d609  (MedChemExpress)

Journal: Frontiers in Immunology

Article Title: Neutrophil depletion at the early stage of Japanese encephalitis virus infection affects CD8+ T cell infiltration into the mouse brain and causes severe encephalitis

doi: 10.3389/fimmu.2025.1748085

Figure Lengend Snippet: Contribution of CD8 + T-cells to control of JEV infection. (A) Schematic representation of experimental plans for CD8 depletion. IP injection of anti-mouse CD8α and Isotype (150 µg/mouse) was given before 48 h of JEV-S1 infection, and antibodies were given as depicted in the schematic figure. (B) Contour plots represent the percentage of CD4 and CD8+ cells. Lymphocyte cell counts (n=4 in each group) in peripheral blood at day 0 and day 6, depicted as a bar graph. P -values are calculated using an unpaired Student’s t-test; ***P<0.001. (C) Kaplan-Meier survival curve of isotype and anti-CD8α-JEV-S1 infected mice (n=7 in each group). (D) The change in the body weight of CD8 + T-cells depleted of JEV-S1 and isotype-infected mice. (E) The Clinical Score is shown in the CD8 + T-cells-depleted group and isotype-infected mice. The color represents a score (0–5) related to the clinical symptoms; ‘0’ represents no symptoms, and 5 represents the most severe symptoms. (F) The expression levels of NS3 and GAPDH were detected in the infected mice brains by immunoblotting.

Article Snippet: Cells were washed multiple times with PBS, Alexa Fluor 488, goat anti-mouse (1:600) (A11029; Invitrogen, Carlsbad, CA, USA) was added for one h at RT, Following multiple wash with 1X PBS, Coverslips were then mounted, and the nucleus was stained with 4′,6-diamidine-2′- pheynylindole dihydrochloride (DAPI) (D1306; Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Infection, Injection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Venom-Derived Proteins from Lonomia obliqua Modulate Cytoskeletal Regulators and Inflammatory Responses in Human Chondrocytes

doi: 10.3390/ijms27020934

Figure Lengend Snippet: Effect of LOCBE, rLOPAP, and rLOSAC on Rac-1 and F-actin. Chondrocytes (8 × 10 3 cells/well in 96-well plate) cultured in DMEM-F12 medium containing 1% FBS were treated or not with 1 ng/mL IL-1β for 1 h, followed by the addition of 50 µg/mL LOCBE, 200 nM rLOSAC, or 200 nM rLOPAP and incubation for 24 h. Cells were fixed and immunolabeled with a primary antibody against Rac-1 and an Alexa Fluor-647-conjugated secondary antibody (red) with nuclear counterstaining by Hoechst 33342 (blue) and F-actin staining by Alexa Fluor 488-phalloidin (green). The stained samples were analyzed by HCS (20× magnification) for quantification of Rac-1 ( A ) and F-actin ( B ). Data represent the mean ± SD from 16 different fields per well. # p > 0.05, * p > 0.05, ** p > 0.01, **** p > 0.0001 (unpaired Student’s t -test, n = 9 independent replicates). CTRL, chondrocytes cultivated in basal DMEM-F12 medium containing 1% FBS (dotted line as a reference). Representative images of F-actin (green), Rac-1 (red), and nuclei (blue) under basal ( C ) or inflammatory conditions ( D ). Control: untreated chondrocytes. The white arrow in the “IL-1β” panel highlights retracted and rounded cells, with yellow color indicating co-localization of Rac-1 and F-actin, an effect that was reduced in the presence of rLOPAP. Scale bar 100 μm.

Article Snippet: After three washes with PHEM-glycine buffer (PHEM containing 100 mM glycine), nuclei were stained with Hoechst 33342 (1/1000, Invitrogen #H3570) and secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen #A11029, Carlsbad, CA, USA) or Alexa Fluor 647 goat anti-rabbit IgG (1:1000, Invitrogen #A21244, Carlsbad, CA, USA) for 1 h at room temperature.

Techniques: Cell Culture, Incubation, Immunolabeling, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Venom-Derived Proteins from Lonomia obliqua Modulate Cytoskeletal Regulators and Inflammatory Responses in Human Chondrocytes

doi: 10.3390/ijms27020934

Figure Lengend Snippet: Effect of LOCBE, rLOPAP, and rLOSAC on RhoA. Chondrocytes (8 × 10 3 cells/well in 96-well plate) cultured in DMEM-F12 medium containing 1% FBS were treated or not with 1 ng/mL IL-1β for 1 h, followed by the addition of 50 µg/mL LOCBE, 200 nM rLOSAC, or 200 nM rLOPAP and incubation for 24 h. Cells were fixed and immunolabeled with a primary antibody against RhoA and an Alexa Fluor-488-conjugated secondary antibody (green), with nuclear counterstaining by Hoechst 33342 (blue). The stained samples were analyzed by HCS (20× magnification), and fluorescence intensities were quantified in nuclear and cytoplasmic compartments under normal ( A , C ) and inflammatory ( B , D ) conditions. Data represent the mean ± SD from 16 different fields per well. * p > 0.05, *** p > 0.001 (unpaired Student’s t -test, n = 6 independent replicates). CTRL, chondrocytes cultivated in basal DMEM-F12 medium containing 1% FBS. Representative images of RhoA (green) and nuclei (blue) of chondrocytes under basal ( C ) or inflammatory conditions ( D ). Control: untreated chondrocytes. Zoomed-in panels highlight the more intense RhoA staining in the cytoplasm. The white arrow in the “IL-1β” zoomed-in panel indicates the presence of RhoA in the cytoplasm. Scale bar 100 μm.

Article Snippet: After three washes with PHEM-glycine buffer (PHEM containing 100 mM glycine), nuclei were stained with Hoechst 33342 (1/1000, Invitrogen #H3570) and secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen #A11029, Carlsbad, CA, USA) or Alexa Fluor 647 goat anti-rabbit IgG (1:1000, Invitrogen #A21244, Carlsbad, CA, USA) for 1 h at room temperature.

Techniques: Cell Culture, Incubation, Immunolabeling, Staining, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Venom-Derived Proteins from Lonomia obliqua Modulate Cytoskeletal Regulators and Inflammatory Responses in Human Chondrocytes

doi: 10.3390/ijms27020934

Figure Lengend Snippet: Effect of LOCBE, rLOPAP, and rLOSAC on β-catenin and Rab9. Chondrocytes (8 × 10 3 cells/well in 96-well plate) cultured in DMEM-F12 medium containing 1% FBS were treated or not with 1 ng/mL IL-1β for 1 h, followed by the addition of 50 µg/mL LOCBE, 200 nM rLOSAC, or 200 nM rLOPAP and incubation for 24 h. Cells were fixed and immunolabeled with a primary antibody against β-catenin and an Alexa Fluor-647-conjugated secondary antibody (red), and a primary antibody against Rab9 with an Alexa Fluor-488-conjugated secondary antibody (green), with nuclear counterstaining by Hoechst 33342 (blue). The stained samples were analyzed by HCS (20× magnification) for quantification of β-catenin ( A ) and Rab9 ( B ). Data represent the mean ± SD from 16 different fields per well. # p > 0.05, * p > 0.05, *** p > 0.001, **** p > 0.0001 (unpaired Student’s t -test, n = 3 independent replicates). CTRL, chondrocytes cultivated in basal DMEM-F12 medium containing 1% FBS (dotted line as a reference). Representative images of β-catenin (red), Rab9 (green), and nuclei (blue) in chondrocytes under basal ( C ), or inflammatory conditions ( D ). Control: untreated chondrocytes. The white arrows in the “rLOSAC” and “rLOPAP” panels highlight retracted and rounded cells, with yellow color indicating the internalization of β-catenin. In samples treated with rLOSAC and rLOPAP, β-catenin is more intense at the cell junctions. Scale bar 100 μm.

Article Snippet: After three washes with PHEM-glycine buffer (PHEM containing 100 mM glycine), nuclei were stained with Hoechst 33342 (1/1000, Invitrogen #H3570) and secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen #A11029, Carlsbad, CA, USA) or Alexa Fluor 647 goat anti-rabbit IgG (1:1000, Invitrogen #A21244, Carlsbad, CA, USA) for 1 h at room temperature.

Techniques: Cell Culture, Incubation, Immunolabeling, Staining, Control

Journal: Journal of Extracellular Vesicles

Article Title: Cholesterol Deficiency Directs Autophagy‐Dependent Secretion of Extracellular Vesicles

doi: 10.1002/jev2.70218

Figure Lengend Snippet: Increased sEV secretion in cholesterol‐depleted conditions is due to autophagy‐driven signalling. (A) Representative images for CD63 and LC3B expression in HEK293T or HEK293T ATG2A/B KO cells after vehicle, AY9944 or rapamycin and chloroquine (Rap & CQN) treatment. Scale bar, 25 µm. (B) Quantified CD63 expression in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8 images analyzed from 2 independent experiments). Two‐way ANOVA (treatment effect: F2,42 = 47.99, p < 0.0001; Autophagy effect: F1,42 = 5.422, p ≤ 0.0248). Sidak's multiple comparisons test (** p ≤ 0.005, **** p < 0.0001). (C) LC3B expression in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8, 4 images analyzed from 2 independent experiments). Two‐way ANOVA (interaction effect: F2,42 = 6.126, p ≤ 0.0046; treatment effect: F2,42 = 5.995, p ≤ 0.0051; Autophagy effect: F1,42 = 12.65, p ≤ 0.0009). Sidak's multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001). (D) Immunoblot for LC3B in isolated sEVs upon cholesterol depletion. (E) CD63 and LC3B colocalization in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8 images analyzed from 2 independent experiments). Two‐way ANOVA (interaction effect: F2,53 = 128.9, p < 0.0001; treatment effect: F2,53 = 157.0, p < 0.0001; autophagy effect: F1,53 = 453.1, p < 0.0001). Sidak's multiple comparisons test (**** p < 0.0001). (F) Quantified sEV secretion in HEK293T or HEK293T ATG2A/B KO cells following vehicle, AY9944, or Rap & CQN treatments (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One‐way ANOVA (F2,6 = 56.17, p ≤ 0.0001), Dunnett's multiple comparisons test (** p ≤ 0.01, **** p < 0.0001). (G) sEV secretion in HEK293T or HEK293T ATG2A/B KO cells following vehicle or D609 treatments (mean ± SEM; n = 3 biological replicates from 3 independent experiments). Unpaired t‐test (HEK293T: t4 = 9.395, *** p ≤ 0.0007; HEK293T ATG2A/B KO: t 4 = 4.069, ** p ≤ 0.0152).

Article Snippet: For FBS rescue experiments, HEK293T cells were treated with vehicle, simvastatin or AY9944 for 24 h. Media was then replaced with 10% EV‐depleted FBS for 24 or 48 h. Other pharmacological agents utilized in these studies include rapamycin (1 μM, Selleck Chemicals; mTOR inhibitor), chloroquine (10 μM, Cayman Chemical; inhibits autophagosome‐lysosome fusion), D609 (100 μM, MedChemExpress; phosphatidyl choline phospholipase C inhibitor), and Q‐VD‐Oph (10 μM, Selleck Chemicals; pan‐caspase inhibitor).

Techniques: Expressing, Western Blot, Isolation