Geneticin g418

The short-hairpin RNA direct against human NFAT5 (NM_138714.3) gene or SBF2-AS1 (NR_036485.1) gene was reconstructed in pGPU6/Neo vector (NFAT5 (−)) or pGPU6/Hygro vector (SBF2-AS1(−)) (GenePharma, Shanghai, China), respectively. The empty vectors were used as NCs (NFAT5(−)NC, SBF2-AS1(-)NC). Human EGFL7 (NM_016215.4) gene coding sequence with or without 3′-UTR was ligated into pIRES2 vector (EGFL7(+)-CDS-3′-UTR, EGFL7(+)-CDS-3′-UTR) (GeneScript, Piscataway, NJ, USA), with the empty vector serving as the NC (EGFL7(+)NC). Stable cell lines were established via Geneticin (G418; Sigma-Aldrich, St Louis, MO, USA) and Hygromycin (Solarbio, China) selection. For transient transfection assays, agomiR-338-3p (miR-338-3p(+)), antagomiR-338-3p (miR-338-3p (−)), and their NC sequence (miR-338-3p (+)NC and miR-338-3p (−)NC) were synthesized (GenePharma). Cells were collected 48 h after transfection. Sequences of shNFAT5, shSBF2-AS1, and shNC were shown in Table S2. The transfection efficiency of NFAT5, SBF2-AS1, EGFL7, and miR-338-3p was shown in Figure S1.

CHO-K1 suspension cells were routinely cultured in growth medium containing 50% of EX-CELL 302 Serum-Free Medium (Sigma), 50% of CHO-K1 growth medium prepared in house, and 2 mM L-glutamine (Gibco). Expi293F (cat. no. A14635, Thermo Fisher Scientific) and Expi293F GnTI^- (cat. no. A39240, Thermo Fisher Scientific) suspension cells were grown in Expi293 Expression Medium (cat. no. A14351, Thermo Fisher Scientific). HEK293S GnTI^- adherent cells (cat. no. CRL-3022, ATCC) were adapted to suspension cultures internally and grown in FreeStyle 293 expression media (cat. no. 12338018, Thermo Fisher Scientific). HEK293-6E suspension cells which were initially developed by the National Research Council of Canada [42 (link)] were cultured in FreeStyle F17 media (cat. no. 1383501, Thermo Fisher Scientific) supplemented with 0.1% of Kolliphor P188 (cat. no. K4894, Sigma), 25 μg/mL of Geneticin G418 (cat. no. 10131027, Thermo Fisher Scientific) and 6 mM of L-glutamine (cat. no. A2916801, Thermo Fisher Scientific). All cells were incubated in a 37°C humidified shaker at 120 rpm with 5% CO₂ and split twice a week.

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.

PM was induced with the syngeneic colon adenocarcinoma CC531 cells (purchased at CLS Cell Lines Service GmbH, Eppelheim, Germany, product number 500,387). The cells were cultured in RMPI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Zwijndrecht, the Netherlands) in a 5% CO₂/air incubator at 37 °C.
CC531 cells were transduced with a neomycin-resistant firefly luciferase gene, as previously described [28 (link)]. Briefly, lentiviral particles were produced by transfecting HEK 293FT with the vectors pLenti PGK V5 luciferase (Addgene plasmid #21,471), pVSV-G, and pCMVd8.74. CC531 cells were transducted with these lentiviral particles, resulting in CC531-Luc cells. Selection with neomycin 2 mg/ml (Sigma-Aldrich geneticin G418, Zwijndrecht, the Netherlands) began 48 h after the transduction process to isolate the resistant colonies positively tested for luciferase activity. After at least three weeks of continuous selection, cells were used in experiments. Before injecting into the animals, the cells were tested for the absence of mycoplasma and rat-specific viral pathogens.

Short-hairpin KCNQ1OT1 (sh-KCNQ1OT1) plasmid and its respective non-targeting sequence (negative control, sh-NC), miR-370 agomir (pre-miR-370), miR-370 antagomir (anti-miR-370) and their respective non-targeting sequence (negative control, NC; pre-NC or anti-NC) were synthesized as previously described (GenePharma, Shanghai, China; Zhou et al., 2009 (link); Wang and Li, 2010 (link); Wang et al., 2012 (link)). CCNE2 full length (with 3′UTR) plasmid, CCNE2 (without 3′UTR) plasmid and their respective non-targeting sequence (negative control, NC) were synthesized as previously described (Life technology, Waltham, MA, USA; Pleet et al., 2016 (link)). We used Opti-MEM and Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA, USA) for transfection when cells were at 50%–70% confluence. Stable cell lines were obtained through the selection by means of Geneticin (G418; Sigma-Aldrich, St. Louis, MO, USA), G418-resistant clones were obtained after 3–4 weeks. We used qRT-PCR to detect the knockdown efficiency.