Modified boyden chamber

Human iPSC lines used in this study are: FUS^WT and FUS^P525L (ref. [39 (link)]); FMR1 KO [40 (link)]; SYN1::HuD [21 (link)]; KOLF WT 2 and P525L16 (LL FUS-eGFP) [49 (link)]. As indicated in the original studies, informed consent had been obtained from all patients involved prior to cell donation. Cells were regularly tested for mycoplasma contamination. Cells were maintained and induced to differentiate into MNs as described [21 (link)]. At day 5, MN progenitors were dissociated with Accutase (Thermo Fisher Scientific, Waltham, MA, USA) and plated on Matrigel (BD Biosciences Franklin Lakes, NJ, USA)-coated dishes or in modified Boyden chambers (Merck, Darmstadt, Germany) 6-well hanging inserts 1.0 µm PET. On day 12, each inner cell culture was washed with PBS w/o Ca²⁺/Mg²⁺ and scraped using a cell lifter to collect the soma compartment. For neurites isolation, the membrane was peeled off with a cutter, pulled into a 2.0 ml tube, lysed with TRK lysis buffer of the Micro Elute Total RNA Kit (VWR International PBI, Milan, Italy) and left rotating on a wheel for 20 min at room temperature before RNA extraction.

To measure the migratory ability of MG63 and U2OS cells, migration assays were performed using modified Boyden chambers (Merck & Co., Inc., Kenilworth, NJ, USA). A total of 1 × 105 cells in 0.2 mL serum-free DMEM treated with various factors were plated in the upper room of each chamber while the lower room was filled with 0.6 mL DMEM supplemented with 10% FBS. After incubating for 24 h at 37 °C, cells on the upper compartments were removed, whereas the migrated cells in the lower parts were stained, observed, and counted under a high-power microscope.

Cells transfected with miR-Ctrl/miR-146a, or nonrelative control moleculars (NC)/antagomiR-146a were plated in 24-well culture plates at a density of 10⁵ or 2 × 10⁵ (Huh-7, hepG2) cells per well, and incubated for 24 h or 48 h (Huh-7, hepG2) to reach confluence. Using a 200 μl tip, a wound was made in the monolayer (at time 0). The cells were then washed with PBS and incubated. The distance between the two sides of the wound was measured with an Olympus CX71 microscope (Olympus). The distance between the two sides of the wound after 20-72 h of migration was divided from the distance at time 0 and represented on a graph.
The invasion abilities of cultured cells were measured using an in vitro transwell assay with modified Boyden chambers containing polycarbonate filters (Millipore, MA), according to the manufacturer’s instructions. Cells transfected with miR-146a/miR-Ctrl, or antagomiR-146a/nonrelated control molecules (NC) were plated 24 h after transfection in serum-free medium and allowed to invade towards a 10% FBS medium for 24 h, or 48 h. Cells that remained on top of the filter were scrubbed off, and those that invaded the underside of the filter were fixed and stained with crystal violet.