X tremegene sirna

Primers, Dynabeads protein A, Superscript VILO cDNA synthesis kit, VCP siRNA, and SVIP siRNA were purchased from Life Technologies (Carlsbad, CA), and X-tremeGENE siRNA and, HP DNA transfection reagent were purchased from Roche Applied Science (Indianapolis, IN). The rabbit polyclonal antibodies against AMFR and ubiquitin and anti-rabbit IgG (conformation specific) were purchased from Cell Signaling (Danvers, MA). The rabbit polyclonal antibody against N-terminus AMFR was purchased from LifeSpan BioScience (Seattle, WA). Polyclonal anti-rabbit antibody against SVIP and mouse monoclonal antibody against actin were purchased from Sigma-Aldrich (St. Louis, MO) Polyclonal anti-rabbit antibodies against p97/VCP and IP lysis buffer were purchased from Thermo Fisher scientific (Waltham, MA). Polyclonal anti-rabbit antibody against AAT was purchased from Dako (Carpentaria, CA).

shRNA was expressed using the CS‐RfA‐FB or CS‐RfA‐EP lentiviral vector as described previously 42. The target sequences of nontargeting control shRNA [NT; CAA CAA GAT GAA GAG CAC CAA (SHC002)] and HIC‐5 #2 [TCT CTG ACT TCC GCG TTC AAA (TRCN0000020103)] were obtained from Mission shRNA (Sigma‐Aldrich) and that of negative control shRNA (NC; GGA ATC TCA TTC GAT GCA TAC) and HIC‐5 #1 (TCA GTT CAA CAT CAC AGA TGA) were from SABiosciences (a Qiagen company, Hilden, Germany).
siRNA (ON‐TARGETplusSMARTpool siRNA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Transfection of siRNA (100 nm) was performed with X‐tremeGENE siRNA (Roche Applied Science, Penzberg, Upper Bavaria, Germany).

RAGE siRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) was transfected into the cells using X-tremeGENE siRNA (Roche Applied Science) according to the manufacturer’s protocol.

Transient transfection of eukaryotic cells was performed using FuGENE^® HD (Promega) and X-tremeGENE™ siRNA (Roche) according to the manufacturers´ instructions. Briefly, cells (1x10⁵ to 4x10⁵ cells/ml) were cultured in 24-well plates and plasmid DNA was added after 15 minutes (min) incubation with transfection reagent in OptiPRO SFM™ (Gibco) at a 1:3 ratio. Transfection of total RNA from VSV-infected cells was performed with X-tremeGENE™ siRNA analogous to FuGENE^® HD transfection.

For circMTCL1 or C1QBP overexpression in vitro, 436 or 858 bp cDNA fragments containing the circMTCL1 or C1QBP were cloned into a pLCDHor Y0009367 vector (GenePharma, Suzhou, China) between EcoRI and BamHI or between BspEI-BglII restriction sites. For circMTCL1 or C1QBP knockdown, cells were transfected with siRNAs (GenePharma, Suzhou, China) against circMTCL1 or C1QBP. siRNA sequences are shown in Tables S8 and S9.The plasmids or siRNAs were transiently transfected into cells following the manufacturer’s instructions using an X-treme GENE siRNA (4,476,093,001, Roche, Basel, Switzerland) or DNA Transfection Reagent (6,366,244,001,Roche, Basel, Switzerland).
For in vivo studies, a lentiviral vector containing circMTCL1 shRNA was synthesised in Hanbio (Shanghai, China), which was used for TU212 cell transfection to establish stable cell lines overexpressing circMTCL1. Lentiviral transfections were performed following the manufacturer’s instructions.

All human breast cancer cell lines and human embryonic kidney cells (HEK293) used in this study were obtained from ATCC (Manassas, VA, USA), and maintained according to ATCC’s instructions. All chemicals were purchased from Sigma (St Louis, MO, USA) unless otherwise stated.
MK‐2206 and rapamycin were purchased from Selleck Chemicals (Houston, TX). All siRNA were purchased from Bioneer (Oakland, CA). All transfections were performed with cells in exponential growth using either X‐tremeGENE siRNA (for siRNA) and X‐tremeGENE HP DNA (for plasmids) (Roche, Indianapolis, IN, USA).