Mz16f fluorescence stereomicroscope

Epifluorescence microscopy was performed with a BIOREVO BZ-9000 (Keyence) and an Axioimager A1 microscope (Carl Zeiss). Bright-field images were taken with an MZ16F fluorescence stereomicroscope (Leica), a BIOREVO BZ-9000 (Keyence) and an Axioimager A1 microscope (Carl Zeiss).

For analyses of cell-to-cell movement, the cassette from plasmids containing all proteins assayed inserted into AMV 3 cDNA, was amplified with specific primer pairs, and the generated amplicons were used directly as templates for in vitro transcription with T7 RNA polymerase (Takara Bio Inc., United States). For MP, N, and P co-inoculation, we balanced the concentration of AMV RNA3 transcripts carrying the heterologous genes. The quantification was performed with agarose gel electrophoresis using an RNA ladder (RiboRuler High Range RNA Ladder, Thermo Scientific) and several dilutions of the transcribed RNAs. Next, transgenic N. tabacum P12 plants that express the polymerase proteins P1 and P2 of AMV (van Dun et al., 1988 (link)) were grown and inoculated with RNA transcripts, as previously described (Taschner et al., 1991 (link)). The chimeric AMV RNA 3 expressing the AMV MP wild type (wt) was obtained from Leastro et al. (2017b) (link). As reviewed by Leastro et al. (2020) (link), three independent experiments were performed, each one included the infiltration of three leaves of P12 plants per construct. The foci images in P12 plants were taken with the aid of a Leica MZ16F fluorescence stereomicroscope.

WT seeds (2,000) were suspended in 0.3% EMS solution for 13 h with rotation, washed with water and planted on soil. Approximately 1,000 independent M2 populations were collected and screened for GFP fluorescence using a Leica MZ16F Fluorescence Stereomicroscope equipped with a GPF Plus filter. Pictures were taken with a DFC300 FX digital camera. For mapping and identification of EMS mutations, mutant #162 was crossed with WT Ler and 10-days-old F2 seedlings grown on media containing 1 × Murashige and Skoog basal salt mixture (MP) and 20 μg ml⁻¹ glufosinate ammonium (Sigma) were analysed for GFP expression. Genomic DNA was isolated from pooled tissue of 50 GFP-positive F2 mutants and analysed by whole-genome re-sequencing for co-segregating single-nucleotide polymorphisms between Col and Ler13 (link). Primer sequences of CAPS markers for co-segregation analyses are shown in Supplementary Table 2.

Freshly fertilized chicken eggs were purchased from McIntyre Farms (Lakeside, CA). The HovaBator Genesis 1588 Egg Incubator Combo Kit was purchased from Incubator Warehouse. 010 PTFE White Teflon O-Rings were purchased from The O-Ring Store. 3 M Tegaderm™ Transparent Dressing was purchased from Moore Medical and the Dremel 7700-1/15 MultiPro 7.2-Volt Cordless Rotary Tool Kit was used to make holes in the eggshell and 33 G acu·needles were purchased from Acuderm inc. Freshly fertilized chicken eggs were incubated at 100°F and with 60% humidity. On day 10 of embryo development, the chorioallantoic membrane (CAM) was dropped to make a window on the eggshell. A Teflon ring was placed on the CAM membrane which was then gently abraded with a stirring rod. 2 × 10⁶ OVCAR-8-GFP ovarian cancer cells were grafted into the ring, and then the window was sealed with Tegaderm film. Tumor formation was observed by a Leica MZ16F fluorescence stereomicroscope. All chicken egg experiments were approved by the UCLA Institutional Biosafety Committee, Office of Research Administration and were performed in compliance with the committee guideline. In ovo experiments do not require any special additional allowance as long as the embryos are sacrificed before hatching as is done in this study.

Embryos carrying myl7:GFP reporter were anesthetized with 0.16% Tricaine at 48 hpf and their hearts dissected in ice-cold PBS under a Leica Mz16 F Fluorescence Stereo Microscope. Pooled hearts (n = 35–70) were lysed in TRIzol reagent (Invitrogen) and total RNA purified by RNA microPrep (Zymo Research) for each biological replicate. The purified RNA eluates were kept at −80 °C prior to sequencing or qPCR. Total RNA from 16 pools (8 biological replicates of control and rbfox-null) were analyzed by TapeStation (Agilent). RNA was processed with poly(A) selection for full length transcripts and libraries generated using the ultra-low input library preparation kit (Illumina). Libraries were sequenced on an Illumina HiSeq platform using the 2 x 150 bp configuration with single index.

The compound 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA, Image-iT LIVE Green Reactive Oxygen Species Detection Kit, Molecular Probes; Invitrogen, I36007) was used to visualize the general in vivo production of ROS, in which fluorescent carboxy-DCF is produced through ROS oxidation after removal of the acetate groups by intracellular esterases. The ROS visualization procedure was performed on Smed-egfr-3 RNAi knockdown animals as well as control- and MEK-inhibited animals either combined or not with the inhibition of ROS production by DPI or light therapy. Animals were exposed to carboxy-H₂DCFDA (25 μM, 1 ml) for 1 h prior to amputation and for 1 day post RNAi. Amputated animals were again incubated in carboxy-H₂DCFDA for 15 min before immobilization in 2% (w/v) low melting point (LMP) agarose (Invitrogen, 16520-050). Imaging of the samples was performed on 30 min, 6- and/or 24 h post amputation (MPA/HPA) using a MZ16F fluorescence stereomicroscope (Leica) combined with a ProgRes C3 camera (Jenoptik, Jena, Germany) or a Ts2-FL inverted microscope (Nikon) combined with a Ds-Fi3 color camera (Nikon). For all pictures the exact same capturing settings were used. Additionally, all experiments were also performed without the carboxy-H₂DCFDA-stain in order to discard possible autofluorescence at the wound sites.