Ab205

For the detection of F-actin, transfected KHOS cells were incubated in the coverslips of six-well plates overnight, followed by mixing with 100 µg/mL PCFE and 1000 µg/mL dacarbazine for 24h. After fixing with methanol, cells were cultured with primary anti-F-actin antibody (ab205, abcam, UK) for 1h, and then incubated with secondary antibodies. A fluorescence microscope (Leica DM4000 B, Germany) was utilized for the visualization.

Specimens were fixed, stained with an anti-phosphorylated myosin light chain (p-MLC) rabbit mAb (#3671, CST, MA, USA) and an anti-F-actin mouse mAb (ab205, Abcam), and then incubated with the corresponding secondary antibodies (Alexa Fluor 488 and Alexa Fluor Plus 555 (ThermoFisher), respectively) at room temperature for 1 h. Samples were visualized using a laser scanning confocal microscope (LSM780, Zeiss).

For immunoblotting of targeted proteins, the following antibodies were used: YAP [1 : 1000, ^#14074, Cell Signaling Technologies (CST), Beverly, MA], WWC2 (1.0 μg/mL, PA5-71284, Invitrogen), E-cadherin (1.0 μg/mL, ab231303, Abcam), N-cadherin (1.0 µg/mL, ab18203, Abcam), Vimentin (1 : 1000, ab137321, Abcam), LATS1 (1 : 1000, ^#3477, CST), phosphorylated (p)-LATS1 (1 : 1000, ^#8654, CST), p-YAP (1 : 1000, ^#4911, CST), cofilin (1.0 μg/mL, ab42824, Abcam), p-cofilin (1 : 1000, ab12866, Abcam), F-actin (1 : 1000, ab205, Abcam), G-actin (1 : 1000, ab123034, Abcam), and GAPDH (1 : 5000, ab8245, Abcam) as well as horseradish peroxidase labeled goat anti-rabbit IgG (ab6721, Abcam) or goat anti-mouse IgG (ab6789, Abcam). ImageJ software was utilized for protein quantitative analysis.

The F-actin of CVECs was observed by immunofluorescence staining. Briefly, CVECs were fixed with 4% paraformaldehyde for 30 min and then were incubated with a 5% FBS for 30 min at 37°C. Cells continued to be incubated with anti-F-actin (Abcam ab205, Shanghai, China) antibodies overnight at 4°C and incubated with FITC-conjugated anti-IgG (Proteintech SA00003-1, Wuhan, Hubei, China) on the next day for approximately 1 h at 37°C and DAPI (Beyotime, #C1002, 1:1500) for 2 min. Images were obtained using a confocal fluorescence microscope (Leica-LCS-SP8-STED).

Cells were seeded onto cover slips, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton x-100 for 10 min. Slides were blocked with 1% bovine serum albumin and incubated with E-cadherin (ab76055, abcam, 1:200), F-actin (ab205, abcam, 1:100) or β-catenin (ab32572, abcam, 1:200) antibodies overnight at 4˚C. After washing in PBS, the cells were stained with secondary antibodies and incubated for 1 h at room temperature, followed by nuclear counterstaining with DAPI.

To characterize embryos on PD and PDCO from IVC day 1 to day 5 and in vivo embryos recovered at E4.5‐E6.5 were characterized. The embryos were fixed overnight with 4% PFA at 4 °C; permeabilized for 1 h in 1% Triton X‐100 in PBS at 37 °C; blocked for 1 h in 0.1% Tween‐20, 0.01% Triton X‐100, and 1% BSA in PBS at 37 °C; and incubated with primary antibodies at 4 °C overnight. The primary antibodies were OCT4 (1:100, sc‐8629, Santa Cruz), F‐actin (1:100, ab205, Abcam), CDX2 (1:100, 3977S, Cell Signaling Technology), Collagen I (1:100, ab90395, Abcam), FOXA2 (1:100, 8186S, Cell Signaling Technology), TFAP2C (1:100, sc‐12762, Santa Cruz), SOX2 (1:100, ab97959, Abcam), EOMES (1:100, ab23345, Abcam), T (1:100, ab209665, Abcam), and Phall (1:500, 40737ES75, Yeasen). The following secondary antibodies were incubated for 2 h at room temperature: Alexa 488 donkey anti‐mouse (1:500, A21202, Invitrogen), Cyanine3 goat anti‐mouse (1:500, A10521, Invitrogen), Alexa Fluor 647 goat anti‐mouse (1:500, A21235, Invitrogen), Alexa 488 goat anti‐rabbit (1:500, A11034, Invitrogen), and Cyanine3 goat anti‐rabbit (1:500, A10520, Invitrogen). Nuclear staining and incubation were performed for 10 min at room temperature in 10 µg mL⁻¹ Hoechst 33 342 (H3570, Invitrogen). The embryos were imaged on a Leica SP8 Zeiss LSM780 confocal microscope. Imaris 9.0.2 software was used to construct the 3D images.