Latrunculin a

For oryzalin treatment, a 30 mg/ml oryzalin (Sigma, 36182) stock solution dissolved with DMSO was prepared (working solution: 30 μg/ml oryzalin containing 0.01% silwet L-77). Floral buds at stage 8 that had flat epidermal cells were immersed in the solution containing 30 μg/ml oryzalin for 5-min treatment. To prevent repolymerization of the microtubules, the same treatment was repeated 24 h later for twice. For Latrunculin A treatment, a 100 μg/mL Latrunculin A (Sigma, L5163) stock solution dissolved with DMSO was prepared (working solution: 0.5 μg/mL Latrunculin A containing 0.01% silwet L-77). Floral buds at stage 8 that had flat adaxial epidermal cells were immersed in the Latrunculin A working solution for 5-min treatment. To prevent repolymerization of the microfilaments, the same treatment was repeated twice24 h later.

To induce EMT in epithelial cells, NBT-II cells were treated in culture medium supplemented with 100 ng ml⁻¹ EGF. For EMT induction in keratinocytes, HaCaT cells were treated in culture medium supplemented with 20 ng ml⁻¹ TGF-β1 (Sigma) and 100 ng ml⁻¹ EGF for 1 day and refreshed with 10 ng ml⁻¹ TGF-β1 and 100 ng ml⁻¹ EGF daily for 3 more days before overnight imaging, or for 4 more days before lysate preparation. To induce chirality in keratinocytes, HaCaT cells were treated with 20–200 nM of latrunculin A (Sigma). For drug inhibition studies, 5–20 nM cytochalasin D (Sigma) or 15 µM SMIFH2 (Sigma) were added to HaCaT cells within 2 h of seeding on micropatterned dishes. For transcriptional inhibition study, cells were treated with 2 µg ml⁻¹ of actinomycin D (Sigma) or pre-treated with 2 µg ml⁻¹ of actinomycin D for 2 h prior to the addition of latrunculin A. All inhibitors remained in the medium during the entire period of observation.

Nocodazole (50μM) (EMD Milipore) and latrunculin A (Sigma-Aldrich) (20μg/ml) were used to destabilize MTs and actin, respectively. When live imaging began, ovaries had been exposed to the drugs in the incubation media for no longer than 15–20 min. The control group was treated with DMSO in the same conditions. We used Mitotracker staining to monitor oocyte health and viability during the treatment.
For analysis of LatA treatment in fixed samples, ovaries from the same fish were divided into latrunculin A (Sigma-Aldrich) (20μg/ml) and DMSO treated groups with two replicates for each condition. We tested incubation times of 6, 12 and 20 hours. After the treatment, ovaries were fixed as above and stained for Buc. We measured the oocyte diameter and analyzed the effect of LatA treatment only in stage I oocytes.
For cold treatment, ovaries were dissected as explained previously; ovaries were kept in culture media, and divided in tubes placed in ice in a cold room (4°C) and controls kept at 28°C, both for 120 min. Then ovaries were fixed and processed for immunostaining.