Ma5 11883

For immunofluorescence analysis of pESLC or EB, cells fixed with 4% paraformaldehyde were washed three times with PBS and permeabilized with 0.5% Triton X-100 for 30 min. Then, cells were coincubated with blocking solution (10% goat serum in PBS) and primary antibody overnight at 4°C. The primary antibodies used were anti-POU5F1 (AB19857; Abcam, Cambridge, United Kingdom, 1:200), anti-SOX2 (AB5603; Millipore, Temecula, CA, 1:200), anti-NANOG (AB70482; Abcam, 1:200), anti-cytokeratin 17 (AB49749; Abcam, 1:200), Desmin (MAB3430; Millipore, 1:200), anti-vimentin (MA5-11883; ThermoFisher, 1:100), anti-neurofilament (MAB1615; Millipore, 1:200), anti-desmin (MAB3430; Millipore, 1:200), and anti-cytokeratin 17 (AB49749; Abcam, 1:200). After washing three times with a washing medium (Tween20, Triton X-100, and PBS), the cells were incubated with the appropriate secondary antibodies. Secondary antibodies were applied, and the cells were incubated at room temperature (20–25°C) for 1 h. Nuclei were stained with Hoechst-33342. Stained cells were examined under a confocal microscope using the ZEN 3.5 Blue Edition software (Zeiss, Oberkochen, Germany).

Paraffin‐embedded sections of placental tissue were prepared as previously reported.¹⁵ After antigen retrieval and blocking, the serial sections were treated with the following primary antibodies overnight at 4°C: rabbit anti‐human NCOA6 serum that was generously provided by Professor Jian‐ming Xu at Baylor College of Medicine in USA and Professor Hong‐mei Wang at Institute of Zoology, CAS in China (1:500),¹⁶ mouse anti‐human Cytokeratin 7 (CK 7, 180234, Invitrogen; 1:200), mouse anti‐human HLA‐G (sc‐21799, Santa Cruz; 1:200) and mouse anti‐human vimentin (MA5‐11883, Invitrogen; 1:200). After incubations with secondary antibodies: Alexa Fluor Plus 647‐conjugated antibody (A32733, Invitrogen) for NCOA6 and Alexa Fluor 488‐conjugated antibody (A‐11001, Invitrogen) for others, the serial sections were finally treated with DAPI (R37606, Invitrogen) in Vectashield^® antifade mounting medium (Vector Laboratories). Florescence images were obtained with a LSM 780 confocal laser‐scanning microscope (ZEISS). All photographs were arranged and processed by PowerPoint (Microsoft Office Professional Plus 2010, Microsoft Corporation) and Photoshop CS6 (Adobe Systems).

Cell pellets were lysed with 1× RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and a phosphatase inhibitor cocktail (Nacalai Tesque). Nuclear proteins were collected using NE-PER Nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteins were subsequently separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunoblotted with the following antibodies: Rabbit anti-NF-κB p65 (phospho S536) antibody (ab28856; 1:500 dilution; Abcam), rabbit anti-NF-κB p65 antibody (#8242; 1:1000 dilution; cell signaling), rabbit anti-RelB antibody (#4922; 1:1000 dilution; cell signaling), rabbit anti-SNAIL antibody (ab180714; 1:1000 dilution; Abcam), rabbit anti-E-cadherin antibody (sc-7870; 1:500 dilution; Santa Cruz), mouse anti-Vimentin antibody (MA5-11883; 1:200 dilution; Invitrogen), mouse anti-GAPDH antibody (ab9484; 1:1000 dilution; Abcam), and rabbit anti-HDAC1 antibody (ab109411 1:1000 dilution; Abcam). The bands were visualized using Molecular Imager Gel DocTMXR+ and ChemiDocTMXRS+ Systems with Image Lab 2.0 software (Bio-Rad). Uncropped scans of the blotted gels with the weight markers are shown in Supplementary Fig. 16.

A 9-year-old girl, without a significant pathologic clinical history, was admitted at the Hospital Infantil de México Federico Gómez with one month´s history of moderate progressive abdominal pain localized in mesogastrium, vomiting and weight loss. A heterogeneous ovarian mass of 17 × 9,7 cm was discovered by CT scan image. The patient underwent surgical excision of the mass. On laparotomy, surgeons found a right ovarian tumor. The pathology specific presented a typical herringbone pattern, high mitogenic index (4 mitoses in 10 high-magnification fields), extensive hemorrhagic and necrotic areas and nuclear pleomorfism. The final histopathology diagnosis was fusocellular sarcoma compatible with fibrosarcoma. Immunohistochemistry was performed against Vimentin (MA5-11883, Invitrogen, CA, USA; 1:100 dilution) and Inhibin (5692, Bio SB, CA, USA; 1:100 dilution) as reported previously^{25 (link)}. The patient had two siblings, her brother was diagnosed with bilateral renal tumors: Wilms´ tumor and a possible metanephric adenoma whereas her sister is healthy at the moment. We were unable to obtain paternity tests.