Journal: Cell Death & Disease

Article Title: A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation

doi: 10.1038/cddis.2012.145

Figure Lengend Snippet: HMQ18–22 inhibited cell viability and decreased phosphorylation of VEGFR2, VEGFR1, Akt, PKC α and PLC γ -1 involved in angiogenesis. ( a ) HMQ18–22 decreased cell survival in lovo and HUVEC cells. ( b ) The AlphaScreen signal indicated HMQ18–22 decreased VEGFR phosphorylation. ( c ) Cell were treated with VEGF (50 ng/ml) for 30 min before extracting proteins with RIPA lysis buffer. HMQ18–22 decreased the phosphorylation of VEGFR2(Tyr 1214 ), VEGFR1(Tyr 1333 ), Akt(Tyr 326 ), PKC α (Tyr 657 ) and PLC γ -1(Tyr 771 ) by western blot analysis. On the contrary, the Raf1(Tyr 341 ) phosphorylation was not altered by HMQ18–22. Results were quantified by densitometry analysis of the bands form and then normalization to GAPDH protein. ( d ) Effect of HMQ18–22 on cells transfected with siRNAs targeting of VEGFR2, VEGFR1, Akt, PKC α or PLC γ -1. Quantification of RT-PCR data showed knockdown of VEGFR2, VEGFR1, Akt, PKC α and PLC γ -1; the bottom right panel showed the effect of HMQ18–22 on cells proliferation was attenuated in knockdown cells. Data were expressed as mean values±S.D. ( n =3). * P <0.05, ** P <0.01 versus the untreated control group.

Article Snippet: VEGFR2 kinase was from Carna Biosciences (Kobe, Japan).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Lysis, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation

doi: 10.1038/cddis.2012.145

Figure Lengend Snippet: HMQ18–22 inhibited tumor growth in nude mice bearing human colon cancer xenografts. ( a ) The representative xenografts of lovo human colon cancer in mice. ( b ) HMQ18–22 decreased the phosphorylation of VEGFR2(Tyr 1214 ), VEGFR1(Tyr 1333 ), Akt(Tyr 326 ), PKC α (Tyr 657 ) and PLC γ -1(Tyr 771 ) in the tumor tissues by western blot analysis. ( c ) Quantitation data of ( b ). Data were expressed as mean values±S.D. ( n =3). ** P <0.01 versus the untreated control

Article Snippet: VEGFR2 kinase was from Carna Biosciences (Kobe, Japan).

Techniques: Western Blot, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1550397

Figure Lengend Snippet: CD123-high HSPC-pDCs are the primary producers of IFNα in response to TLR9 stimulation. (A) Gating strategy used to identify HSPC-pDCs. The pDC-related markers CD303 and CD123 are analyzed within viable, lineage-negative, and CD11c-negative cells. (B) Representative flow cytometry plots showing IFNα secretion in HSPC-pDCs after TLR9 stimulation. (C) Bar graph showing the percentage of IFNα-positive cells within the hLin - ; CD11c - ; population at 5, 12, and 24 hours following TLR9 stimulation with CpG-A. (D) Bar graph depicting the proportion of IFNα-positive cells within each subset of hLin - ; CD11c - ; cells. The data shown represent the mean ± SEM of cells from four donors. Statistically significant differences between groups were determined using Two-Way ANOVA followed with Geisser-Greenhouse correction. *p < 0.05 (E) UMAP projections of pDC markers and IFNα gene expression in HSPC-pDCs, using an integrated dataset from HSPC-pDCs derived from multiple donors and stimulated with the TLR9 agonist CpG-A for 5, 12, and 24 hours. ns, non-significant.

Article Snippet: The following fluorochrome-conjugated antibodies were used for staining cell surface markers: anti-human Lineage Cocktail (APC, BioLegend, Cat. No:348803), anti-human CD11c (APC, BioLegend, Cat. No:301614), anti-human CD123 (PE, eBioscience, clone 6H6, Cat. No:12-1239-41), anti-human CD303 (PE-Cy7, eBioscience, clone 201A, Cat. No: 25-9818-41; BV421, BioLegend, Cat. No: 354212; PE-Vio770, Miltenyi Biotec, Cat. No: 130-113-655).

Techniques: Flow Cytometry, Gene Expression, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1550397

Figure Lengend Snippet: Characterization of cell subsets derived from in vitro pDC differentiation by RNA-seq (A) Schematic diagram illustrating the experimental design for this study. Following 16 days of HSPC-to-pDC differentiation, HSPC-pDCs obtained from three donors were primed with IFN-β and IFN-γ for 24 hours and then stimulated with the TLR9 agonist CpG-A for 12 hours. A primed but unstimulated condition was included for each donor. Based on the expression of CD303 and CD123, the three subsets (DNeg, DPos, and CD123H) were sorted from unstimulated and stimulated cells. RNA was extracted from the sorted samples for RNA-seq analysis (B) Heat map illustrating RNA-Seq expression data for Type I and Type III IFNs within the three sorted subsets. (C) Bar graph displaying Transcripts Per Million (TPM) counts for various genes involved in the TLR9-mediated immune signaling pathway. Upon ligand binding, TLR9 initiates downstream signaling via MyD88, leading to the activation of IRAK4. Subsequently, IRAK4 recruits IRAK1 and TRAF6, which further promotes the type I IFN, NF-κB, and MAPK signaling pathways.

Article Snippet: The following fluorochrome-conjugated antibodies were used for staining cell surface markers: anti-human Lineage Cocktail (APC, BioLegend, Cat. No:348803), anti-human CD11c (APC, BioLegend, Cat. No:301614), anti-human CD123 (PE, eBioscience, clone 6H6, Cat. No:12-1239-41), anti-human CD303 (PE-Cy7, eBioscience, clone 201A, Cat. No: 25-9818-41; BV421, BioLegend, Cat. No: 354212; PE-Vio770, Miltenyi Biotec, Cat. No: 130-113-655).

Techniques: Derivative Assay, In Vitro, RNA Sequencing, Expressing, Ligand Binding Assay, Activation Assay, Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1550397

Figure Lengend Snippet: Differentiation potential of subsets obtained during HSPC-to-pDC differentiation. (A) Schematic representation of the experimental design: After 16 days of differentiation, unstimulated cells were sorted into DNeg, DPos, and CD123H subsets based on CD303 and CD123 antibody staining. These subsets were then cultured in differentiation medium for up to eight additional days followed by priming and TLR9 stimulation. Their phenotype and TLR9 responsiveness were assessed through cell surface staining and intracellular IFN-α staining. (B) Flow cytometry plots showing CD123 and CD303 expression in bulk cells prior to sorting (left), and in sorted cells immediately post-sorting, after 8 additional days of differentiation, and following TLR9 stimulation (middle panels). The right panels show IFN-α expression in sorted cells after 8 additional days of culture and subsequent TLR9 stimulation. (C-E) Bar graphs showing the frequencies of DNeg, DPos, and CD123H subsets within sorted subsets after an additional four and eight days of culture in differentiation media and subsequent TLR9 stimulation. Each symbol corresponds to a different donor. Phenotypic analysis was performed at both time points after sorting for some donors, whereas for others, analysis was restricted to only one time point due to the limited number of available cells. Data are presented as mean ± SEM. (F) Bar graph showing the proportion of IFN-α-positive cells within each fraction of DNeg, DPos, and CD123H cells that emerged from the sorted DPos subset after 8 days of further culturing, priming, and CpG-A stimulation. For example, the bar with the darkest shade (IFNa + > CD123H) means that 81% of all IFN + cells were located in the CD123H subpopulation that had emerged from the sorted DPos population. IFNα intracellular staining was performed 5 hours after stimulation with CpG-A. Data are presented as mean ± SD.

Article Snippet: The following fluorochrome-conjugated antibodies were used for staining cell surface markers: anti-human Lineage Cocktail (APC, BioLegend, Cat. No:348803), anti-human CD11c (APC, BioLegend, Cat. No:301614), anti-human CD123 (PE, eBioscience, clone 6H6, Cat. No:12-1239-41), anti-human CD303 (PE-Cy7, eBioscience, clone 201A, Cat. No: 25-9818-41; BV421, BioLegend, Cat. No: 354212; PE-Vio770, Miltenyi Biotec, Cat. No: 130-113-655).

Techniques: Staining, Cell Culture, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1550397

Figure Lengend Snippet: Yield and phenotypic impact of knockout of TFs conventionally associated with pDCs and TFs with potential significance for pDCs during HSPC-to-pDC differentiation. (A) Knockout efficiencies in HSPC-pDCs for each TF associated with pDCs. Genomic DNA samples were obtained 3-5 days after nucleofection, and indel frequencies were determined by ICE analysis. (B) Graph illustrating the fold expansion of HSPC-pDCs cultured over a 16-day differentiation period. Data points represent mean values ± SEM of three donors. (C) Representative flow cytometry histograms illustrating the surface expression of CD303 and CD123 on knockout HSPC-pDCs. HSPC-pDCs were previously primed with IFN-β and IFN-γ for 24 hours. (D, E) Bar graphs illustrating the percentages of DPos and CD123H cells following priming with IFN-β and IFN-γ for 24 hours. Data shown represent mean ± SEM of three donors. One-way ANOVA was used to analyze differences between groups. (F, G) Levels of IFN-α2a and IFN-λ1/IL-29 in gene-edited HSPC-pDCs after stimulation with CpG-A. Data shown represent the mean ± SEM of a minimum of three donors. Differences between groups were analyzed using the Kruskal-Wallis test. * p < 0.05.

Article Snippet: The following fluorochrome-conjugated antibodies were used for staining cell surface markers: anti-human Lineage Cocktail (APC, BioLegend, Cat. No:348803), anti-human CD11c (APC, BioLegend, Cat. No:301614), anti-human CD123 (PE, eBioscience, clone 6H6, Cat. No:12-1239-41), anti-human CD303 (PE-Cy7, eBioscience, clone 201A, Cat. No: 25-9818-41; BV421, BioLegend, Cat. No: 354212; PE-Vio770, Miltenyi Biotec, Cat. No: 130-113-655).

Techniques: Knock-Out, Cell Culture, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells

doi: 10.3389/fimmu.2025.1550397

Figure Lengend Snippet: Comparison of Fc receptor blocking methods in the cell surface staining of subsets derived from HSPC-to-pDC differentiation. (A) Bar graph displaying Transcripts Per Million (TPM) counts of the human pDC markers CD123 (IL3RA) and CD303 (CLEC4C) in each subset. (B) Representative flow cytometry plots showing the cell surface expression of CD123 and CD303 in cells on day 16 of differentiation, employing human IgG alone, TruStain alone, or a combined blocking approach. (C) Flow cytometry histograms illustrating the CD303 expression profile obtained following the indicated blocking method. (D) Bar graphs showing the proportion of CD303 positive cells within hLin and CD11c negative cells (left) and the CD303 surface expression levels (MFI) (right) using various blocking methods during surface staining. Data are presented as mean ± SD from two donors. (E) Bar graph showing Transcripts Per Million (TPM) counts of the Fc gamma receptor I (FcγRI; FCGR1A).

Article Snippet: The following fluorochrome-conjugated antibodies were used for staining cell surface markers: anti-human Lineage Cocktail (APC, BioLegend, Cat. No:348803), anti-human CD11c (APC, BioLegend, Cat. No:301614), anti-human CD123 (PE, eBioscience, clone 6H6, Cat. No:12-1239-41), anti-human CD303 (PE-Cy7, eBioscience, clone 201A, Cat. No: 25-9818-41; BV421, BioLegend, Cat. No: 354212; PE-Vio770, Miltenyi Biotec, Cat. No: 130-113-655).

Techniques: Comparison, Blocking Assay, Staining, Derivative Assay, Flow Cytometry, Expressing

Journal: Cells

Article Title: Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction

doi: 10.3390/cells10081871

Figure Lengend Snippet: Mechanism of action of triazinetriones on TrkA. ( a ) Full-length TrkA with a HA-tag (TrkA-HA) fused to the C -terminus was purified through immunoprecipitation using anti-HA agarose beads. Purified TrkA-HA was incubated with DMSO (blue circles and hatched line), ACD855 (5 µM) (black triangles and solid line), or ACD856 (1 µM) (red squares and solid line) for approx. 5 min. Thereafter, ATP was added to yield the indicated concentration. Each data point is the mean ± SEM ( n = 3). The solid lines are the curve-fit using the Michaelis–Menten equation used to calculate the apparent Vmax(app) and km(app), and the dotted lines are the 95% confidence band for each curve fit. ( b – d ) Affinity labeling and streptavidin adsorption of Trka. ( b ) Western blot of streptavidin adsorbed TrkA-HA non-covalent labeled with NHS-biotinylated triazinetrione compound (lane 2) or covalent labeled by UV-crosslinking of 100 µM sulfo-SBED biotinylated compound (lane 3). Detection of immunoreactive band was performed with anti-TrkA antibody. Supernatant loaded to the left (lane 1) was used as positive control for the Western blot. Arrows on the left indicate the migration of molecular weight markers corresponding to 198, 98, and 62 kDa. ( c ) Anti-HA agarose immunoprecipitation of cross-linked or non-crosslinked sulfo-SBED compound (AC27019-SBED) from cell lysate incubated with 100 µM AC27019-SBED. Lane 1, non-UV-crosslinking, and lane 2, UV-crosslinking of sulfo-SBED labeled TrkA-HA, both detected with streptavidin-HRP. Lanes 3 and 4 are loading controls of lanes 1 and 2, respectively, were TrkA-HA was detected by immunoblotting using the anti-TrkA antibody. Both blots were part of the same gel, but the membrane was cut in two pieces, and the proteins were detected by streptavidin-HRP (left panel) or by an anti-TrkA antibody (right panel). ( d ) Streptavidin adsorption of biotinylated compound bound to TrkA-HA. Lane 1, cell lysate used for positive control of immunodetection; lane 3, cell lysate without biotinylated compound was adsorbed to streptavidin-agarose as negative control; lanes 5 and 7, cell lysates containing sulfo-SBED compound UV-crosslinked to TrkA (from two different experiments) were adsorbed to streptavidin-agarose and immunoblotted using anti-TrkA antibody. Lanes 2, 4, and 6 are empty lanes to avoid cross-contamination between lanes.

Article Snippet: Recombinant intracellular domain (ICD) of TrkA (08-186) and single site biotinylated activated (08-486-20N) or non-activated TrkA (08-486-23N) were obtained from Carna Biosciences, Odense, Denmark.

Techniques: Purification, Immunoprecipitation, Incubation, Concentration Assay, Labeling, Adsorption, Western Blot, Positive Control, Migration, Molecular Weight, Membrane, Immunodetection, Negative Control