Ix71 microscope

To measure fusion-from-without (FFWO) between CEM.CCR5/shRNA cells, cells were suspended in OPTI-MEM. One-half of cells were labeled with 2 μM CellTracker^TM Orange (CMRA), while the second half was loaded with 1 μM CellTracker^TM Green (CMFDA). Differently labeled cells were washed to remove residual dye, mixed at a 1:1 ratio, and transferred to a U-bottom 96-well plate (1.5 × 10⁵ cells/well). Viruses (MOI = 10) were bound to cells by centrifugation at 1550× g at 4 °C for 30 min. Unbound virus was removed, and the samples were incubated in a growth media for 2 h at 37 °C, 5%CO₂. The cells were then placed on ice, washed with cold PBS, suspended in live-cell imaging buffer/2% FBS, and adhered to poly-l-lysine-coated 8-chamber cove slips (Lab-Tek, Nunc, Waltham, MA, USA) for 10 min at 4 °C prior to imaging on an Olympus IX-71 microscope equipped with an EM-CCD camera (Hamamatsu, C9100-12, Shizuoka, Japan). The fraction of effector and target cells that fused was determined by counting the number of cells positive for both dyes using ImageJ software (National Institutes of Health, Bethesda, Rockville, MD, USA).

Human induced pluripotent stem cell (iPSC)-derived motor neurons were purchased from iXCells Biotechnologies and plated at a density of ∼86 000 cells/cm² in 8-well chamber slides coated with 80 µg/µL Matrigel in DMEM/F-12. Motor neurons were cultured in Motor Neuron Maintenance Medium (iXCells Biotechnologies) for 8 days. On Day 8, human ApoB (Millipore), human MOG (Sigma-Aldrich), human haptoglobin (abcam), human ApoC-III (Athens Research & Technology), and human ApoE (Novus Biologicals) proteins were applied at 0.01 µg/µL or 0.05 µg/µL in media, or 50% sALS CSF and ApoB-depleted sALS CSF diluted in media were applied. In a separate ApoB titration experiment, lower doses of 0.005 ng/µL or 0.01 ng/µL ApoB were applied. At 24 h pre- and post-treatment, cells were visualized using a 10X objective on an Olympus IX71 microscope and bright field images were captured on a Hamamatsu Orca-Spark camera using Cell Sens Dimension Programme. Motor neurons were fixed with 4% PFA for immunocytochemistry at 24 h post-treatment. Using ImageJ, areas of ChAT⁺ motor neuron clusters were quantified from three images per well and then averaged.

The cells were fixed with 4% paraformaldehyde for 15 min. After several washes, they were blocked with 0.01M phosphate-buffered saline supplemented with 1% BSA and 0.3% Triton X-100 for 30 min. Primary antibodies (mouse anti-LAP2α, 1:500, Abcam, UK; mouse anti-betaIII tubulin, 1:500, Abcam; mouse anti-NeuN, 1:1000, Abcam; rabbit anti-NeuN, 1:200, Abways; rabbit anti-Tbr1, 1:500, Abcam; mouse anti-γH2AX, 1:250, Millipore, USA; mouse anti-Lamin A/C, 1:200, Abcam; rabbit anti-H3K9me3, 1:4000, Abcam; mouse anti-6E10, 1:200, Abcam) were diluted with blocking buffer and applied overnight at 4° C. The cells were stained with suitable Alexa Fluor–labeled secondary antibodies (Invitrogen) in blocking buffer at a concentration of 1:500 for 1.5 h at room temperature. 4', 6-Diamidino-2-phenylindole (Thermo Fisher, IL, USA) was applied to counterstain cells for visualizing nuclei at 1:1000 in Milli Q water (Biocel, Millipore, USA). The images were obtained with an Olympus IX71 microscope using a Hamamatsu ORCA CCD camera and Leica TCS SP5.

HEK293T cells transfected with sodium channels (rNa_V1.4 and fNa_V1.4) were incubated with F-BmK-M9 for 1 h in 2 mM Ca²⁺ Ringer's solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 10 mM Glucose, 2 mM CaCl₂, and 10 mM HEPES, pH = 7.4) before fluorescence imaging recordings. Fluorescence images of HEK293T cells incubated with F-BmK-M9 were acquired by using an Olympus IX-71 microscope with a Hamamatsu R2 camera controlled by MetaMorph software. F-BmK-M9 was excited by a LED light source (X-Cite 120LED, Lumen Dynamics) with a 560 nm excitation filter, while fluorescence emission was detected by a 590 nm emission filter.

MDA-MB-231, MDA-MB-468, SUM1315, HCC1937 or MDA-MB-468-PTX^R cells were seeded at a density of 5–7 × 10³ cells per well in 96-well plates and treated with vehicle or 0.1–100 nM PTX. To assess the effect of RAD6 inhibition on PTX sensitivity, cells were treated with 0.75 µM SMI#9 alone or together with PTX. Cell viability was assessed at 72 h posttreatment by MTT assays. Experiments were performed in triplicates and results presented are representative of three independent experiments. Phase contrast images were taken at 24–48 h posttreatment with the Olympus IX71 microscope equipped with a Hamamatsu digital camera. PTX and SMI#9 interaction was determined using CompuSyn software (Combosyn Inc., Paramus, NJ, USA) and combination index (CI) values calculated: CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively [43 (link), 44 (link)].