Fig 2: The levels of P. aeruginosa intake or accumulation are similar among wild-type, tbh-1(n3247), and tdc-1(n3419) animals. (A) Pharyngeal pumping rates of animals on P. aeruginosa lawns were counted as pumps per 30 s. Counting was conducted in triplicate, and results were averaged to give a pumping rate. The graph represents combined results of three independent experiments. Bars represent means ± SEM. n = 30 animals per strain. P values represent results of comparisons to WT animals as follows: tbh-1(n3247) (P = 0.1270); tdc-1(n3419) (P = 0.1231). (B) Animals were exposed to P. aeruginosa expressing GFP for 24 h and then visualized using a Leica MZ10F fluorescence stereomicroscope. Scale bar indicates 500 µm. (C) Animals were exposed to P. aeruginosa expressing GFP for 24 h at 25°C, and CFU counts were performed. Ten animals were used for each condition. The graph represents combined results of three independent experiments. Bars represent means ± SEM. P values represent results of comparisons to WT animals as follows: tbh-1(n3247) (P = 0.3803); tdc-1(n3419) (P = 0.2426). (D) Defecation rates of animals on P. aeruginosa lawns were measured as averages of 10 intervals between two defecation cycles. Bars represent means ± SEM. n = 12 animals per strain. P values represent results of comparisons to WT animals as follows: tbh-1(n3247) (P = 0.2575); tdc-1(n3419) (P = 0.1838).

Fig 3: The inhibition of STAT3/miR-21 pathway abrogated AngII-induced angiogenesis in vivo. A Matrigel mixture containing VEGF (50 ng/ml) and AngII (100 nM) alone or in combination with either Stattic (50 µM) or miR-21 antagomir (10 nM) was injected subcutaneously into the ventral side of the mice. After 14 days, the Matrigel plugs were recovered. Each group consisted of six mice. (a) Representative images of macroscopic visualization of Matrigel plugs at 14 days. (Leica MZ10 F; Leica, Wetzlar, Germany), ×80. (b, d) Plugs from implanted mice were subjected to H&E staining (b) and immunohistochemical staining for CD31 (c, d); representative images (c) for quantitative analysis (d; n = 6). Scale bars: 500 µm in (a), 19.6 µm in (b) and (c). New vessel formation (black arrowheads) was observed at ×400. *p < 0.05 vs. con. **p < 0.01 vs. con. # p < 0.05 vs. AngII. ## p < 0.01 vs. AngII. AngII: angiotensin II; H&E: hematoxylin and eosin; STAT3: signal transducer and activator of transcription 3 [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Inventory of tools and equipment required. (A) (1) PCR tubes, if possible, with individual lids (Eppendorf), (2) two black SYLGARD coated Petri dishes (10 cm and 15 cm), (3) mouth pipette with a syringe filter and valve to control the flow rate, (4) dissection forceps (FST size 55), (5) sterile micropipettes (ORIGIO MBB-FP-M-0), and (6) clear 6 cm Petri dishes for PBS washes. (B) (1) Heating lamp to maintain culture temperature, (2) thermometer, and (3) sandstone connected via a plastic tube and valve regulator to a pressurized oxygen bottle. (C) Image of embryos in culture during retrograde labeling experiment. The sandstone provides oxygenation, and the temperature is monitored during the entire incubation time. (D) Fluorescent stereomicroscope (Leica MZ10 F, with 1.6× ApoPlan lens, connected to a Leica EL6000 fluorescent light source) used for cell picking

Fig 5: Creation of Csbpc2 transgene-free cucumber plants using the CRISPR/Cas9 system. A Schematic of the CRISPR/Cas9 binary vector pKSE402. The vector was modified by inserting a GFP cassette, which was used to screen for transgenic plants. BCsBPC2 gene structure and target site. Black lines and red boxes represent introns and exons, respectively. The 5'- and 3'-untranslated regions are indicated by green boxes. The target site (vertical black bar) is located at 175 bp in the first exon of CsBPC2. The PAM and target sequence is provided in red and black letters, respectively. C–FAgrobacterium tumefaciens-mediated cucumber transformation. C Preparation of explants, scale bar = 2 cm; D 16-day-old explants cultured on shoot regeneration medium, scale bar = 2 cm; E GFP fluorescence of regenerated shoots, scale bar = 0.2 mm; F Transgenic plant, scale bar = 2 cm. G–J Identification of GFP-positive plants, scale bar = 0.5 mm. The GFP fluorescence in the tendrils (G), male flowers (H), ovaries (I), and germinated seeds (J) of T0 plants was detected using the Leica MZ10F stereomicroscope. Plants with green fluorescence (left side) were transgenic lines, whereas plants with red fluorescence (right side) were wild-type lines. K Edited CsBPC2 target site in T0 plants. The wild-type sequence (i.e. control) is presented at the top, whereas the remaining sequences were from individual clones. Sequences were aligned using the Geneious software. The target and PAM sequences are indicated above the sequence chromatograms. Red dashes represent inserted or deleted nucleotides. L Identification of Csbpc2 transgene-free homozygous mutants on the basis of Sanger sequencing. WT, wild-type; Lines 1–12, transgene-free homozygous mutants. Red dashes represent deleted nucleotides (the number of deleted nucleotides is indicated on the right).