Jetprime transfection reagent

Human ADAM17-directed shRNA was obtained from Sigma. To prepare lentiviral particles, 293T human embryonic kidneys were transfected using Jetprime transfection reagents (Polyplus transfection) as per the manufacturer's instructions. PC3 cells were infected with the lentivirus from transfected 293T human embryonic kidneys and were subsequently selected using puromycin (2 μg ml⁻¹). shRNA: 5′-CCGGCCTATGTCGATGCTGAACAAACTCGAGTTTGTTCAGCATCGACATAGGTTTTTG-3′ (Clone ID:NM_003183.3-2002s1c1).
Human CUX1a sequence: 5′-AACAGGAGGACACAAGGCAAAGCUG-3′and CUX1b sequence: 5′-CAGGGUUUGUUUAAUACACUCCAUU-3′ were custom siRNA synthesized (Dharmacon). siRNA transfection was performed using Jetprime transfection reagents (Polyplus transfection) as per the manufacturer's instructions.

Vectors containing the cDNA of WT IKKβ were obtained by amplifying mouse cDNA with Phanta® Super-Fidelity DNA Polymerase (cat. No. P511-01; Vazyme Biotech co., ltd, Nanjing, China). The IKKβ S177A mutant was obtained by amplifying the cDNA of WT IKKβ, followed by insertion into a pLVX-IRES-ZsPuro lentiviral expression vector (Clontech, Shiga, Japan). Lentivirus was produced by co-transfection of lentivirus packing plasmids with psPAX2 and pMD2.G using the Jet PRIME transfection reagents (PolyPlus) into HEK293 cells according to the manufacturer’s instructions. The medium was changed after 24 h, and the medium containing the virus was collected after another 72 h, followed by 10 min of centrifugation at 2000 × g and the supernatant was collected and stored at −80 °C. The supernatant was used to infect NIH3T3 cells with 10 μg/ml polybrene (cat. No. H9268; Sigma-Aldrich), and resistant colonies were selected after 8 h using 50 μg/ml puromycin (cat. No. 58-58-2; Sangon Biotech). miR-146a and anti-miR-146a vectors were purchased from GeneCopoeia (Guangzhou, China), and the miR-146a and anti-miR-146a lentivirus were obtained by co-transfection of lentivirus packing plasmids with psPAX2 and pMD2.G using the Jet PRIME transfection reagents (PolyPlus) into HEK293 cells according to the manufacturer’s instructions as described above.

For overexpression of miR-146a mimics or inhibitors, NIH3T3, MRC-5 cells, or HEK293 cells were transfected with RNA at a final concentration of 50 nM using Jet PRIME transfection reagents (PolyPlus, Illkirch, France) according to the manufacturer’s instructions. Negative control mimics or inhibitors (GeneCopoeia) were transfected to serve as matched controls. For overexpression of NAMPT with its 3′-UTR, cDNA encoding NAMPT with the WT or MUT 3′-UTR was amplified and inserted into pZX-FR01 (GeneCopoeia), followed by transfection using Jet PRIME transfection reagents (PolyPlus) into NIH3T3 cells according to the manufacturer’s instructions.

The human FBXL10 expression plasmid was used as previously described [43 (link)]. The GFP-SNAI1, HA-SLUG, Flag-ZEB1, Myc-HDAC1 and Myc-HDAC2 were constructed from our lab [58 (link)]. p300 plasmid was mentioned in our previous study [56 (link)]. For HEK293T, polyetherimide was used for transfection reagent. Breast cancer cells were transfected with jetPRIME transfection reagent (Polyplus, 114-15) according to the manufacturer’s specifications. The siRNAs for human FBXL10 were obtained from Hippobiotec, the sequences were listed: 5′-GCAAACAGAGUGACAUCUUTT-3′ (#1 sense), 5′-AAGAUGUCACUCUGUUUGCCT-3′ (#1 antisense); 5′-GGACCCAUCUCACUGAGUUTT-3′ (#2 sense), 5′-AACUCAGUGAGAUGGGUCCAT-3′ (#2 antisense); 5′-CCAUCUGCAAUGAAAUCAUTT-3′ (#3 sense), 5′-AUGAUUUCAUUGCAGAUGGAG-3′ (#3 antisense). The sequences of siRNAs targeted for SNAI1 were listed: 5′-CCACUCAGAUGUCAAGAAGUA-3′ (#1 sense), 5′- UACUUCUUGACAUCUGAGUGG-3′ (#1 antisense); 5′-CCAAUCGGAAGCCUAACUACA-3′ (#2 sense), 5′-UGUAGUUAGGCUUCCGAUUGG-3′ (#2 antisense). The targeted sequence of shRNA for mouse FBXL10 was 5′-GCTGTGGAAATATCTGTCATA-3′, shRNAs were constructed using pRNAT-U6.1/Hygro.

lnc-AROD, CUEDC2, and lnc-AROD-ORF1/ORF2-FLAG were cloned into the pCDH-CMV-MCS-EF1-copGFP plasmid (P0268; MiaolingBio, Wuhan, China); these constructs were named pCDH-AROD, pCDH-ORF1, pCDH-ORF2, and pCDH-CUEDC2. The full-length lnc-AROD, lnc-AROD-del, CUEDC2–3′-UTR, and CUEDC2–3′-UTR-del were inserted into the pGLO plasmid (Promega, Madison, WI, USA) and were named pGLO-AROD, pGLO-AROD-DEL, pGLO-CUEDC2–3′-UTR, and pGLO-CUEDC2–3′-UTR-del. Argonaute2 (AGO2) was cloned into the PCDH-3flag-EGFP plasmid (catalog number 21538; Addgene), and lnc-AROD was cloned into the pMS2 plasmid, which was gifted from Myriam Gorospe (47 (link)). lnc-AROD-specific siRNAs and CUEDC2-specific siRNAs were designed by Thermo RNAi Designer and transfected into human lung adenocarcinoma epithelial (A549) cells using jetPRIME transfection reagent (Polyplus, France) according to the manufacturer's instructions. The siRNA sequences used in this study are listed in Table S1. The miRNA mimics or inhibitors were synthesized by GenePharma (Shanghai, China), and their sequences are listed in Table S1.