Ehuegfp

For All‐Trans RA (ATRA, Sigma Aldrich) experiments, cells were treated for 48 hr with 0.1 mM of ATRA. For siRNA experiments, cells were transfected with 20nM of control nonspecific siRNA (EHUEGFP, Sigma) or with specific siRNA targeting ZYG11B (EHU022091, Sigma) using DharmaFECT™ Transfection Reagent following manufacturer's instructions (Dharmacon Horizon Discovery). Cell proliferation was assessed by Crystal Violet staining.

siRNAs for EGFP (EHUEGFP, Sigma) and Mmp2 (EMU04861, Sigma) were transfected using RNAiMAX transfection reagent (13778, Life Technologies) according to the manufacturer’s instructions.

SKOV-3 cells were plated at 4 × 10⁴ cells per well in 24-well plates and allowed to attach overnight. Endonuclease prepared small interfering RNAs (esiRNAs) were commercially synthesized (Sigma-Aldrich, St. Louis MO, USA) using either 420 bp length of P2Y₂ receptor (EHU156731; P2Y₂-esiRNA) or 472 bp length of K_Ca3.1 channel gene (EHU035251; K_Ca3.1-esiRNA), targeting human sequences NM_002564 and NM_002250, respectively. Then, for P2Y₂ receptor or K_Ca3.1 channel knockdown, cells were transfected with 150 ng per well of P2Y₂-esiRNA or K_Ca3.1-esiRNA, respectively, by using Lipofectamine 3000 (Invitrogen, Grand Island NY, USA) to deliver esiRNA into the cells following the method indicated by the manufacturer. As a non-targeting control (referred to as CNT), was transfected in the same condition the esiRNA of enhanced green fluorescent protein (EHUEGFP, Sigma-Aldrich), all responses of transfected SKOV-3 cells with either P2Y₂-esiRNA or K_Ca3.1-esiRNA were compared versus those of the CNT group. Twenty-four to 72 h after transfection, esiRNA-treated SKOV-3 cells maintained in culture were recorded electrically, or used for immunocytochemistry or fluorometric assays, as well as for migration quantification using the methods described above.

Fibroblasts were transfected with following siRNAs: control siRNA targeting GFP (EHUEGFP, Sigma Aldrich), siRNA targeting WWTR1/TAZ (EHU08032, Sigma Aldrich), siRNA targeting YAP1 (EHU113021, Sigma Aldrich). Final siRNA amount used per well was 1pmol per well. Transfection complexes were prepared with Lipofectamine RNAiMax (Life Technologies) according to the manufacturer’s instructions. Transfection mixture was added to the bottom of the well and cells were plated on the top in 100 μl complete medium to perform reverse transfection. Fibroblasts were incubated 24h with the transfection complexes and then subjected to the experimental treatments.

Mission esiRNAs targeting murine Smg7 (EMU150861), murine Pvt1 (EMU193181), murine Cyld (EMU031111), human SMG7 (EHU007301), and EGFP (EHUEGFP) were purchased from Sigma. Briefly, 5 × 10⁴ MF or NIH 3T3 cells or 1 × 10⁵ MCF‐7 cells in 500 µL medium were preseeded in 24‐well plates 1 day before. About 30 pmol siRNAs were mixed with 2 µL Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in 100 µL serum‐free medium, incubated at room temperature for 10 min, and directly added onto the cells. Seventy‐two hours after the transfection, cells were harvested or seeded for subsequent experiments.

Mission esiRNAs targeting human TMEM33 (EHU035611), EGFP (EHUEGFP), murine Tmem33 (Emu078331), murine Fads2 (EMU027741), murine Scd1 (EMU023031) and murine Hsd17b12 (EMU064031) were purchased from Sigma. 1.5 × 10⁵ cells were typically seeded in six-well plates 1 day before. Prior to transfection, 200 ng of siRNA and 3 μl Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) were mixed and incubated at room temperature for 15 min in serum-free media, then added dropwise on top of the cells. After 48 h transfection, cells were harvested for subsequent experiments.