Htb 4

HEK293T (female), HeLa (female), and U251 (male) were maintained in HyClone Dulbecco’s High Glucose Modified Eagles medium with L-glutamine (GE #SH30081.01) plus 10% fetal bovine serum (Gibco #10437028) and 1% penicillin/streptomycin (Gibco #15140122). HEK293T was obtained from ATCC (ATCC #CRL-3216) and similarly for HeLa (ATCC #CCL-2). U251 was a kind gift from Roger Abounader. T24 bladder cancer cell line (female) was obtained from ATCC (ATCC #HTB-4) and maintained in MyCoy’s 5A medium with L-glutamine (Corning #10-050-CV) plus 10% FBS and 1% penicillin/streptomycin. Mycoplasma contamination was routinely checked by PCR kit (SouthernBiotech #13100-01). Cells were grown in humidified incubators with 5% CO₂ at 37 °C.
To construct stable HEK293T cells expressing Flag-HA-Ago2, HEK293T cells were first co-transfected with pLJM1-Flag-HA-Ago2-WT, pMD2.G and psPAX2 to package lentivirus. 48 h after transfection, supernatant containing virus was harvested and used to transduce HEK293T cells. Puromycin selection was performed for 5 days. pLJM1-Flag-HA-Ago2-WT (Addgene plasmid #91978) was a gift from Joshua Mendell^{70 (link)}. pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) were gifts from Didier Trono. The expression of tagged Ago2 was confirmed by western blotting against pan-Ago (Millipore #MABE56, clone 2A8) (Fig. 5a) and against FLAG (Sigma #F-1804) (Fig. 5c).

The human urinary bladder cell carcinoma line T24 (ATCC, HTB-4) was purchased from ATCC and was cultured in McCoy’s 5A (Modified) medium (Cat.Nr.: 16600082, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cat.NR.:10082147, Thermo Fisher Scientific) and 1% HEPES buffer (1M) (Cat.Nr.: 15630056, Thermo Fisher Scientific) and 1% PenStrep (Cat.Nr.: 15140122, Thermo Fisher Scientific). Cells were cultured at both 1% and 21% atmosphere oxygen pressure in an InVivO2 400 hypoxic chamber (Baker Ruskinn, Sanford, MA, USA) and maintained at 37 ${}^{\circ }$ celsius, 5% CO ${}_2$ and in a humidified atmosphere. Cell irradiation was carried out using a Multi Rad 225kV irradiator. Cells were initially synchronized (see next section for protocol) prior to irradiation with 6 Gy at room temperature. The cells that received 0 Gy (control) and 6 Gy were then plated into T25 flask to allow colony formation over 10 to 12 days. A total of 220 and 7500 cells were platted per flask, respectively for the 0 Gy and 6 Gy dose values. A total of 5 flasks were made per dose point. Cell survival was evaluated using a standard colony forming assay, where colonies were counted after 10 to 12 days.

Human bladder transitional cell carcinoma T24 (ATCC® HTB4™), TCCSUP (ATCC® HTB5™), and non-tumorigenic human bladder epithelial cells HBlEpC (938-05a) (Cell Applications Inc.) were cultured in McCoy’s 5A and Eagle’s Minimum Essential Medium (Millipore-Sigma), respectively, with 1% penicillin–streptomycin containing 10% fetal bovine serum (Gibco, Life Technologies) at 37 °C, in a 5% CO₂atmosphere. It was demonstrated that in vitro models can adequately reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of bladder cancer (Vallo et al. 2015 (link)).

The T24 cells used in functional validation of METTL3 were purchased from ATCC (HTB-4) and grown in McCoy’s 5A medium (Gibco, 16600) supplemented with 10% FBS (Gibco), and 1% penicillin–streptomycin (Gibco, 15140). The 5637 cells were purchased from ATCC (HTB-9) and grown in the RPMI-1640 medium (Gibco, 11875) supplemented with 10% FBS and 1% penicillin–streptomycin. Both cell lines were not tested for mycoplasma contamination and authenticated. Construction of the pcDNA3 plasmids for the expression of mutated METTL3 in mammalian cells was achieved by using a Q5^® Site-Directed Mutagenesis Kit (NEB) following the manufacturer’s protocols.
Sequencing primer used are provided in Supplementary Note 7. All siRNAs were ordered from QIAGEN. All stars negative control siRNA (1027281) was used as a siRNA control. Sequences for METTL3 is 5′-CGTCAGTATCTTGGGCAAGTT-3′. Transfection was achieved by using Lipofectamine RNAiMAX (Invitrogen) for siRNA, or Lipofectamine 2000 (Invitrogen) for the plasmids following manufacturer’s protocols.

Human bladder epithelial SV40 immortalized cell line, SV-HUC-1 (ATCC® CRL-9520), was obtained from ATCC (Manassas, VA, USA) and cultured in F-12K Medium (Catalog No. 30-2004, ATCC) supplemented with 10% FBS. A bladder cancer cell line, T24 (ATCC® HTB-4), was obtained from ATCC and cultured in McCoy's 5a Medium Modified (Catalog No. 30-2007, ATCC) supplemented with 10% FBS. All cells were cultured at 37° C in 5% CO₂.
The expression of miR-140-5p was modulated by transfection of miR-140-5p or anti-miR-140-5p (GenePharma, Shanghai, China). FGF9 or LINC01140 was knocked down by transfection of si-FGF9 or si-LINC01140 (GenePharma). All transfections were performed using the transfection agent Lipofectamine 3000 (Invitrogen). The relative sequences are shown in Supplementary Table 2.

Four bladder cancer cell lines, J82 (ATCC^® HTB-1^™), HT-1376 (ATCC^® CRL-1472^™), RT4 (ATCC^® HTB-2^™), and T24 (ATCC^® HTB-4^™) and a normal human urothelial cell line, SV-HUC-1 (ATCC® CRL-9520™), was obtained from ATCC (Manassas, VA, USA). J82 and HT-1376 cell lines were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma). RT4 and T24 cells were cultured in McCoy's 5a Modified Medium (ATCC, Catalog No. 30-2007) supplemented with 10% FBS. All cells were cultured at 37°C with 5% CO₂.