13 cis ra

VeroE6/TMPRSS2 cells (JCRB #1819) [16 (link)] and Calu-3 cells were cultured in DMEM containing 10% FBS. SARS-CoV-2 Pango lineage A (JPN/TY-WK-521/2020 EPI_ISL_408667), alpha strain (JPN/QK002/2020 EPI_ISL_768526), beta strain (JPN/ TY8-612-P1/2021 EPI_ISL_1123289), gamma strain (JPN/TY7-501/2021 EPI_ISL_833366), and delta strain (JPN/TY11-927-P1/2021 EPI_ISL_2158617) were obtained from NIID, JAPAN, and handled in biosafety level 3 (BSL3). While performing SARS-CoV-2 infection experiments, DMEM containing 2% FBS was used. The chemical compound library was obtained from Tokiwa Phytochemical (Chiba, Japan) and retinoids including ATRA, all-trans retinal, retinol, 9-cis RA, and 13-cis RA were obtained from Sigma Aldrich (St. Louis, MO, USA). GC376 was purchased from Selleck chemicals (Houston, TX, USA).

The chemicals and reagents used were of analytical grade. 13-cis-RA, all-trans-RA, 4-oxo-13-cis-RA, all-trans-retinol, all-trans-retinal, retinol acetate, l-ascorbic acid, N-ethylmaleimide (NEM), 2, 4-dihydroxy-benzophenone (BP-1), ethanol, (sulfate sodium salt)-β-cyclodextrin (Sulfate-β-CD) and heptakis (2,3,6-tri-o-methyl)-β-cyclodextrin (Heptakis-β-CD) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl alcohol, disodium tetraborate decahydrate (borate), glycine, tris (hydroxy-methyl)aminomethane (tris), sodium dodecyl sulfate (SDS), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Methanol and acetone were purchased from Macron Fine Chemicals (Center Valley, PA, USA). Acetonitrile was purchased from J. T. Baker (Phillipsburg, NJ, USA). Perchloric acid and hexane were purchased from Honeywell (Seelze, Germany). Ultrapure water was obtained from the Milli-Q^® system and was used to prepare the experimental solution. α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), Hydroxypropyl-β-cyclodextrin (HP-β-CD) were purchased from Tokyo Chemical Industry CO., LTD (Tokyo, Japan). The standard solutions (13-cis-RA, all-trans-RA and 4-oxo-13-cis-RA) and internal standard (BP-1) were dissolved in methanol and stored at −20 °C.

ATRA, 9cisRA, 13cisRA, endrin, dieldrin, and sterile Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The stock solutions were prepared in DMSO: ATRA, 9cisRA and 13cisRA at 0.1, 1 and 10 mM, endrin and dieldrin at 10 mM.

To block apoptosis, embryos were incubated with either 200 or 400 µM of the pan-caspase inhibitor Z-VAD-FMK (Promega) from 14 to 31 hpf (Williams et al., 2000 (link)). The drug was washed out and embryos were either fixed or incubated in fish water until 3 dpf and then fixed.
To activate RA pathway activity, embryos were soaked in 25 nM AM580 (Sigma), a pan-retinoic acid receptor (RAR) agonist, beginning at 24 hpf for the times indicated in figure legends. To inhibit the pathway, embryos were soaked in either 15 µM BMS493 (Sigma), an RARα inverse agonist, or 15 µM BMS614 (Tocris), an RARα antagonist, for 14-36 h from the 12-14 somite stage, which is after initial dorsal-ventral patterning of the eye and brain. After drug treatments, embryos were washed twice with embryo medium and then incubated until fixation with 4% paraformaldehyde at 28, 40 or 60 hpf. For optical manipulation of RA activity in the retina, Tg[RARE:YFP]^id1 embryos were soaked in 5 nM 13-cisRA (Sigma; Xu et al., 2012 (link)) at 24 hpf for 1 h in the dark and then mounted in 1.2% low melting point agarose. Photoactivation was performed with a single pulse of UV light (360-375 nm) illuminating the eye for 30 s, using a Zeiss 510 NLO two-photon microscope with 5 mW of power. Embryos were fixed at 33 hpf in 4% paraformaldehyde and immunostained for GFP.

Chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. ATRA and 13-cis-RA (Sigma-Aldrich, St. Louis, MO) were dissolved in either ethanol or DMSO at 10 mM or 50 mM, respectively, and protected from light at –20 °C. RAMBAs methyl 2,2-dimethyl-3-[4-(naphthalen-2-ylamino)phenyl]-3-(1H-1,2,4-triazol-1-yl)propanoate (compound 17; C17; molecular weight 400.5) and methyl 3-(1H-imidazol-1-yl)-2,2-dimethyl-3-(4-(naphthalen-2-ylamino)phenyl)propanoate (compound 2; C2; molecular weight 399.5) were generated as described [19 (link)] and were dissolved in ethanol at 50 mM (C17) and 1 mM (C2; maximal solubility in this solvent). Antibodies were sourced as follows: anti-N-myc (B8.4.B; Insight Biotechnology, Wembley, UK); anti-phospho-Ser472-AKT (ab4060; Cell Signaling Technology, Danvers, MA); anti-AKT (ab9272; Cell Signaling Technology, Danvers, MA); anti-actin (A5316; Sigma-Aldrich, St. Louis, MO); anti-GAPDH (14C10; Cell Signaling Technology, Danvers, MA). HRP-linked secondary antibodies were purchased from DAKO Ltd. (Bucks, UK).