Szx10 microscope

To collect fruit flies, we used sets of three traps made of modified urine sample cups (described in Leblanc et al. 2015a ), separately baited with the fruit fly lures methyl eugenol, cue-lure and zingerone, with a 10×10mm piece of dichlorvos insecticide strip to kill trapped flies. These traps were maintained for three to five days at each of 220 sites in forest reserves and national parks in 2015 and 2017 (Figure 1). Specimens retrieved from the traps were preserved in 95 % ethanol and stored in a -20 °C freezer when returned to the laboratory. A selection of specimens was dried and pinned. We pinned these specimens fresh out of the ethanol each with a minuten pin through the scutum, then soaked them in ethyl-ether for 3–12 hours to preserve the coloration, and double-mounted them. We photographed specimens using a Nikon D7100 camera attached to an Olympus SZX10 microscope. Pictures from different focal plains were merged using Helicon Focus pro v6.7.1. Initially, we performed identifications based on morphology, using available keys (Drew and Hancock 1994 (link), Drew and Romig 2016 ). Measurements were taken using an ocular grid mounted on an Olympus SZ30 dissecting microscope. The collecting and taxonomy information for all specimens can be found at BOLD http://dx.doi.org/10.5883/DS-VIETDACI.

Samples were fixed overnight in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Sorenson phosphate buffer at pH 7.3. For bright-field imaging, samples were washed in PBS and imaged using an Olympus SZX10 microscope. For light microscopy, samples were post-fixed in 1% osmium tetraoxide and dehydrated in gradient ascending series of ethanol concentrations prior to Epon 812 resin embedding overnight. 1 µm sections were prepared using a Leica EM UC6 microtome and glass knife, mounted on glass slides and stained with toluidine blue. Prepared sections were imaged by a Leica DMLB bright field illumination microscope and Leica DFC 480 camera. The number of dying cells in the ciliary marginal zone (CMZ) was quantified by recording pyknotic nuclei present in sections 5 µm apart surrounding the optic nerve. The area of the CMZ was measured in central retinal sections using the polygonal selection tool in imagej, morphology data was analysed using an ordinary one-way ANOVA. The area of the CMZ was determined according to the criteria in Raymond, et al.^{69 (link)}. For transmission electron microscopy (TEM), 0.1 µm sections were prepared using a Leica EM UC6 microtome and diamond knife, transferred to a support grid, contrasted with uranyl acetate and lead citrate, and analysed on a FEI-Tecnai 12 BioTwin transmission electron microscope (FEI Electron Optics).

Adult wings were mounted in Gary’s Magic Mount (Canada balsam [Sigma] dissolved in methyl salicylate [Sigma]). Images were taken with an Olympus SZX10 microscope with an Olympus DP21 camera using the CellSens Dimension software (Olympus) or with an Echo Revolve R4 microscope and camera.
All adult wings that were 75% or 100% the size of a normal wing were used to quantify the loss of the wing margin. The wing margin was divided into five segments defined by where the wing veins intersect the margin. Each wing was scored for the number of segments with missing margin to assess the extent of the patterning defect. Percentages from the three independent experiments were used to calculate averages plotted in the graphs. The area of undamaged and regenerated wings was measured using ImageJ (NIH). ImageJ was also used to measure the percentage of linear length of margin lost for the entire perimeter of the wing. Graphs were plotted using Excel and Graphpad Prism 7.

MRC-5 and A-549 cells were incubated with 0.4–0.7 μM auranofin for 24, 48, and 72 h (at a density of 5 × 10⁵ cells/mL). Cells were trypsinized for cell cycle analysis and then washed twice with cold PBS. Cells were centrifuged for 5 min at 1500 rpm. Next, cells were resuspended with 580 μL PBS solution containing 10 μg/mL of DAPI (solution 10, Chemometec), incubated for 5 min, and analyzed by flow cytometry. Cell cycle analysis was performed using Nucleo Counter^® NC-3000^TM system (Chemometec).
Cells (2 × 10⁵ cells/well) were seeded in 24-well plates to grow in a monolayer for 24 h. Then, a sterile 2–20 μL pipette tip was held vertically to scratch a cross in each well. The detached cells were removed by washing with 500 μL PBS and shaking at 500 rpm for 5 min. 500 μL of fresh medium with auranofin (in the range of 0.4–0.8 µM to achieve a concentration equal to IC50 and in the range of 0.8–1.6 µM to achieve a concentration equal to 2 × IC50) was added to each well and incubated for 72 h. Before the image acquisition, the plate was washed with 500 μL pre-warmed PBS [62 (link)] and gently shaken for 30 s. Then, a medium was added again, and pictures were taken. The scratch closure was monitored and imaged in 24 h intervals using an SZX10 microscope (Olympus, Tokyo, Japan). Images were analyzed using ImageJ software with Wound Healing Tool.