Dragonfly 200

Generally, imaging was performed at 12‒36 h post-transfection. Images were acquired with the Dragonfly 200 (Andor, Dragonfly 200) using a Sona camera (Andor, SONA-4BV6U) and a Plan APO λ 100× / 1.45 Oil ∞ / 0.17 WD 0.13 objective (Nikon, CFI Plan Apochromat Lambda 60XC) mounted on a Leica DMi8inverted 20 microscopes. A 200-mW solid-state 405-nm laser and 445/46-nm BP emission filter were applied for BFP. GFP images were acquired with a 150-mW solid-state 488-nm laser and 521/38-nm BP emission filter. Red (tdTomato) images were acquired with a 150-mW solid-state 561-nm laser and 594/43-nm BP emission filter. Purple (Halo) images were acquired with a 150-mW solid-state 637-nm laser and 698/77-nm BP emission filter. Unless specifically indicated, Z-stack images were captured at a 0.2 µm step size to include all foci of each nucleus examined. For XYZ-t 4D imaging, cells were incubated in a Bold Line Cage Incubator (Okolab, H201-PRIOR-H117) equipped with a Dragonfly 200 at 37 °C and 5% CO₂.

Cy3-labeled KCNQ1OT1 and FAM-labeled miR-421-3p probes were designed and produced by GenePharma (Shanghai, China). RNA-FISH was performed using an RNA FISH SA-Biotin Amplification System Kit (GenePharma, China) according to the manufacturer's instructions. Nuclei were stained using DAPI. Finally, using the Spinning Disc Laser Confocal Microscope (Dragonfly 200, Oxford Instruments Andor, USA), it was possible to determine the locations of the lncRNA KCNQ1OT1 and the miR-421-3p in MC3T3-E1 cells.

Human bronchial ring sections were imaged using a Leica DMi8 microscope equipped with an Andor Dragonfly 200 spinning disk confocal and a 40× water objective (NA 0.80). From every stained ring section, 3 to 8 FOVs with a size of 2048 × 2048 pixels were recorded. Rat samples, other human airway sections, and in vitro cultures were imaged using a Leica confocal scanning microscope or a Zeiss Axioscope 7 fluorescence microscope equipped with a 40× oil DIC objective (NA 1.4). Six FOVs with a size of 2048 × 2048 pixels were recorded per sample.

Organoids were thoroughly removed from Matrigel with Cell Recovery Solution (Corning) on ice before centrifugation at the speed of 800 rpm. 4% paraformaldehyde was used to fix the organoids overnight at 4 °C. Immunofluorescence proceeded as outlined above. Microscopic observation was performed using a high-speed confocal platform (Andor, Dragonfly 200).

We performed cell imaging with an inverted microscope (Ti-2, Nikon) equipped with a confocal unit (Dragonfly 200, Andor Technology), a microscope objective (CFI Plan Apochromat λ 60 × Oil; numerical aperture, 1.40; Nikon), and a stage-top incubator with 5% CO₂ supply (STXG-WSKMX, Tokai Hit). We used a 445 nm laser for excitation and collected the fluorescence emission through bandpass filters (ET480/40 nm and ET540/30 nm for mT and mV, respectively; Chroma). We captured the fluorescence images with an EMCCD camera (iXon Ultra, Andor Technology). The exposure time was 500 ms, and the binning size was 2 × 2 pixels. We measured fluorescence ratios of mV/mT cell by cell to calculate the average values and the standard deviation.

PARylation reactions were set up as described above and transferred onto 35mm-diameter glass-bottom dishes (MatTek) and sealed with a glass coverslip to limit evaporation. Images were acquired on a Nikon Ti-2E microscope with an Andor Dragonfly 200 unit using a 40x oil immersion objective (DIC channel).

Embryos were laid on apple juice agar plates for approximately 1 h and embryos were collected and dechorionated in 50% bleach solution (2.5% final concentration of sodium hypochlorite solution diluted in water). Preparation of embryos for live imaging was performed as described,⁷⁷ (link) with embryos mounted onto a heptane glue coated coverslip (Scientific Laboratory Supplies, Cat# MIC3110) and inverted over a coverslip bridge in a 7:1 ratio mix of 700:27 halocarbon oil (Sigma, Cat# H8773 and Cat# H8898) on the membrane of a Lumox dish (Sarstedt AG & Co, Cat# 94.6077.305). Images were collected on an Andor Dragonfly200 spinning disk upright confocal microscope with a 40x/1.30 HCL pL Apochromat objective. Samples were excited using 488nm (11%; sogMS2, gtMS2 and mewMS2 or 13%; hntMS2) and 561nm (6%) diode lasers via Leica GFP and RFP filters respectively. Images were collected simultaneously using dual camera imaging with Zyla 4.2 Plus sCMOS (2048 X 2048) and iXon EMCCD camera (1024 X 1024) with a gain of 180 and binning [2X and 1X respectively] for 130ms. For each movie a total of 50 Z stacks at 0.7μm spacing were collected using the fastest setting yielding a total Z size of 35 μm at a time resolution of between 20 and 25 s on average.