Las x

The full-length CDSs of SlWRKY80 and SlJAZ1 (excluding the termination codon) were constructed in the pSPYNE and pSPYCE vectors, respectively. Supplementary Data Table S1 provides the primers. Subsequently, the constructed vector was sequenced and validated, and the plasmid was transferred into Agrobacterium GV3101. The empty plasmids pSPYNE and pSPYCE, along with the recombinant plasmid-containing Agrobacterium, were diluted with MES (OD₆₀₀ = 1.0). Then, Agrobacterium containing pSPYNE-X (Vec or SlWRKY80) and pSPYCE (Vec or SlJAZ1) was mixed in a 1:1 volume ratio and left to stand in a dark environment at 28°C for 2 h before injection into tobacco leaves. After 48 h, the infected tissue was examined using a confocal microscope (LAS X, Leica, Mannheim, Germany). The images were then subjected to postprocessing using Leica LAS X software (v.3.7.2). The scale used in this experiment was 20 μm.

ImageJ 1.53, and LAS X (Leica version 3.5.7.23225) were used for image analysis. Excel (v1108) and GraphPad Prism 9 (9.5.1) were used for data and statistical analysis. Bio-Rad Image Lab (6.1) was used for western blot analysis. Cytoscape (3.9.0) and R package were used for proteomic data analysis and representation. ClusterProfiler (3.8) was used for Gene Ontologies term enrichment analysis. Motif-x algorithm proposed through the MoMo tool included in the MEME Suite was used for motif enrichment analysis. Spectronaut v.15 (Biognosys) was used for DIA analysis. Mascot (Matrix Science, London, UK; version 2.5.1) was used for the peak list file searches against the PlasmoDB_P.berghei ANKA database (PlasmoDB.org, release 38). iTEM FEI (Olympus), ZEN 2.6 (Zeiss) and LAS X (Leica version 3.5.7.23225) were used for image acquisition.

For clarity, images were rotated, colorized, and the intensity was adjusted using Adobe Photoshop. All images show a single optical section (Z), except for Figs. 5, 7, 10 and Supplementary Figs. 2 and 4. In Fig. 5, six Zs with 1.2 μm steps were maximum projected using Leica LAS X software. In Fig. 7, three Zs with 1.2 μm steps were maximum projected using Leica LAS X. In Fig. 10, two Zs with 1.2 μm steps were maximum projected using Fiji (NIH). In Supplementary Fig. 2, images where maximum projected to span a region of 2.5 μm using Fiji (NIH). In Supplementary Fig. 4a, four Zs with 1.2 μm steps were maximum projected using Fiji (NIH). In Supplementary Fig. 4c, two Zs with 1.2 μm steps were maximum projected using Fiji (NIH). In Supplementary Fig. 4e, six Zs with 1.2 μm steps were maximum projected using Fiji (NIH). Figures 5c, f and Supplementary Movies were rotated, colorized, and the intensity was adjusted using Imaris. Several Zs were maximum projected in Imaris for Supplementary Movies. For the left movie in Supplementary Movie 4, the brightness was adjusted in each frame using Photoshop to compensate for photobleaching.

Tissues were cryopreserved by incubation in 2-Methylbutane at −25 °C (Merck, Germany). The brains were cut into coronal sections 10 µm thickness with cryostat (MICROM HM560, Fisher Scientific GmbH, Schwerte, Germany) and kept at −25 °C. Immunostaining was performed after fixation in PFA 4% for 20 min at 4 °C of the slides. Detection of the sig1R protein was performed by overnight incubation at 4 °C of the slides with the primary mouse monoclonal antibody (1:500 in blocking buffer 5% normal goat serum, B-5: sc-137075, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing with a solution of 1% BSA in PBS, the slides were incubated for 1 h at room temperature with the secondary polyclonal goat anti-mouse antibody (1:200 in dilution buffer 1% BSA, Alexa Fluor ^® 488; ab150117, Abcam, Berlin, Germany). A Hoechst counterstaining, 10 min at room temperature, was performed to visualize the nuclei of the cells (1:1000 in PBS, Hoechst 33258, Life Technologies, Carlsbad, Ca, USA). After a step of washing and drying, slides were cover up with mounting medium. (Aquapolymount, Polysciences Europe GmbH, Hirschberg an der Bergstrasse, Germany). Visualization of the slides was performed by fluorescence microscopy (Leica, DMi8, software Leica LASX, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany).

Aliquots (20 μl) of each microalgal culture, obtained as described before, were submitted to microscopic observations in order to visually assess phenotypical differences, as well as to take measures of each strain. Microscopic observations were performed by using a Dialux 22 light microscope (Leitz, Stoccarda, Germany) in immersion - under mineral oil – at 100× magnification. The microscope was equipped with a Leica DC450 C (Leica, Wetzlar, Germany) digital camera. Images were handled with the software Leica LASX (Leica Application Suite X).

Random forest classifiers for both platelets and megakaryocytes were trained in Napari using APOC. These classifiers were used in an initial semantic segmentation step, which was followed by connected component labeling using sci‐kit‐image. For platelet segmentation, objects smaller than 50 voxels and larger than 80 000 voxels were filtered out, and the remaining objects were counted by determining the largest‐area slice in the 3D volume and measuring the length of the long axis of this 2D slice. For megakaryocyte segmentation, objects smaller than 100 000 voxels were removed. Object Volume was determined by multiplying the number of voxels by the voxel size. The surface area was determined by mapping a surface using the marching cubes algorithm and then calculating the surface area of the resulting mesh using sci‐kit‐image. Alternatively, 3D reconstruction and image processing were performed using Leica LasX (Leica) and Arivis Vision 4D (Zeiss). Sphericity and volumes were measured by Arivis Vision 4D (Zeiss). The analyses of pathological samples have been performed blinded to patients’ diagnoses.

PASMC proliferation was assessed using the Click-iT Edu Alexa Fluor 488 Imaging Kit (ThermoFisher Scientific, Waltham, Massachusetts, United States, Cat#10337). Isolated PASMCs (passage 0) cultured in 24-well plates were exposed to chronic hypoxia for 96 h (1% O₂, 5% CO₂, balanced with N₂), incubated with 5-Ethynyl-2′-Desoxyuridin (EdU, 10 µM) for further 24 h under chronic hypoxia and finally fixed with acetone-methanol (1:1; Sigma-Aldrich, St. Louis, Missouri, United States, Cat#32201 and Cat#32213) for 5 min at room temperature. Fixed PASMCs were blocked with 3% BSA (Sigma-Aldrich, St. Louis, Missouri, United States, Cat#A7030) and stained according to manufacturer’s protocol. Afterwards, PASMC nuclei were visualized with Hoechst^®33342 (4µM; ThermoFisher Scientific, Cat# 62249). Fluorescence emission was detected using Leica DMI6000 CS fluorescence microscope (Leica, Wetzlar, Germany). Hoechst-positive (Hoechst⁺; total cell number) and EdU-positive (EdU⁺; proliferation cell number) PASMCs were counted using the Leica Las X (Leica, Wetzlar, Germany) software. Finally, proliferation rate was calculated by assessing the ratio between EdU⁺ and Hoechst⁺ PASMCs as previously described (Malczyk et al., 2013 (link)).