Journal: Molecular Medicine

Article Title: Downregulation of HMGB1 carried by macrophage-derived extracellular vesicles delays atherosclerotic plaque formation through Caspase-11-dependent macrophage pyroptosis

doi: 10.1186/s10020-023-00753-z

Figure Lengend Snippet: Macrophage-EV/siHMGB1 suppresses the release of HMGB1 expression to inhibit foam cell formation and inflammatory responses in macrophages. A Expression of HMGB1 in macrophages treated with ox-LDL and intervened with MEV/siHMGB1 was detected by Western blotting. B HMGB1 content in the macrophage culture supernatants after macrophage-EV/siHMGB1 or ox-LDL treatment measured by ELISA. C Lipid droplet in macrophages to reflect foam cell formation analyzed by Oil red O staining. D Uptake of Dli-labeled ox-LDL by macrophages determined by IF (400 × , 25 μm). E Foam cell formation in plaque tissues of AS mice after macrophage-EV/siHMGB1 treatment assessed by Oil red O and F4/80 staining (400 × , 25 μm). F Levels of TNF-α and IL-6 in the macrophage culture supernatants studied by ELISA. G Macrophage polarization measured by flow cytometry. H Macrophage polarization in the plaque tissues of AS mice tracked by IF. I Expression of LOX-1, SR-A, and CD36 in macrophages treated with ox-LDL and intervened with MEV/siHMGB1 by Western blot analysis. iNOS is the M1-type macrophage marker, while CD206 is an M2-type macrophage marker. *Indicates p < 0.05. Data among multiple groups are compared using one-way ANOVA, followed by Tuckey’s post hoc test

Article Snippet: The membrane was incubated with primary antibodies against HMGB1 (ab79823, 1:10,000, Rabbit, Abcam), Caspase-11 (ab180673, 1:1000, Rabbit, Abcam), LOX-1 (11837-1-AP, 1:1000, Rabbit, Proteintech), CD36 (18836-1-AP; 1:1000, Rabbit, Proteintech), SRA (ab151707, 1:1000, Rabbit, Abcam), FL-GSDMD (# 39754, 1:1000, Rabbit, CST, Danvers, MA, USA), and N-GSDMD (#39754, 1:1000, Rabbit, CST) overnight at 4 °C.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Flow Cytometry, Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Dysfunction of brain pericytes in chronic neuroinflammation

doi: 10.1177/0271678x15606149

Figure Lengend Snippet: Figure 2. Diminished pericyte coverage of BBB in HIV-1-infected patients paralleling macrophage/microglia activation. (a,b) Strong contiguous staining for PDGF-Rb (a, blue) or CD13 (b, blue) was detected in control brains, while only resting microglia were identified by minimal immunoreactivity to HLA-DR (a,b, red, arrows). (c,d) HIV cases with ART showed decreased/uneven CD13 (c, arrowheads) and PDGF-Rb (d, blue) labeling of pericytes and featured varying degree of microglial/macrophage activation and perivascular macro- phages (HLA-DR, red, arrows). (e–g) HIV cases without ART demonstrated diminution of CD13 (e,f, blue) and PDGF-Rb (g, blue) and HLA-DR-positive microglia (red, arrows). (h,i) Further increase in HLA-DR (red) was found in a HIV-1 encephalitic case (without ART) accompanied by substantially decreased CD13 labeling (blue). (j–l) a-SMA immunostaining was similar in control (j) and HIV-infected brain tissues highlighting arterioles (k,l). Original magnification: (a–l) 200. The scale bars on the lower left are 50 mm in length for the 20 (objective magnification) images. (m) Quantitative assessment of CD13 and CD31 immunostains was performed as described in Materials and methods section in controls (n ¼ 4), HIV cases without ART (n ¼ 9) and HIV cases with ART (n ¼ 5). The results designated as pericyte coverage, are shown as the average ratio SEM of CD13 to CD31 (****p < 0.0001; ***p ¼ 0.0003).

Article Snippet: One section from the frontal cortical lobe and the hippocampus from each case was used for the evaluation of neuroinflammation and BBB structure by immunohistochemistry using the following antibodies: CD68 for macrophage/microglia (1:100, Dako, Carpinteria, CA) and Iba-1 (1:100, Wako Chemicals USA, Richmond, VA), Human Leukocyte Antigen-DR (HLA-DR) (1:50, Dako) for microglia activation, HIV-1 p24 antigen (1:10, Dako), CD31 endothelial cell marker (1:50, Cell Marque, Rocklin, CA), platelet derived growth factor (PDGF)-Rb (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA), aSMA (1:50, Ventana Medical Systems Inc., Tucson AZ), and CD13 for pericytes (1:50, R&D Systems, Minneapolis, MN).

Techniques: Infection, Activation Assay, Staining, Control, Labeling, Immunostaining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Dysfunction of brain pericytes in chronic neuroinflammation

doi: 10.1177/0271678x15606149

Figure Lengend Snippet: Figure 3. Downregulation of pericyte cell markers by cytokines. Decreased expression of aSMA (a) and a1 integrin (b) upon inflammatory insult in quiescent pericytes. Pericytes (grown in 5% medium) were stimulated with TNFa or IL-1b (75 ng/ml) for 4 h, stained intracellularly for aSMA (a), surface stained for a1 integrin (b) and analyzed by FACS. Expression of PDGF-Rb (c) and gap junction connexin 43 (CX43) (d) was downregulated in response to inflammatory cytokines. Western blots of human brain pericyte whole cell lysates from cells treated for 24 h with TNFa or IL-1b (25–75 ng/ml). Actin served as a control for whole cell lysate loading.

Article Snippet: One section from the frontal cortical lobe and the hippocampus from each case was used for the evaluation of neuroinflammation and BBB structure by immunohistochemistry using the following antibodies: CD68 for macrophage/microglia (1:100, Dako, Carpinteria, CA) and Iba-1 (1:100, Wako Chemicals USA, Richmond, VA), Human Leukocyte Antigen-DR (HLA-DR) (1:50, Dako) for microglia activation, HIV-1 p24 antigen (1:10, Dako), CD31 endothelial cell marker (1:50, Cell Marque, Rocklin, CA), platelet derived growth factor (PDGF)-Rb (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA), aSMA (1:50, Ventana Medical Systems Inc., Tucson AZ), and CD13 for pericytes (1:50, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Staining, Western Blot, Control

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Genetic strategy used to disrupt NMDAR expression within a cohort of postnatal OPCs. St. = stop. B, Extent of Cre activity in P-NR1+/+ mice as revealed by EGFP expression in forebrain. The region of corpus callosum highlighted by the dashed box is shown at higher magnification in the panel at right. Mice were injected with 0.1mg 4HT at P4 and examined at P30. Ctx = cortex, CC = corpus callosum, HC = hippocampus. C, Response of callosal OPCs in acute brain slices from P-NR1+/+ and P-NR1fl/fl mice to photolysis of caged glutamate (MNI-L-glutamate, 500 μM). Currents were elicited at a holding potential of 40 mV and in the presence of antagonists for AMPA/kainate receptors (NBQX 50μM, GYKI53655 100μM) and voltage-gated sodium channels (TTX 1μM). NMDAR-mediated currents (sensitive to CPP, 20μM) were not observed in OPCs in P-NR1fl/fl mice (n = 14 cells). Mice were injected with 4HT at P16-30 and recorded 10–15 days post injection. D, EGFP+ PDGFαR+ cortical OPCs from P-NR1+/+ (top) and P-NR1fl/fl (bottom) mice; Mice injected with 4HT at P4 and examined at P30. E, 3D reconstruction of OPCs shown in D. F, Response of representative callosal OPCs in P-NR1+/+ and P-NR1fl/fl mice to depolarization. Red trace = injection of 640pA. Mice were P40-45 and were > 19 days post 4HT injection.

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Expressing, Activity Assay, Injection

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Immunostaining for EGFP, PDGFαR (pseudocolored red), and CC1 in the cortex and corpus callosum illustrating the strategy used to identify PDGFαR+ OPCs and CC1+ mature oligodendrocytes in P-NR1+/+ and P-NR1fl/fl mice. Mice were injected with 4HT at P4 and examined at P30. B, Density of EGFP+ cells in cortex (Ctx) and corpus callosum (CC); n for each group indicated at the base of the column. The density of EGFP+ cells was not significantly different between genotypes (Ctx p = 0.46, CC p = 0.75) but was significantly different between brain regions (Ctx vs. CC p = 0.0003). C, The identity of EGFP+ cells in P-NR1+/+ and P-NR1fl/fl mice expressed as a percentage of the total number of EGFP+ cells. PD = PDGFαR. The percentage of EGFP+ cells that had become mature oligodendrocytes (CC1+) was not significantly different between genotypes (Ctx p = 0.24, CC p = 1), but was significantly different between brain regions (Ctx vs. CC p = 0.0002).

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Immunostaining, Injection

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Response of representative OPCs, pre-oligodendrocytes (Pre-OLs), and oligodendrocytes (OLs) from P-NR1+/+ mice (top panel) and P-NR1fl/fl mice (lower panel) to depolarization. Resting potential of each cell is indicated to the left. Red trace = injection of 320pA. Green trace = injection of 40pA. Gray trace = injection of 1000pA. B – D, Membrane resistance (Rm), membrane capacitance (Cm), and resting potential (Vm) of OPCs, Pre-OLs, and OLs in P-NR1+/+ and P-NR1fl/fl mice. In both genotypes, Pre-OLs exhibited increased Rm and OLs exhibited decreased Rm relative to OPCs. In both genotypes, Pre-OLs and OLs displayed increased Cm and positive shifts in Vm relative to OPCs.

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Injection, Membrane

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Extent of Cre activity in O-NR1+/+ mice (bred to Z/EG reporter mice) as revealed by EGFP expression in the forebrain. Ctx = cortex, CC = corpus callosum, HC = hippocampus. The region of corpus callosum highlighted by the dashed box is shown at higher magnification at right. Inset shows the genetic strategy used to delete the NR1 subunit from oligodendrocyte lineage cells. B, Immunostaining for EGFP, PDGFαR, and NG2 reveals that the majority of oligodendrocyte progenitor cells (OPCs) in corpus callosum were EGFP+. Red arrows highlight several examples. C, EGFP expression within Olig2+ oligodendrocyte lineage cells in the corpus callosum of O-NR1+/+ mice. D, Graph showing the percentage of PDGFαR+ or Olig2+ cells that expressed EGFP in the corpus callosum and cortex of O-NR1+/+ mice (mice were P30). E, NMDAR-mediated currents recorded from OPCs in the corpus callosum of young adult (P20-40) O-NR1+/+ and O-NR1fl/fl mice (bred to NG2-DsRed mice). Currents were elicited by UV-uncaging of MNI-L-glutamate (500μM) at a holding potential of 40 mV in antagonists of AMPA/kainate receptors (NBQX 50μM, GYKI53655 100μM) and voltage-gated sodium channels (TTX 1μM). CPP-sensitive currents were not observed in OPCs in O-NR1fl/fl mice (n = 10 cells).

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Activity Assay, Expressing, Immunostaining

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Representative images of PDGFαR+ (top) and Olig2+ cells (bottom) in the corpus callosum of early postnatal (P7 – P14) O-NR1+/+ mice. B, Graph showing the density of PDGFαR+ cells in the cortex (Ctx) and corpus callosum (CC) of P7 O-NR1+/+ and O-NR1fl/fl mice. Differences between genotypes are not significant (Ctx p = 1, CC p = 1), while differences between brain regions are significant (Ctx vs. CC p = 0.005). C, Graph showing the density of Olig2+ cells in the cortex and corpus callosum of P14 O-NR1+/+ and O-NR1fl/fl mice. Differences between genotype are not significant (Ctx p = 1, CC p = 0.38), while differences between brain regions are significant (Ctx vs. CC p = 0.0007).

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques:

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, BrdU labeling of proliferating cells in representative forebrain section of early postnatal (P7-8) O-NR1+/+ mouse. Regions of cortex (Ctx) and corpus callosum (CC) highlighted by dashed boxes are shown at higher magnification at right, and illustrate co-immunostaining for BrdU and OPC marker PDGFαR. B, BrdU labeling of proliferating cells in representative forebrain section of P13-14 O-NR1+/+ mouse. Regions of cortex (Ctx) and corpus callosum (CC) highlighted by dashed boxes are shown at higher magnification at right, and illustrate co-immunostaining for BrdU and oligodendrocyte lineage marker Olig2. C, Quantification of proliferating PDGFαR+ cells in P7-8 O-NR1+/+ and O-NR1fl/fl mice (n = 3 mice per genotype). Differences between genotypes were not significant (Ctx p = 0.66, CC p = 1), but differences between brain regions were significant (Ctx vs. CC p = 0.03). D, Quantification of proliferating Olig2+ cells in P13-14 O-NR1+/+ (n = 6) and O-NR1fl/fl (n = 4) mice. Differences between genotypes were not significant (p = 0.11, CC p = 0.86), but differences between brain regions were significant (Ctx vs. CC p = 0.0008).

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Labeling, Immunostaining, Marker

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination

doi: 10.1523/JNEUROSCI.2455-11.2011

Figure Lengend Snippet: A, Response of representative callosal OPCs to hypertonic solution in mature (P40-45) O-NR1+/+ (top) and O-NR1fl/fl (bottom) mice. Duration of hypertonic solution (HS) application indicated by the black bar. Regions indicated by the gray bars are shown at an expanded time scale to the right. B, Response of representative callosal OPCs to HS in mature (P40-45) P-NR1+/+ (top) and P-NR1fl/fl (bottom) mice. Recordings were performed in mice at least 19 days post 4HT injection. HS application indicated by the black bar. Regions indicated by the gray bars are shown at expanded time scale to the right. C, Quantification of the number of HS-evoked miniature excitatory post synaptic currents (mEPSCs) in OPCs from control (O-NR1+/+, P-NR1+/+), constitutive (O-NR1fl/fl), and inducible (P-NR1fl/fl) NMDAR ablation mice. n for each group indicated at the base of each column. p = 0.22, K-W ANOVA. D, Cumulative probability distribution of the amplitudes of HS-evoked mEPSCs recorded in callosal OPCs from O-NR1+/+ and O-NR1fl/fl mice. p = 0.16, K-S test. Inset shows average amplitude of HS-evoked mEPSCs recorded in callosal OPCs from O-NR1+/+ and O-NR1fl/fl mice. p = 0.055. E, Response of representative callosal OPCs from mature (P40-50) O-NR1+/+ and O-NR1fl/fl mice to photolysis of caged glutamate (MNI-L-glutamate,500 μM) at holding potentials of −80 mV and 40 mV. Currents were recorded in the presence of TTX (1μM), CPP (20μM), and 7-chlorokynurenic acid (100 μM) and spermine (100μM) was included in the recording pipette. Inset shows response of a representative callosal OPC (P36 O-NR1+/+ mouse) to glutamate uncaging (black dot) before (black trace) and after the addition of AMPAR antagonists NBQX (50μM) and GYKI53655(100μM) (gray trace) to the bath (n = 19 cells).

Article Snippet: For morphological analysis of OPCs in P-NR1 +/+ /P-NR1 fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP + PDGFαR + cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification.

Techniques: Injection, Control, Transferring

Journal: The FASEB Journal

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

doi: 10.1096/fj.12-214353

Figure Lengend Snippet: Binding of C. albicans Saps 4–6 to cell surface integrin. A) Left panel: confocal microscopic image of an isolated human platelet is virtually without fluorescence. Center and right panels: a platelet that was incubated with Alexa-Fluor 488-labeled Sap 6 (center panel) and a deconvolved fluorescence image from confocal microscopy of an Alexa-Fluor Sap 6-treated platelet (right panel) show fluorescence concentrated at the cell surface. Scale bars = 2 μm. B) Fluorescence intensity of platelets after incubation with Alexa-Fluor Sap 6 and different concentration of ADP. C) Fluorescence images from confocal microscopy of an A549 cell after incubation with Saps at 10°C. Integrin β1 chain on the cell surface was revealed by Cy3-conjugated antibody (left panels). Fluorescence images of Alexa-Fluor modified Saps 4–6 are seen on the surface of A549 cells (center panels); thus, Alexa-Fluor-conjugated Sap 4mut, Sap 5mut, and Sap 6mut did not bind the cells and appear as dark fields. Merged images (right panels) show colocalization of integrin β1 chain and Alexa-Fluor Saps 4–6 on the cell surface (arrows). Scale bar = 20 μm. D) Platelet- and A549-associated fluorescence from bound Alexa-Fluor Sap 6 was inhibited by RGD-containing peptides RGDS and integrilin. E) Highly purified Saps 4–6 inhibited the binding of integrin β1 antibody to A549 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell lines and platelets Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line SCC-15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Isolation, Fluorescence, Incubation, Labeling, Confocal Microscopy, Concentration Assay, Modification, Purification

Journal: The FASEB Journal

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

doi: 10.1096/fj.12-214353

Figure Lengend Snippet: Binding of Saps 4–6 but not Sap 2 or mutant Saps 4–6 to A549 cells. Isolated Saps 2 and 4–6 and mutant Saps (Sap 4mut, Sap 5mut, and Sap 6mut were mutated to change the integrin-binding motifs) were individually conjugated to Alexa Fluor-488 and incubated with A549 cells at 10°C for 1 h. After washing 3 times with PBS, the cells were observed in a Zeiss LSM510 confocal fluorescence microscope to detect cell-bound Alexa-Fluor Saps. Green fluorescence from Alexa-Fluor Saps 4–6 was found to associate with the cells; however, Alexa-Fluor-conjugated Sap 2, Sap 4mut, Sap 5mut, and Sap 6mut displayed only minimal association with the cells. Scale bar = 20 μm.

Article Snippet: Cell lines and platelets Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line SCC-15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Mutagenesis, Isolation, Incubation, Fluorescence, Microscopy

Journal: The FASEB Journal

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

doi: 10.1096/fj.12-214353

Figure Lengend Snippet: Internalization of cell surface bound Sap 6. A) A549 cells were incubated with Alexa-Fluor Sap 6 for 1 h at either 10°C (top panels) or 37°C (bottom panels), washed, and then observed by confocal microscopy. Left panels show fluorescent images from Alexa-Fluor Sap 6; center panels show cell images from transmission light; right panels show merged images. At 10°C, Alexa-Fluor Sap 6 bound predominantly at the cell surface, while at 37°C, Alexa-Fluor Sap 6 was localized inside the cell. B) Internalized Alexa-Fluor Sap 6 colocalizes with endosome and lysosome markers and integrin β1 chain. A549 cells were incubated with Alexa-Fluor Sap 6 and stained with Cy3-labeled antibodies against either endosome marker EEA1 (top panels), lysosome marker LAMP1 (middle panels), or integrin β1 chain (bottom panels). Left panels: confocal microscopic images of the cells were obtained for EEA1, LAMP1, and integrin β1 chain, and Alexa-Fluor Sap 6 from respective antibodies conjugated to Cy3. Center panels: fluorescence images of Alexa-Fluor Sap 6. Right panels: merged images. Arrows at vesicle-like images indicate examples of colocalization of Alexa-Fluor Sap 6 with EEA1, LAMP1, and integrin β1 chain, respectively. Scale bar: 20 μm. C) Confocal microscopic image of internalized Sap 5 and integrin. Experimental conditions were the same as in Fig. 2C, except that after binding at 10°C, the temperature was raised to 37°C to allow internalization. Since Sap 5mut did not bind, only Sap 5 images are shown. Arrows indicate defined spots of colocalization. Scale bars = 20 μm.

Article Snippet: Cell lines and platelets Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line SCC-15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Incubation, Confocal Microscopy, Transmission Assay, Staining, Labeling, Marker, Fluorescence, Binding Assay

Journal: The FASEB Journal

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

doi: 10.1096/fj.12-214353

Figure Lengend Snippet: Effect of Saps 4–6 on cell viability and lysosome permeability. A) Viability of A549 cells after incubation with isolated Sap 6 at 37°C for a time period as indicated. Cell viability was analyzed by counting trypan blue-stained cells. B–E) Sap 6 induced lysosome permeability and changed distribution of acridine orange (AO). B) In control A549 cells, AO accumulated in lysosomes and produced red fluorescence. C) Cells pretreated with Sap 6 had marked decrease of red fluorescence of lysosomes and increased green fluorescence from the cytosol as compared with the control cells in B. D) Sap 2 did not significantly change lysosomal AO. E) Ratios of green to red fluorescence from B–D. F–J) Sap 4-induced changes in cellular distribution of AO were reversed by GRL-110C, a specific inhibitor of Sap 4. F) Confocal microscopic image of AO in untreated A549 cells. G) AO in cells pretreated with Sap 4 showed increased cytosolic green fluorescence, as compared with cells in F. H) AO fluorescence in cells pretreated with Sap 4 in the presence of Sap 4 inhibitor GRL-110C was significantly redder than those cells in G and approached that in F. I) Image of AO fluorescence in H2O2-treated cells (positive control). J) Ratios of green to red fluorescence in experiments F–I. K–Q) Saps 4–6, but not Saps 4mut, 5mut, and 6mut, induced changes in cellular distribution of AO. K) Image of AO in untreated A549 cells. L, N, P) AO in cells treated separately with Saps 4–6 showed increased cytosolic green fluorescence as compared with untreated cells in K. M, O, Q) Image of AO in cells treated separately with Saps 4mut, 5mut, and 6mut showed lysosomal red fluorescence similar to the untreated cells in K. R–W) Confocal microscopic images of Sap-treated A549 cells immunochemically stained for cathepsin D. R) An untreated cell contains cathepsin D-positive lysosomes (arrow). S) An H2O2-treated cell showed no cathepsin D-positive vesicles. T, U, W) Cells separately treated with Saps 4–6 showed no cathepsin D-positive lysosomes as in S. V) Cell treated with Sap 5mut clearly retained cathepsin D-positive lysosomes (arrow). Quantitation on the ratios of green to red fluorescence in experiments K–Q between Saps and Sapmuts are statistically significant in the range of P < 0.05 to P < 0.01 but are not statistically significant between untreated control and the Sapmuts. ns, not significant. *P < 0.05, **P < 0.01.

Article Snippet: Cell lines and platelets Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line SCC-15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Permeability, Incubation, Isolation, Staining, Control, Produced, Fluorescence, Positive Control, Quantitation Assay

Journal: The FASEB Journal

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

doi: 10.1096/fj.12-214353

Figure Lengend Snippet: Saps 4–6 induce apoptosis of A549 cells. Cells were incubated with individual Saps at 37°C for 23 h and studied for apoptosis. A) Typical flow cytometric 2-dimensional dot plots of A549 cells after incubation with individual Saps, Sap mutants, or controls. Early apoptosis of the cells incubated with Saps, Sap mutants, or the controls were analyzed by annexin V-PE staining and flow cytometry. Right panels: Saps 4–6 significantly increased the number of annexin-positive cells (bottom right quadrants) over the untreated control cells. Center panels: Sap 4mut-, 5mut-, and 6mut-treated cells did not show significant increase in the population of the annexin-positive cells. Data are representative of 4 independent experiments. B) Top panel: results from two experiments in A; quantitated percentage of cells in early apoptosis as determined by annexin staining and flow cytometry analysis. Camptothecin treatment was an apoptotic-positive control. Bottom panel: relative number of cells in late apoptosis after incubation with Saps. Quantitation was done with Hoechst 33342 staining and confocal microscopy. Apoptosis reagent camptothecin was used as a positive control. Statistical significance (in the range of P < 0.01 to P < 0.001) was observed between individual solid columns with respective controls (open columns) and between Sap 5 and Sap 5mut. C) Time course of Sap 4–6 induction of caspase 3 activity in A549 cells from two separate experiments (top and bottom). Pretreatments: black open circles, none; black solid circles, H2O2 (positive control); red open circles, Sap 2; brown solid circles, Sap 4; violet solid circles, Sap 5; open violet circles with broken lines, Sap 5mut; turquoise solid circles, Sap 6.

Article Snippet: Cell lines and platelets Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line SCC-15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Incubation, Staining, Flow Cytometry, Control, Positive Control, Quantitation Assay, Confocal Microscopy, Activity Assay

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A-B) Uniform Manifold Approximation and Projection (UMAP) plots of cell types in the combined single-cell RNA-sequencing (scRNA-seq) dataset from human pancreatic ductal adenocarcinoma (PDAC) (n=5) and chronic pancreatitis (n=4) tissues. Different conditions (A) and cell types (B) are colour coded. scRNA-seq data is from Dimitrieva et al. (C) Cell type contribution in human PDAC and pancreatitis, represented as bar plot showing proportions of the different cell clusters in each condition. (D) Significantly upregulated pathways identified by gene set enrichment analysis (GSEA) of PDAC malignant cells compared to pancreatitis ductal cells, as assessed by Model-based Analysis of Single-cell Transcriptomics (MAST) from the scRNA-seq dataset, and genes were ranked based on log2 fold change. Inflammat, inflammatory; Proliferat., proliferative. (E) Significantly upregulated pathways identified by GSEA of PDAC cancer-associated fibroblasts (CAFs) compared to pancreatitis fibroblasts, as assessed by MAST from the scRNA-seq dataset. (F) Schematic of models and techniques used for analysis of normal pancreas (i.e. from untreated or 6-week PBS-treated mice), pancreatitis (i.e. from 6-week caerulein-treated mice) and PDAC (i.e. from KPC genetically engineered mouse models, GEMMs) tissues from C57BL/6J mice. (G-N) Representative images and quantification of the ductal marker keratin 19 (CK19) (G-H) , the fibroblast marker PDGF receptor alpha (PDGFRα) (I-J) , Masson’s trichrome for collagen deposition (K-L) and the myofibroblast marker alpha smooth muscle actin (αSMA) (M-N) immunohistochemical stains of normal pancreas, pancreatitis and PDAC tissues. Results show mean ± SEM. * P adj < 0.05; ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. Scale bars, 50 μm.

Article Snippet: Primary antibodies for IHC were αSMA (ab5694; Abcam; RRID:AB_2223021), PDPN (127403; BioLegend; RRID: AB_1134221), PDGFR-α (3174; Cell Signaling Technology; RRID: AB_2162345), CK19 (TROMA-III; DSHB; RRID: AB_2133570), Ly6G ( 127602; BioLegend; RRID: AB_1089180), and F4/80 (MCA497; Serotec; RRID: AB_323279).

Techniques: RNA Sequencing, Single-cell Transcriptomics, Marker, Immunohistochemical staining

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Dot plot of scaled expression of cell type-specific markers in each cell cluster of human chronic pancreatitis (n=4) and pancreatic ductal adenocarcinoma (PDAC, n=5) tissues, as analysed by single-cell RNA-sequencing (scRNA-seq). The colour intensity represents the expression level, and the size of the dots represents the percentage of expressing cells. scRNA-seq analysis is from Dimitrieva et al. (B) Sample contribution to different cell types in human pancreatitis and PDAC analysed by scRNA-seq, represented as bar plot showing proportions of the different samples in each cell cluster. (C) Heatmap showing large-scale copy number variation (CNV) profile of the fibroblast and malignant cell clusters identified by scRNA-seq of human PDAC. The colour coding represents the CNV level based on a sliding window of 250 gene expression. Amplifications are shown in red, and deletions are shown in blue. The fibroblast cluster was used as reference cell cluster. (D) Schematic of models and techniques used for the analysis of murine acute pancreatitis tissues. (E) Lipase activity levels in plasma from normal pancreas, acute pancreatitis and pancreatitis mouse models. Results show mean ± SEM. * P adj < 0.05, Kruskal-Wallis test. (F) Amylase activity levels in plasma from normal pancreas, acute pancreatitis and pancreatitis mouse models. Results show mean ± SEM. ** P adj < 0.01, Kruskal-Wallis test. (G) Weights of pancreata from normal pancreas, acute pancreatitis and pancreatitis mouse models. Results show mean ± SEM. * P adj < 0.05; ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. (H-I) Representative images and quantifications of the ductal marker keratin 19 (CK19), the fibroblast marker platelet-derived growth factor receptor alpha (PDGFRα), the myofibroblast marker alpha smooth muscle actin (αSMA), the fibroblast marker podoplanin (PDPN), the macrophage marker F4/80, the neutrophil marker Ly6G and Masson’s trichrome (for collagen deposition) stains of acute pancreatitis murine tissues. Results show mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, Mann-Whitney test, indicate significant downregulation compared to pancreatitis tissues from and S1. # P < 0.05; ### P < 0.001, Mann-Whitney test, indicate significant upregulation compared to normal pancreas tissues from and S1. Scale bars, 50 μm. (J-M) Representative images and quantifications of F4/80 (J-K) , Ly6G (L-M) and PDPN (N-O) stains of normal pancreas, pancreatitis and PDAC murine tissues. Results show mean ± SEM. * P adj < 0.05; ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. Scale bars, 50 μm.

Article Snippet: Primary antibodies for IHC were αSMA (ab5694; Abcam; RRID:AB_2223021), PDPN (127403; BioLegend; RRID: AB_1134221), PDGFR-α (3174; Cell Signaling Technology; RRID: AB_2162345), CK19 (TROMA-III; DSHB; RRID: AB_2133570), Ly6G ( 127602; BioLegend; RRID: AB_1089180), and F4/80 (MCA497; Serotec; RRID: AB_323279).

Techniques: Expressing, RNA Sequencing, Gene Expression, Activity Assay, Clinical Proteomics, Marker, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Representative images of PDGFRα stains of P and PDAC organoid/pancreatic stellate cell (PSC) co-cultures after 7 days in culture. Scale bars = 100 μm. (B) Bright field images of P and PDAC organoid/PSC co-cultures after 7 days in culture. Scale bars = 100 μm. (C) Fibroblast (PDGFRα + )/epithelial cell (CK19 + ) ratio as assessed by immunohistochemical analysis of pancreatitis or PDAC murine tissues (from ). Results show mean ± SEM. *** P < 0.001, Mann-Whitney test. (D) Ratio of fibroblast to epithelial cell numbers per sample as determined by snRNA-seq analysis of pancreatitis and PDAC murine tissues (from ). Results show mean ± SEM. ** P < 0.01, Mann-Whitney test. (E) Summary table of the conditions analysed by RNA-seq in monoculture (i.e. culture of a single cell type) or co-culture (i.e. culture of two or more cell types). (F) PCA of P organoids in monocultures (i.e., P monocx, n=5 biological replicates, each with 2 replicates of different passages) or in co-culture with PSCs (i.e., P cocx, n=10 biological replicates), PDAC organoids in monocultures (i.e., PDAC monocx, n=4 biological replicates, of which three samples with 2 replicates of different passages) or in co-culture with PSCs (i.e., PDAC cocx, n=7 biological replicates), and PSC monocultures (i.e., PSC monocx, n=2 biological replicates, each with 2 replicates of different passages) and in co-culture with PDAC organoids (i.e., PDAC PSCs, n=7 biological replicates) or P organoids (i.e., P PSCs, n=10 biological replicates), as assessed by RNA-seq. (G) Significantly upregulated and downregulated pathways identified by GSEA of P or PDAC organoids in co-culture compared to their respective monocultures, as assessed by RNA-seq. (H) Heatmap showing GSVA enrichment scores (averaged per group) of selected pathways in P and PDAC organoid monocultures and co-cultures, as assessed by RNA-seq. Pathways displayed correspond to pathways significantly altered in GSEA ( and/or S4G). (I) GSEA of the murine pancreatitis Ductal 1 (top) and Ductal 2 (bottom) signatures in P organoids in co-culture with PSCs compared to P organoids in monoculture. The pancreatitis Ductal 1 signature was significantly enriched in P organoids in co-culture. The pancreatitis Ductal 2 signature was not significantly different between conditions. (J) Venn diagrams of differentially expressed curated protein-coding ortholog genes significantly ( P adj < 0.05) upregulated in P organoids in PSC co-cultures compared to PDAC organoids in PSC co-cultures (hereon, ‘P organoid orthologs from PSC co-cultures’), as assessed by RNA-seq, murine pancreatitis ductal orthologs, and human pancreatitis ductal orthologs. Selected genes common to all or two datasets are indicated. Significance of the overlap between P organoid orthologs from PSC co-cultures and human pancreatitis ductal orthologs was defined by hypergeometric test (with denominator = 16,076 orthologs). (K) Venn diagrams of differentially expressed curated protein-coding ortholog genes significantly ( P adj < 0.05) upregulated PDAC organoids in PSC co-cultures compared to P organoids in PSC co-cultures (hereon, ‘PDAC organoid orthologs from PSC co-cultures’), as assessed by RNA-seq, murine PDAC malignant orthologs, and human PDAC malignant orthologs. Selected genes common to all or two datasets are indicated. Significance of the overlap between PDAC organoid orthologs from PSC co-cultures and human PDAC malignant orthologs was defined by hypergeometric test (with denominator = 16,076 orthologs). (L) Western blot of p19 in N and PDAC organoids, as well as PDAC organoids that have not undergone loss of heterozygosity of the wild-type Trp53 allele (KPC organoids) and pre-cancerous pancreatic intraepithelial neoplasia organoids from KC mice (KC organoids) cultured in complete media for 3 days. ACTIN, loading control. (M) Venn diagrams of curated protein-coding significantly ( P adj < 0.05) upregulated genes in P organoids in co-culture with PSCs compared to PDAC organoids in co-culture with PSCs (hereon, ‘P organoid markers from PSC co-cultures’) and curated protein-coding significantly upregulated genes in P organoids in co-culture with PSCs compared to P organoids in monoculture. (N) Venn diagrams of curated protein-coding significantly ( P adj < 0.05) upregulated genes in PDAC organoids in co-culture with PSCs compared to P organoids in co-culture with PSCs (hereon, ‘PDAC organoid markers from PSC co-cultures’) and curated protein-coding significantly upregulated genes in PDAC organoids in co-culture with PSCs compared to PDAC organoids in monoculture.

Article Snippet: Primary antibodies for IHC were αSMA (ab5694; Abcam; RRID:AB_2223021), PDPN (127403; BioLegend; RRID: AB_1134221), PDGFR-α (3174; Cell Signaling Technology; RRID: AB_2162345), CK19 (TROMA-III; DSHB; RRID: AB_2133570), Ly6G ( 127602; BioLegend; RRID: AB_1089180), and F4/80 (MCA497; Serotec; RRID: AB_323279).

Techniques: Immunohistochemical staining, MANN-WHITNEY, RNA Sequencing, Co-Culture Assay, Western Blot, Cell Culture, Control

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Schematic of analyses of P or PDAC organoid/multi-stroma co-cultures in reduced media (i.e. 5% FBS/DMEM). (B) Representative images of H&E, PDPN and CK19 stains of P or PDAC organoid/multi-stroma co-cultures after 7 days in culture. Scale bars = 100 μm. (C) UMAP plot of cell subsets in the combined scRNA-seq dataset of PDAC (n=2) and P (n=2) organoid/multi-stroma co-cultures after 3.5 days in culture. Different cell subsets are colour coded. (D) Significantly upregulated and downregulated pathways identified by GSEA of PDAC organoids compared to P organoids 1 or P organoids 2, as assessed by MAST from the scRNA-seq dataset of organoid/multi-stroma co-cultures. (E) Venn diagrams of curated protein-coding genes significantly ( P adj < 0.05) upregulated in P organoids in multi-stroma co-culture compared to PDAC organoids in multi-stroma co-culture (hereon, ‘P organoid markers from multi-stroma co-cultures’), as assessed by scRNA-seq, and pancreatitis Ductal 1 or Ductal 2 markers. Significance of the overlap between datasets was defined by hypergeometric test (with denominator n=18,000). Selected genes common to all datasets are indicated. (F) Venn diagrams of curated protein-coding genes significantly upregulated in PDAC organoids in multi-stroma co-culture compared to P organoids in multi-stroma co-culture (hereon, ‘PDAC organoid markers from multi-stroma co-cultures’), as assessed by scRNA-seq, and PDAC malignant markers. Significance of the overlap between datasets was defined by hypergeometric test (with denominator n=18,000). Selected genes common to all datasets are indicated. (G) Representative multiplex immunofluorescence (IF) images of MUC5AC (yellow), CK19 (red) and DAPI (nuclear stain, blue) in murine pancreatitis and PDAC tissues. Scale bars = 50 μm. (H) Quantification of MUC5AC + CK19 + DAPI + αSMA - PDGFRα - cells relative to DAPI + CK19 + αSMA - PDGFRα - epithelial cells in murine pancreatitis and PDAC tissues, as assessed by multiplex IF analysis. Results show mean ± SEM. ** P < 0.01, Mann-Whitney test. (I) Representative multiplex IF images of SOX9 (cyan), CK19 (red) and DAPI (nuclear stain, blue) (top panels), and STAT1 (cyan), CK19 (red) and DAPI (blue) (bottom panels) in murine pancreatitis and PDAC tissues. Scale bars = 50 μm. (J-K) Quantification of SOX9 + CK19 + DAPI + (J) and STAT1 + CK19 + DAPI + (K) αSMA - PDGFRα - cells relative to DAPI + CK19 + αSMA - PDGFRα - epithelial cells in murine pancreatitis and PDAC tissues, as assessed by multiplex IF analysis. Results show mean ± SEM. ** P < 0.01, Mann-Whitney test.

Article Snippet: Primary antibodies for IHC were αSMA (ab5694; Abcam; RRID:AB_2223021), PDPN (127403; BioLegend; RRID: AB_1134221), PDGFR-α (3174; Cell Signaling Technology; RRID: AB_2162345), CK19 (TROMA-III; DSHB; RRID: AB_2133570), Ly6G ( 127602; BioLegend; RRID: AB_1089180), and F4/80 (MCA497; Serotec; RRID: AB_323279).

Techniques: Co-Culture Assay, Multiplex Assay, Immunofluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Significantly upregulated and downregulated pathways identified by GSEA of murine multi-stroma (i.e. PSCs, fibroblasts and mesothelial cells) cultured with PDAC organoids compared to multi-stroma cultured with P organoids, as assessed by MAST from the scRNA-seq dataset of PDAC and P organoid/multi-stroma co-cultures. (B) Significantly upregulated and downregulated pathways identified by GSEA of PSCs, fibroblasts or mesothelial cells cultured with PDAC organoids compared to PSCs, fibroblasts or mesothelial cells cultured with P organoids, as assessed by MAST from the scRNA-seq dataset of PDAC and P organoid/multi-stroma co-cultures. (C) Venn diagrams of curated protein-coding genes significantly ( P adj < 0.05) upregulated in multi-stroma cultured with P organoids compared to multi-stroma cultured with PDAC organoids (hereon, ‘P multi-stroma markers’), as assessed by scRNA-seq, and pancreatitis fibroblast markers. Significance of the overlap between datasets was defined by hypergeometric test (with denominator = 18,000). Selected genes common to both datasets are indicated. (D) Venn diagrams of significantly upregulated ( P adj < 0.05) curated protein-coding DEGs in murine multi-stroma cultured with PDAC organoids compared to multi-stroma cultured with P organoids (hereon, ‘PDAC multi-stroma markers’), as assessed by scRNA-seq, and PDAC CAF markers. Significance of the overlap between datasets was defined by hypergeometric test (with denominator = 18,000). Selected genes common to both datasets are indicated. (E) Representative multiplex IF images of PDPN (white), αSMA (green), PDGFRα (purple) and DAPI (nuclear stain, blue) in murine pancreatitis and PDAC tissues. Scale bars = 50 μm. (F-G) Quantification of PDGFRα + αSMA - PDPN + DAPI + (F) and αSMA + PDGFRα - PDPN + DAPI + (G) CK19 - cells relative to PDPN + CK19 - DAPI + fibroblasts in murine pancreatitis and PDAC tissues, as assessed by multiplex IF analysis. Results show mean ± SEM. *** P < 0.001, Mann-Whitney test. (H) Representative multiplex IF images of FN1 (cyan), αSMA (green), PDGFRα (purple) and DAPI (nuclear stain, blue) in murine pancreatitis and PDAC tissues. Scale bars = 50 μm. (I) Quantification of FN1 + αSMA - PDGFRα + DAPI + CK19 - cells relative to PDGFRα + CK19 - DAPI + fibroblasts in murine pancreatitis and PDAC tissues, as assessed by multiplex IF analysis. Results show mean ± SEM. No significant difference was found, as assessed by Mann-Whitney test. (J) Quantification of FN1 + PDGFRα - αSMA + DAPI + CK19 - cells relative to αSMA + CK19 - DAPI + fibroblasts in murine pancreatitis and PDAC tissues, as assessed by multiplex IF analysis. Results show mean ± SEM. ** P < 0.01, Mann-Whitney test. (K) Heatmap showing the fractional contribution of in vitro scRNA-seq PSCs, mesothelial cells and fibroblasts from P organoid/multi-stroma co-cultures (columns) to in vivo snRNA-seq pancreatitis fibroblast subtypes (rows), derived from averaged scANVI soft label probabilities. (L) PAGA illustrating the inferred pseudotemporal trajectory among in vitro P stromal cells and in vivo pancreatitis fibroblast subtypes. Nodes (i.e., circles) represent cell subsets, and their size is scaled to the number of cells per cluster. Edges (i.e., line) represent the strength of connectivity (i.e., thickness). (M) Heatmap showing the fractional contribution of in vitro scRNA-seq PSCs, mesothelial cells and fibroblasts from PDAC organoid/multi-stroma co-cultures (columns) to in vivo snRNA-seq PDAC CAFs (row), derived from averaged scANVI soft label probabilities. (N) PAGA illustrating the inferred pseudotemporal trajectory among in vitro PDAC stromal cells and in vivo PDAC CAFs. Nodes (i.e., circles) represent cell subsets, and their size is scaled to the number of cells per cluster. Edges (i.e., line) represent the strength of connectivity (i.e., thickness).

Article Snippet: Primary antibodies for IHC were αSMA (ab5694; Abcam; RRID:AB_2223021), PDPN (127403; BioLegend; RRID: AB_1134221), PDGFR-α (3174; Cell Signaling Technology; RRID: AB_2162345), CK19 (TROMA-III; DSHB; RRID: AB_2133570), Ly6G ( 127602; BioLegend; RRID: AB_1089180), and F4/80 (MCA497; Serotec; RRID: AB_323279).

Techniques: Cell Culture, Multiplex Assay, Staining, MANN-WHITNEY, In Vitro, In Vivo, Derivative Assay

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques:

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Marker, Expressing, Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Human and porcine aortic valve endothelial and interstitial cell isolation and characterization

doi: 10.3389/fcvm.2023.1151028

Figure Lengend Snippet: Human valvular endothelial cell (hVEC) isolation and characterization. ( A ) Workflow of VEC isolation: (1) Explanted aortic valve cusps were incubated for 10 min in collagenase type II solution. (2) VECs were carefully scraped with a scalpel into a dish filled with VEC medium, which (3) was transferred into a 15 ml tube and washed twice for 15 min at 1,000 rpm at room temperature. (4) Cells were incubated with CD105 magnetic beads for 15 min, and (5) the CD105 positive selection was carried out with MACS® separator, according to the manufacturer's protocol. (6) The specific VEC population was seeded into one well of a fibronectin-coated 6-well plate. ( B ) Gene expression of isolated hVECs and human valvular interstitial cells (hVICs) showed a positive expression of the endothelial markers von Willebrand factor ( vWF ), platelet adhesion molecule 1 ( PECAM1 ), endoglin ( CD105 ), and nitric oxide synthase 3 ( NOS3 ) in hVECs. ( C ) Brightfield and immunofluorescence images of VECs positive for vWF and CD31 and negative for alpha smooth muscle actin (α-SMA) and vimentin (VIM). ( D ) Migration analysis by scratch wound healing assay resulted in a scratch width of 15% after 8 h. Treatment of hVECs with H 2 O 2 for 24 h resulted in decreased ( E ) cell viability, increased ( F ) caspase 3/7 activity, and decreased ( G ) cell proliferation. ( H ) Life span of hVECs in cell culture at different passages (p). ( I ) Inducing EndMT by incubating VECs with tumor necrosis factor alpha (TNFα) for 7 days. TNFα led to an upregulation of α - SMA, VIM , cadherin 2 ( CDH2 ) and vascular adhesion molecule 1 ( VCAM1 ) and a downregulation of PECAM1, vWF and NOS3. ( B ) n = 5 donors, ( D ) n = 3 donors, ( E–I ) n = 3 donors with technical replicates indicated by one color per donor, * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed by Student t -test, 2-tailed, unpaired, ( A ) created with BioRender.com.

Article Snippet: RT-PCR-based gene expression profiling was performed using TaqManTM Gene Expression Master Mix (Thermo Fisher Scientific, #4370074) and the following human TaqManTM primers (Thermo Fisher Scientific): GAPDH (Hs02758991_g1), α-SMA (Hs00426835_g1), vWF (Hs01109446_m1), PECAM1 (Hs01065279_m1), VIM (Hs00418522_m1), CD105 (Hs00164438_m1), NOS3 (Hs01574665_m1), CDH2 (Hs00983056_m1), VCAM1 (Hs00365486_m1), SNAI2 (Hs00161904_m1), ALPL (Hs01029144_m1), BGLAP (Hs01587814_g1) and RUNX2 (Hs01047973_m1).

Techniques: Isolation, Incubation, Magnetic Beads, Selection, Gene Expression, Expressing, Immunofluorescence, Migration, Wound Healing Assay, Activity Assay, Cell Culture

Journal: Frontiers in Cardiovascular Medicine

Article Title: Human and porcine aortic valve endothelial and interstitial cell isolation and characterization

doi: 10.3389/fcvm.2023.1151028

Figure Lengend Snippet: Porcine valvular endothelial cell (pVECs) isolation and characterization. ( A ) Gene expression analysis of pVECs revealed a significant upregulation of von Willebrand factor ( vWF ), platelet adhesion molecule 1 ( PECAM1 ), endoglin ( CD105 ), nitric oxide synthase 3 ( NOS3 ), and vimentin ( VIM ) and a downregulation of alpha smooth muscle actin ( α-SMA ) and cadherin 2 ( CDH2 ) compared to porcine valvular interstitial cells (pVICs). ( B ) Brightfield images and immunofluorescence staining of pVECs showing a positive signal for vWF and an absent signal for α-SMA. ( C ) Analysis of migration properties by scratch wound healing assay revealed a scratch closure to 70%. pVECs treated with 100 µM H 2 O 2 showed a decrease in ( D ) cell viability, ( E ) caspase 3/7 activity, and ( F ) cell proliferation. ( G ) Life span of pVECs in cell culture at different passages (p). ( H ) In vitro EndMT induction by TNFα for 7 days. TNFα displayed downregulated endothelial marker expression, ( PECAM1, vWF, NOS3 ) and mesenchymal markers, ( α-SMA, VIM, CDH2 ). CDH2 and vascular cell adhesion molecule 1 ( VCAM1 ) was upregulated. ( A ) n = 5 donors, ( C ) n = 3 donors, ( D–H ) n = 3 donors with technical replicates indicated by one color per donor, * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed by Student t -test, 2-tailed, unpaired, (a) created with BioRender.com.

Article Snippet: RT-PCR-based gene expression profiling was performed using TaqManTM Gene Expression Master Mix (Thermo Fisher Scientific, #4370074) and the following human TaqManTM primers (Thermo Fisher Scientific): GAPDH (Hs02758991_g1), α-SMA (Hs00426835_g1), vWF (Hs01109446_m1), PECAM1 (Hs01065279_m1), VIM (Hs00418522_m1), CD105 (Hs00164438_m1), NOS3 (Hs01574665_m1), CDH2 (Hs00983056_m1), VCAM1 (Hs00365486_m1), SNAI2 (Hs00161904_m1), ALPL (Hs01029144_m1), BGLAP (Hs01587814_g1) and RUNX2 (Hs01047973_m1).

Techniques: Isolation, Gene Expression, Immunofluorescence, Staining, Migration, Wound Healing Assay, Activity Assay, Cell Culture, In Vitro, Marker, Expressing

Journal: International Journal of Oncology

Article Title: Targeting CALR reduces energy metabolism of esophageal cancer cells and inhibits tumor-associated fibroblast infiltration

doi: 10.3892/ijo.2025.5755

Figure Lengend Snippet: CALR regulates tumor growth and the expression of tumor-associated fibroblast activation marker proteins in mice. (A) Subcutaneous graft. (B) Size of the subcutaneous graft. (C) Staining and (D) quantification of collagen fibers by Masson's staining. (E) Staining and (F) quantification of reticular fibers. (G) Expression of (H) CALR, (I) CANX and (J) PDIA3 detected by immunofluorescent staining. Scale bar, 100 µ m. (K) Expression of (L) CALR, (M) CANX and (N) PDIA3 detected by western blotting. (O) Representative western blots. Expression levels of tumor-associated fibroblast activation marker proteins (P) α-SMA, (Q) FAP, (R) FSP1, (S) PDGFR and (T) TGF-β were detected by western blotting. ** P<0.01, *** P<0.001. CALR, Calreticulin; CANX, calnexin; PDIA3, protein disulfide isomerase A3; SMA, smooth muscle actin; FAP, fibroblast Activation Protein; FSP1, fibroblast specific protein 1; PDGFR, platelet derived growth factor receptors; sh, short hairpin; NC, negative control.

Article Snippet: A total of 20 μ g protein/lane was separated by 10% SDS-PAGE, transferred to PVDF membranes and blocked with 5% non-fat dry milk at room temperature for 1 h. The blocked PVDF membranes were incubated overnight at 4°C with primary antibodies against CALR (1:1,000; cat. no. 27298-1-AP; Proteintech Group, Inc.), CANX (1:500; cat. no. BF0515; Affinity Biosciences), PDIA3 (cat. no. 15967-1-AP; Proteintech Group, Inc.), vimentin (cat. no. bs-8533R), N-cadherin (cat. no. bs-1172R), glucose regulatory protein 78 (GRP78) (cat. no. bs-1219R; all BIOSS), α-smooth muscle actin (SMA; cat. no. Bs70000; Biogot Technology Co., Ltd.), fibroblast activation protein (FAP; all 1:1,000; cat. no. bs-5758R; BIOSS), ferroptosis suppressor protein 1 (FSP-1) (1:4,000; cat. no. 20886-1-AP), Platelet-derived growth factor receptors (PDGFR) (cat. no. 13449-1-AP; both Proteintech Group, Inc.), TGF-β (both 1:1,000; cat. no. Ab66043, Abcam).

Techniques: Expressing, Activation Assay, Marker, Staining, Western Blot, Derivative Assay, Negative Control