Cytochalasin d

After preliminary experiments to determine cell toxicity (Supplementary MaterialsFigure S2), final concentrations of cytochalasin D (Sigma-Aldrich), an inhibitor of actin polymerization, were employed at 0.25, 0.5 and 1 µM. Each concentration of cytochalasin D was incubated in the culture medium 30 min before the addition of secreted melanosome clusters [43 (link)]. After 2 days of incubation, the cells were washed twice with D-PBS and were harvested by trypsinization. The cells were centrifuged and the contents of internalized secretory melanosome clusters adjusted per cell number were measured spectrophotometrically [13 (link)].

ATTO-550-labeled α-Synuclein monomer was prepared as previously described [12 (link)]. 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), was purchased from Sigma and prepared as 10 mM stock in dimethyl sulfoxide (DMSO, FUJIFILM Wako Pure Chemical). Cultured neurons were either treated or not treated with 1 μM ATTO-550-labeled α-Synuclein monomer and 10 μM MPP⁺ for 48 h. To examine the sensitivity of FABP3-dependent uptake of α-Synuclein to cytochalasin D, which is an inhibitor of pinocytosis [23 (link)], we exposed cultured mesencephalic neurons to 2 μM cytochalasin D (Merck, Sigma-Aldrich, St. Louis, MO, USA).

To assess parasite transfer between parasite-exposed cells and naive cells, B cells or macrophages were isolated or differentiated from both wild-type C57BL/6 mice expressing CD45.2 and from CD45.1 congenic mice as described above. The cells were seeded in 24-well plates at a concentration of 1 × 10⁶, and the wells containing cells from one allelic variant were exposed to PKH67-labeled LV9 amastigotes for 1 h for B cells or overnight in the case of macrophages, thoroughly washed using sterile PBS for a minimum of three times until there was no presence of free parasite visible under the microscope, and then further incubated with naive cells of the other allelic variant for 2.5 or 5 h before preparation of samples for flow cytometry or confocal microscopy. For experiments using cytochalasin D, the parasite-exposed cells were treated with 1 or 10 μM cytochalasin D (Sigma) for 1 h before further coincubation with the initially nonexposed cells of the other allelic variant for 2.5 or 5 h. The absence of free parasites in the supernatant was further investigated by subjecting naive cells to the supernatants of exposed cells after washing.

For SCFS in presence of a β1 integrin blocking antibody (clone AIIB2, DSHB) or β1 integrin activating antibody (clone 12G10, ab30394, abcam) cells were incubated with a 1:100 (vol/vol) dilution (in SCFS medium) for at least 30 min prior to the experiments in SCFS medium. For cell–ECM SCFS AIIB2 was present throughout the experiments at a 1:1000 (vol/vol) dilution in SCFS medium. For cell–cell adhesion experiments 12G10 was present throughout the experiments at a 1:1000 (vol/vol) dilution in SCFS medium. For SCFS in the presence of Mn²⁺ or EGTA, suspended cells were incubated with 0.5 mM MnCl₂ or 7.5 mM EGTA in SCFS medium for at least 30 min and the chemicals were present throughout the experiments. For inhibition of RhoA cells were incubated with 0.5 µg ml^–1 Rho inhibitor I (# CT04, Cytoskeleton) in the culture flask for 4 h prior to their detachment. The inhibitor was present during the recovery of the detachment and during SCFS. For cytochalasin D, suspended cells were incubated with 0.1, 0.5, or 1 µM cytochalasin D (C8273, Sigma Aldrich) in SCFS medium for at least 30 min prior to the experiments. The inhibitor was present throughout the experiments.

Vesicle shuttle experiments were carried out as previously reported 6 (link). In brief, caput epididymis from WT (n=5) and CB1^-/- (n=5) mice were separately pulled in PBS (pH 7.6) and cut to let SPZ flow out from the ducts. Then, caput SPZ samples were filtered throughout cheesecloth to eliminate fragments of epididymal tissue and centrifuged at 1500 × g for 30 min at 4°C. The resulting caput fluid was further clarified via centrifugation (16000 × g for 30 min at 4°C) with the supernatant yielding the ELF. The resulting caput SPZ pellet and caput ELF were used for in vitro treatments as follows.
For each experimental group, 10 × 10⁶ of WT SPZ from caput epididymis were incubated for 15 min at 37°C in 1 ml of: 1) WT Caput ELF (CTRL group); 2) WT Caput ELF combined with Cytochalasin-D (C8273; Sigma-Aldrich, Milano, Italy) at the concentration of 10 μM; 3) CB1^-/-Caput ELF; 4) CB1^-/-Caput ELF combined with Cytochalasin-D 10 μM. After treatment, SPZ were centrifuged at 1500 × g for 20 min at 4°C and washed twice with PBS. Sperm pellets were stored at -80°C for RNA extraction and dried on slides to be finally stored at -20°C for immunofluorescence analysis.

Ficoll-Paque was obtained from Pharmacia (Uppsala, Sweden). Fibronectin was from Calbiochem (La Jolla, San Diego, CA, USA). Bicarbonate-free Hank’s solution, Ca2⁺-free Dulbecco PBS, cytochalasin D, minoxidil, doxycycline, wortmannin, Akt 1/2 inhibitor, cytochalasin D, latrunculin A, blebbistatin, staurosporine, 4-bromophenacyl bromide, and E64 were obtained from Sigma (Steinheim, Germany). Analytical chromatography conditions: eluent MCI Buffer L-8800-PH-1–4 and ninhydrin coloring solution kit for Hitachi 29,970,501 (Wako Chemicals, North Chesterfield, VA, USA). Coomassie Brilliant Blue G-250 was obtained from Serva, PMSF from MP Biomedical, trypan blue from Fluka AG, trypsin from Promega, glutaraldehyde from Ted Pella, carboxy-H₂DCF-DA from Molecular Probe, (Eugene, OR, USA).