Ltx and plus reagent

The short-hairpin RNA direct against human XIST (NR_001564.2) gene or FOXC1 (NM_001453.2) gene was reconstructed in a pGPU6/GFP/Neo vector (XIST(−), FOXC1(−); GenePharma, Shanghai, China), respectively. Its empty vector was used as a NC (XIST(−)NC, FOXC1(−)NC). Human FOXC1 gene coding sequence was ligated into pIRES2-EGFP vector (FOXC1(+)) (GeneScript, Piscataway, NJ, USA), and its empty vector was used as a NC (FOXC1(+)NC).
ECs were seeded in 24-well plates and transfected using LTX and Plus reagent (Life Technologies) when the confluence reached at ~80%. Stable cell lines were selected through the medium containing Geneticin (G418; Sigma-Aldrich, St Louis, MO, USA), G418-resistant clones were obtained after 4 weeks.
Agomir-137 (miR-137(+)), antagomir-137 (miR-137(−)) and their NC sequence (miR-137(+)NC and miR-137(−)NC; GenePharma) were transiently transfected into ECs, ECs which stably transfected shXIST or FOXC1 overexpression, respectively, according to the manufacturer’s instructions using lipofectamine 3000 reagent. Cells were collected 48 h after transfection.
Sequences of shXIST, shFOXC1 and shNC were shown in Supplementary Table 2. The transfection efficiency of XIST, FOXC1 and miR-137 was shown in Supplementary Figure 1.

Knockdown plasmids of TARBP2, SNHG7, NFATC3 were constructed on pGPU6/GFP/Neo vector (GenePharma, Shanghai, China) and were named as TARBP2(−), SNHG7(−), NFATC3(−) groups. Overexpression plasmids of TARBP2, SNHG7, NFATC3 were constructed on pcDNA3.1 and were named as TARBP2(+), SNHG7(+), NFATC3(+) groups. Respectively, the non-targeting sequences were used as NC groups. LTX and Plus reagent (Life Technologies, Carlsbad, CA, USA) were used to stably transfect plasmids mentioned above into ECs. The primers and probes used in this study were shown in Supplementary Table S1B.

Four short-hairpin MCM3AP-AS1 (sh-MCM3AP-AS1) plasmids and the respective non-targeting sequences (negative control, NC) (sh-NC) were synthesized (Geenseed Biotech Co., Guangzhou, China). KLF5 full length (with 3′-UTR) (KLF5(+) or KLF5) plasmid, short-hairpin KLF5 (sh-KLF5) plasmid, KLF5 (without 3′-UTR) (KLF5 (non-3′UTR)) plasmid and their respective non-targeting sequences (negative control, NC) (KLF5-NC or sh-NC), short-hairpin AGGF1 (sh-AGGF1) plasmid and the respective non-targeting sequences (negative control, NC) (sh-NC) were constructed (Life Technologies, Waltham, MA, United States). After seeded into 24-well plates (Corning), ECs were transfected with the plasmids via LTX and Plus reagent (Life Technologies) when they were at 70–80% confluence. Geneticin (G418; Sigma–Aldrich, St. Louis, MO, United States) was utilized to select the G418-resistant clones after 3∼4 weeks. miR-211 agomir (miR-211(+)), miR-211 antagomir (miR-211(-)) and their respective non-targeting sequences (negative control, NC) (miR-211(+)-NC or miR-211(-)-NC) (GenePharma, Shanghai, China) were transiently transfected into ECs using Opti-MEM and Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA, United States) when the confluence reached 70∼80%. Cells were collected at 48 h after transfection.