Cytochalasin d cytod

Hyaluronidase (Hyal; Worthington Biochemical Corp., Lakewood, NJ, USA) was diluted to 7,500 U/mL in sterile PBS. Bovine cartilage sections were incubated in 50 μL of 7,500 U/mL Hyal solution for 30 min. Hyaluronic acid (HA) digestion was confirmed by fluorescent localization of HA using a biotinylated Hyaluronic Acid Binding Protein (HABP; Calbiochem, Billerica, MA, USA). Control and Hyal-incubated sections were labeled with HABP (1:300) and streptavidin-488 (1:500; Life Technologies, Grand Island, NY, USA) to visualize HA content and DAPI (1:500; Roche, Mannheim, Germany) to identify nuclei.
Cytochalasin D (Cyto D; 5 mg/mL in dimethyl Sulfoxide, Sigma-Aldrich, St. Louis, MO, USA) was diluted to 0.5 mg/mL in PBS. 50 μL of the diluted CytoD was used to treat the bovine cartilage for 30 min. Filamentous actin disruption was confirmed by fluorescence microscopy for phalloidin-555 (1:100; Life Technologies, Grand Island, NY, USA) on undigested control and digested sections, in addition to DAPI (1:500) to identify cell locations.
Control and treated sections were imaged with a Zeiss LSM 710 microscope at Purdue University’s Life Science Fluorescence Imaging Facility using a 63× oil objective. All the imaging parameters were exactly the same for the control and treated sections.

The pH-sensitive green fluorophore-tagged Escherichia coli (E. coli) bioparticles (pHrodo Green E. coli BioParticles Conguates, # P35366, Invitrogen Corporation, Frederick, MD, USA) were used to measure the phagocytosis activity. The E. coli bioparticles are nonfluorescent at neutral pH. When the microglia engulf the bioparticle, phagocytic cargo presents early phagosomes of higher pH, which sequentially mature into late phagosomes, becoming increasingly acidic and finally fusing with the lysosomes for degradation and clearance. Briefly, BV2 microglial cells (5 × 10⁵/well) were plated in 6-well plates and cultured for 24 to 72 h. A total of 100 μg of pHrodo Green Escherichia coli (E. coli) BioParticles Conguates were added per condition and incubated with BV2 cells for 30 min at 37 °C. Phagocytosis was inhibited with 10 μM cytochalasin D (Cyto. D; #SI-C8273, Sigma-Aldrich), which was added 30 min before the addition of PHrodo E. coli bioparticles as a negative control. Cells were gated based on forward scatter (FSC)/side scatter (SSC) properties by Novocyte flow cytometry (ACEA Biosciences, CA, USA). Then adjusted threshold to eliminate debris, and the percentage of the pHrodo + cell was determined.

An antibody to DLD peptide sequence of gB (anti-DLD) [10 (link)], rabbit antibodies to the RGD-containing sequence of gB (anti-RGD) [19 (link)], rabbit antibodies to the C-terminal domain in gB (anti-gB-C) [19 (link)], rabbit anti-gB antibodies [11 (link)], and rabbit anti-gH antibodies [20 (link)] were used in this study. Polyclonal sheep antibodies to PIKfyve (R&D systems, Minneapolis, MN) and polyclonal rabbit antibodies to β-actin (Cell Signaling, Beverly, MA) were used in the Western blotting experiments. Cytochalasin D (Cyto-D) and Rac-1 inhibitor, NSC23766, purchased from Sigma-Aldrich, St. Louis, MO were used in this study. His-tagged, recombinant and soluble KSHV gBΔTM [21 (link)], gBΔTM lacking the RGD (gBΔTM-RGA; referred to as gBΔTMΔR) [21 (link)], and gBΔTM lacking the DLD (gBΔTMΔD) [10 (link)] were expressed and purified from Sf9 cells as per earlier studies [10 (link)]. Vascular endothelial growth factor (VEGF) purchased from R&D Systems [18 (link)] was used as a positive control in MTT assay.

Ammonium chloride (NH₄Cl), chloroquine (CQ), dynasore, and cytochalasin D (Cyto D) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), nystatin, Cat Bi (Cathepsin B inhibitor, CA-074Me), chlorpromazine (CPZ), Cat Li (3-epiursolic acid), and methyl-β-cyclodextrin (MβCD) were purchased from MCE Corporation (New Jersey, USA). Antibodies against dynamin II, clathrin heavy chain (CHC), and caveolin-1 were obtained from Cell Signaling Technology (Danvers, MA, USA). The rabbit polyclonal antibody against the BPIV3 HN protein was prepared in our laboratory. The rabbit monoclonal antibody against vesicular stomatitis virus (VSV) G protein was purchased from Abways Technology (Shanghai, China). Alexa Fluor 555-conjugated cholera toxin subunit B (CTB-AF555) was purchased from Invitrogen (Carlsbad, CA, USA).

Human promyelocytic leukemia HL60 cells were used as a model cell line for monocytes. The cells were obtained from the European Collection of Cell Cultures (ECACC) and cultured in RPMI 1640 medium (Invitrogen, The Netherlands) with 2 mM L-glutamine (Lonza, Belgium) and 10 % Fetal Bovine Serum (Greiner, The Netherlands). Cell cultures were maintained at a concentration between 1−9 × 10⁵ cells/ml at 37 °C in a humidified atmosphere containing 5 % CO₂. Cell differentiation towards monocytic lineage was stimulated by treatment with 0.4 mM Sodium Butyrate (Sigma, The Netherlands), for 4 days at culturing conditions prior to experiment. Cell activation was induced by incubation with 2 μg/ml Lipopolysaccharides (LPS, Sigma, The Netherlands) for 15 min at 37 °C.
For the validation experiment, non-treated HL60 cells (NT) were stimulated by diffusion of Cytochalasin-D (CytoD, Sigma, The Netherlands) through the porous membrane from the upper stimulus channel to the lower cell channel. For the demonstration of the application of the device, both activated cells (LPS) and monocytes from patients affected by atherosclerosis were stimulated by diffusion of Pentoxyfilline (PTX) through the porous membrane.

Palmitate (Sigma) was dissolved in ethanol and was stored as 50 mmol/L solution at −20°C. To assess the effects of FFAs, Palmitate was pre-added to 50 µL culture medium, heated to 70°C for complete dissolution, and then added the whole 50 µL mixture to the cells. Cytochalasin D (CytoD) (Sigma) was dissolved in DMSO (dimethyl sulfoxide) and stored as 2 mmol/L solution at −20°C. Latrunculin B (LatB) (Millipore) was dissolved in DMSO and stored at 4 mg/mL solution at −20°C. A final concentration of 1 µg/mL of recombinant human CTGF protein (Gemini) was used to treat β-cells under cultured conditions.