + NK cells plotted for IFN-γ ( A and B ), TNF-α ( C and D ), and NK cells’ degranulation (CD107a surface expression) ( E and F ) from 6 independent experiments performed with PBMCs from 6 donors are shown. All results are shown as mean ± SEM. ** P < 0.01, *** P < 0.001, as calculated by repeated measures (RM) 1-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Staining, Expressing

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: Purified CD56 + NK cells, cultured alone ( A and B ), with purified CD14 + cells ( C and D ), or with CD14 – CD56 – fraction ( E and F ) and the parent PBMCs ( G and H ) were stimulated with or without EBOV VP40 (5 μg/mL) for 24 hours (IFN-γ) or 48 hours (CD107a). Cells were analyzed flow cytometrically. Representative dot blots with CD3 – CD56 + NK cells plotted for IFN-γ ( A , C , E , and G ) and CD107a ( B , D , F , and H ) are shown. Corresponding summary data from 5 independent experiments performed with cells isolated from 5 donors are shown in I and J , respectively. Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, as calculated by 2-tailed paired Student’s t test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Purification, Cell Culture, Isolation

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were cultured in complete media unstimulated or stimulated with EBOV VP40 (5 μg/mL) for 24 hours. Where indicated blocking monoclonal antibodies for IL-1β, IL-10R, IL-12, IL-15, IL-18, IFN-αβR2 or the corresponding isotype controls were included in the cultures (final concentration of 3 μg/mL). Cells were analyzed flow cytometrically as described earlier. Representative dot blots for CD3 – CD56 + NK cells secreting IFN-γ in the presence of the blocking antibodies that showed statistically significant effect compared with the EBOV VP40 alone are shown ( A ). Corresponding summary data from 5 independent experiments performed with PBMCs from 5 donors are shown ( B ). Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, as calculated by RM 1-way ANOVA followed by Holm-Šidák multiple comparisons test. IL-10R, IL-10 receptor; IFN-αβR2, IFN-αβ receptor 2.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Cell Culture, Blocking Assay, Concentration Assay

Journal: JCI Insight

Article Title: Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

doi: 10.1172/jci.insight.158902

Figure Lengend Snippet: PBMCs were cultured, then stimulated with EBOV VP40 (5 μg/mL) for 48 hours in the presence of blocking monoclonal antibodies for IL-1β, IL-10R, IL-12, IL-15, IL-18, and IFN-αβR2 or the corresponding isotype controls (all at a final concentration of 3 μg/mL) and analyzed flow cytometrically as described earlier. Representative dot blots with CD3 – CD56 + NK cells plotted for surface CD107a are shown ( A ). Corresponding summary data from 5 independent experiments performed with PBMCs from 5 donors are shown ( B ). Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, as calculated by RM 1-way ANOVA followed by Holm-Šidák multiple comparisons test.

Article Snippet: Purity of the isolated cell populations was assessed by flow cytometry using APC-conjugated mouse anti-human CD56 (clone CMSSB, MBL International) and V500-conjugated mouse anti-human CD14 (clone M5E2, BD Biosciences) antibodies.

Techniques: Cell Culture, Blocking Assay, Concentration Assay

Journal: Cytotechnology

Article Title: Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

doi: 10.1007/s10616-009-9247-3

Figure Lengend Snippet: Assessment of myogenic purity of human primary skeletal muscle cell cultures using flow cytometry. a These representative graphs from myoblasts stained with anti-desmin or anti-NCAM antibodies, show that 89% of the cells were desmin-positive and 58% of the cells were NCAM positive. b To support the flow cytometry data are representative images of bona fide proliferating myoblasts and multinucleated myotubes where the desmin was stained green (FITC), nuclei were stained blue (DAPI) and the cytoskeleton was stained red (Alexa Fluoro 568 Phalloidin); white scale bar represents 75 μm. (please refer to online manuscript for colour images)

Article Snippet: Direct immunofluorescence was performed on fresh cells to detect NCAM (CD56) with a FITC-conjugated mouse anti-CD56 antibody (#CBL510F, Cymbus Biotechnology; diluted 1:10 in PBS plus 1% BSA).

Techniques: Flow Cytometry, Staining