Glass bottomed confocal dishes

Laser microirradiation was carried out on an inverted two-photon microscope (LSM780; Carl Zeiss) equipped with an inverted Axio Observer.Z1 stand, motorized scanning stage, and integrated laser microbeam system, with total UV laser output set to 750 nm (8%). Cells cultured on glass-bottomed confocal dishes (SPL Life Sciences) were subjected to laser microirradiation in a temperature-controlled (37 °C) environmental chamber supplied with 5% CO₂ at 24 h after transient transfection with GFP-tagged indicated plasmids. Time-lapse images were acquired by ZEN 2012 (Carl Zeiss) software with a Plan Apochromat 40×/1.4 oil differential interference contrast (DIC) M27 objective and further processed by ImageJ software to analyze mean fluorescence intensity (MFI) across the laser-microirradiated regions. MFI was quantified as the difference between the average fluorescence intensity in the laser-microirradiated regions versus the average fluorescence intensity from adjacent undamaged regions of the same size in the same nucleus.

Nascent transcription at laser-microirradiated sites was detected by a Click-iT RNA Alexa Fluor 594 Imaging Kit (C10330; Thermo Fisher Scientific). Briefly, cells grown on glass-bottomed confocal dishes (SPL Life Sciences) for 24 h were subjected to laser microirradiation by a live Carl Zeiss LSM780 inverted confocal microscope (10× objective) with a 750-nm laser (8% output). Subsequently, cells were cultured with complete media containing 1 mM 5-EU for 1 h after laser microirradiation. 5-EU labeling was performed following manufacturer instructions. Cells were immunostained for γH2AX and nuclei were counterstained with DAPI before mounting. Images were captured by Olympus BX51 fluorescence microscope (PlanApo N 60×/1.42 oil-immersion objective). ImageJ was used to analyze relative fluorescence intensity across the laser-microirradiated stripes. An analyzing line drawn by a line tool was perpendicular to the damaged stripes, centered at the stripes with two ends at undamaged regions, and the fluorescence intensity was multiplotted. Relative fluorescence intensity was normalized to each end from the same cell.

Primary culture of hippocampal neurons was prepared from embryonic day 18–19 Sprague-Dawley rats (Laboratory Animal Unit, The University of Hong Kong, accredited by Association for Assessment and Accreditation of Laboratory Animal Care International) as previously described. All experimental protocols involving animals were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) at LKS Faculty of Medicine, HKU. Briefly, hippocampi were isolated and mechanically dissociated in ice-cold phosphate buffered saline (Life Technologies) supplemented with glucose (18 mM, Sigma-Aldrich). Neurons were plated onto poly-L-lysine (25 μg/ml, Sigma-Aldrich) coated glass-bottomed confocal dishes (SPL Life Science) and MatTek (MatTek) culture dishes at a density of 1.5×10⁵ cells per dish, or at 5×10⁴ and 4.5–6×10⁵ cells per well for 12-well and 6-well plates (Iwaki), respectively. Hippocampal neurons were cultured with Neurobasal^® medium (Life Technologies) supplemented with B-27^® supplement (2%, Life Technologies), GlutaMAX™ (2 mM, Life Technologies), Penicillin/Streptomycin (100 U and 100 mg/ml, Life Technologies) and β-mercaptoethanol (10 μM, Sigma-Aldrich) for 7–9 days at 37 °C in a humidified 5% CO₂ atmosphere. 5-Fluoro-2′deoxyuridine (2 μM, Sigma-Aldrich) was added after one day in culture to inhibit glial proliferation.

For hydrogel droplet formation, the recombinant proteins of mCherry-linked LC domains of hnRNPH1, FUS, TAF15, hnRNPA2, hnRNPA1, hnRNPF, and DHX9 were dialyzed in gelation buffer (20 mM Tris⋅HCl pH7.5, 200 mM NaCl, 20 mM BME, and 0.1 mM PMSF). Dialyzed protein solution was subjected to brief sonication for 1 s at 3% power for three times. Upon removal of the precipitates by centrifugation for 3 min at maximum speed, the protein solution was concentrated to 80 to 100 mg/mL using Amicon Ultra (Merck Millipore). Small droplets (0.5 µl) of the protein solution were deposited on to a glass-bottomed confocal dishes (SPL, Korea). The confocal dishes were sealed with parafilm (Bemis) and incubated for 1 wk at room temperature until protein solution adopted a gel-like state. For hydrogel-binding assays, solutions of GFP-linked proteins at low concentration (1 or 3 µM) were applied to the confocal dishes containing different kinds of mCherry hydrogel droplets. Upon overnight incubation at 4 °C, trapping of the GFP-linked proteins by mCherry hydrogel droplets was analyzed by confocal microscopy (LSM 510, Zeiss, Germany).