Sc 659

Male C57BL6/J mice weighing 20–25 g were housed at the Ethics Committee for Animal Experiments of the First Hospital of China Medical University under a 12 h:12 h light/dark cycle with a constant temperature (22 ± 1 °C) and free access to food and water. All animal experiments were performed according to the National Institutes of Health guide for the care and use of Laboratory animals, and were approved by the Animal Care and Use Committee of the First Hospital of China Medical University.
The detailed information of antibodies are as follows: Anti-ILEI antibody (ab72182, Abcam, Cambridge, U.K.), Anti-α‐SMA antibody (ab5694, Abcam), Anti-vimentin antibody (ab137321, Abcam), Anti-E‐cadherin antibody (ab133597, Abcam), Anti-p‐Akt antibody (ab18206, Abcam), Anti-Akt antibody (ab126811, Abcam), Anti-p‐ERK antibody (ab223500, Abcam), Anti- ERK antibody (ab17942, Abcam), Anti-collagen Ι antibody (ab34710, Abcam), Anti-LIFR antibody (sc-659, Santa cruz, Dallas, U.S.A.),Anti-collagen ΙII antibody (WL03186,wanleibio,Shenyang, China), Anti-β‐actin antibody (WL01845,wanleibio).

Blastocysts were fixed in 3.7% paraformaldehyde in PBS for 20 min at room temperature and washed twice in PBS containing BSA (BPBS). Embryos were permeabilized with 0.2% Triton X-100 in PBS for 30 min, then washed twice in BPBS. Embryos were incubated in blocking solution (0.1% Tween 20 in BPBS) for 1 h and then incubated with primary antibodies against HIF-2α (Abcam, USA; ab199) (1:100), MnSOD (Abcam; ab16956) (1:200), and LIFR (Santa Cruz, USA; sc-659) (1:200) overnight at 4 °C. On the following morning, embryos were rinsed three times in BPBS and incubated in the appropriate secondary antibody conjugated with Alexa 488–labeled goat antimouse IgG (Invitrogen; A-11029) (1:100) or Alexa 568–labeled goat antimouse IgG (Invitrogen; A-11036) (1:100) for 1 h in the dark. After washing in PBS, the stained embryos were mounted in Fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, ab104139) and observed by confocal laser-scanning microscopy (FV1000; Olympus, Japan) to detect the fluorescence. For image analysis, the intensities of green fluorescence (Alexa 488) and red fluorescence (Alexa 568) were measured using Olympus Fluoview® software (FV10-ASW).

Western blotting for LIFR was performed as previously described^{41 (link)} with minor modifications. Briefly, proteins (30 µg) were separated on a 7% Tris-Acetate polyacrylamide gel (Thermo Fisher, UK) and transferred onto an Immobilon-Fl membrane (Merk Millipore, UK). Blots were probed with primary antibodies against LIFR (sc-659; 1:400; Santa Cruz Biotechnology, Germany) and TUBBA (ab6160; 1:5000; Abcam, UK). Primary antibodies were detected using Goat anti-Rabbit IRDye® 800CW (925-32211, 1:10,000; LI-COR Biosciences, UK) and Goat anti-Rat IRDye® 680RD (925-68076; 1:10,000; LI-COR Biosciences, UK) secondary antibodies. Blots were imaged using the LI-COR Odyssey imaging system (LI-COR biosciences, UK). Full blot images are included in supplementary materials.