Cellsens dimension 3

Candida albicans yeast cells (1 × 10⁵ per well) were suspended in 100 μl of RPMI 1640 medium (Biowest) containing peptides at concentrations in the range of 3.1–50 μM and 0.8–6.2 μM for ΔM3 and ΔM4, respectively, and placed in triplicates into the wells of 96-well black/clear flat bottom polystyrene high binding microplate (Corning Inc., Corning, NY, United States). Fungal cells were also suspended in medium without peptide as a control of the correct morphological transition. The incubation was carried out at 37°C in an atmosphere of 5% CO₂ and 95% humidity for 90 min without shaking. Subsequently, fungal cells were stained with Calcofluor White (Sigma-Aldrich) at a final concentration of 1 μg/ml and Sytox Orange (ThermoFisher Scientific, Waltham, MA, United States) at a final concentration of 1 μM for 10 min in the dark. C. albicans cells were visualized using an Olympus IX73 microscope (Olympus, Tokyo, Japan) equipped with a Hamamatsu Orca Spark camera (Hamamatsu, Hamamatsu City, Japan) and a UPLXAPO60XO lens (Olympus), and the images were analyzed using Olympus CellSens Dimension 3.1 software. To determine the length of the hyphae, three images were taken in each well (40 × magnification) and for each image the length of the hyphae was measured for 20 cells.

For tethering yeast cells to glass-bottom dishes (35 mm, catalog no. 81218-200; ibidi GmbH) for live-cell imaging, we coated the dishes with 6% concanavalin A type 6 (catalog no. C2010; Sigma-Aldrich). Yeast cells were precultured overnight, subcultured for 3 to 4 h to mid-log phase (OD₆₀₀ = 0.5 to 0.8) in SC broth at 30°C with rotation at 180 rpm, and plated onto concanavalin A-coated dishes. Live-cell imaging was performed using an Olympus FV3000 laser point scanning confocal microscope equipped with a 100× oil objective, high-sensitivity GaAsP photomultiplier tube (PMT) detectors, and solid-state lasers (488 nm and 561 nm). Images were acquired using Olympus FluoView 3000 (2.4.1.198) software. All time-lapse movies consisted of 7 z-slices at a step size of 0.5 μm for a period of 60 to 180 min with a 1-min interval. Three-dimensional (3D) deconvolution of raw images was done using Olympus CellSens Dimension (3.1) software, followed by maximum-intensity projection using Fiji for representation in a montage. All images in Fig. 2c and Fig. S1e were acquired at a 2,048-by-2,048 resolution under the conditions described above.

The specimens were deposited at the Department of Plant Protection of Sichuan Agricultural University, Chengdu, China (SICAU). The ambers containing the tree crickets are yellow and transparent specimens from the Hukawng valley at 99 Ma, and many Grylloidea samples were collected from the site [23 ]. A recent study using U-Pb zircon dating determined the age to be 98.79 ± 0.62 Ma or at the Albian/Cenomanian boundary [24 (link)]. The amber containing the specimen was ground and polished on the right size.
Photographs were taken with an SZX16 microscope system and cellSens Dimension 3.2 software (Olympus, Tokyo, Japan). In most instances, incident and transmitted light were used simultaneously. All images are digitally stacked photomicrographic composites of approximately 20 individual focal planes obtained using Helicon Focus 6 (http://www.heliconsoft.com accessed on 12 May 2022) for a better illustration of the 3D structures.
The terminology of the fore wing venation follows Gorochov and Tan [25 (link)]. Sc, subcosta; R, radius; M, media; MP, posterior media, CuA, anterior cubitus; CuP, posterior cubitus; A, anals; d, dividing vein; di, diagonal vein; ch, chord vein; and o, oblique vein.

Using an 8-well plate on glass (Sarstedt, Nümbrecht, Germany), 4000 and 8000 cells of A431 and 12,000 and 16,000 cells of CAL-39 were seeded and incubated for 24 h. After incubation with formaldehyde (3.7%; Sigma-Aldrich) for 15–30 min, the cells were washed twice with fluorescence staining solution/DPBS (Pan Biotech), 2% BSA (Carl Roth, Karlsruhe, Germany) and 0.25% Triton X-100 (Sigma-Aldrich). The cells were incubated overnight with the rabbit antihuman GPER1 antibody at 1:100 dilution (Thermo Fisher Scientific) in a wet chamber. After washing, fluorescence staining solution containing DAPI at 1:1000 dilution (Novus Biologicals) and Alexa Fluor 488 antirabbit IgG antibody solution (Invitrogen) at 1:10 dilution were added. After 30 min incubation, the cells were washed twice with fluorescence staining solution and once with DPBS. Fluorescence mounting medium (Dako North America) was added and covered with a cover slip (Menzel-Gläser, Braunschweig, Germany) and allowed to dry. The stained cells were photographed using an Olympus IX83 microscope (Olympus Life Science Solutions) and cellSens Dimension 3.2 software (Olympus Life Science Solutions).