Quickmutation site directed mutagenesis kit

Wild type (WT) LTBR gene promoter at different sites and the mutated (wt) sites were subcloned into the pGL3 luciferase reporter vector (GeneCopoeia). Mutation was performed using a QuickMutation™ Site-Directed Mutagenesis Kit (#D0206; Beyotime). HEK-293 T tool cells at the concentration of 5 × 10⁶ cells/well were transfected with pcDNA 3.1-CREB1 and the abovementioned pGL3 vectors containing LTBR gene promoter at different sites. Cell transfection was conducted using Lipofectamine 3000 reagent. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System (Promega).

The luciferase reporter assay was determined using the psi-CHECK2 dual-luciferase system (Promega, Madison, WI, USA) following the standard manual. The QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime, Shanghai, China) was applied for construction of EIF4G3 3′-UTR reporter plasmids with a mutant miR-375 binding site. Primer sequences used for construction of these plasmids were as follows: 5′-GCGCGATCGCAACTTCAAATACACAAAATG-3′ (EIF4G3-WT, forward), 5′-GCGTTTAAACCTGTCCAAAGGAGAAGTCAC-3′ (EIF4G3-WT, reverse); 5′-AGGCTTGTAAATACATACTTGTTTTATTTAAAAAAAC-3′ (EIF4G3-Mut1, forward), 5′-GTTTTTTTAAATAAAACAAGTATGTATTTACAAGCCT-3′ (EIF4G3-Mut1, reverse); 5′- CACTTTGAAAATATAAACTTGTTTTAAAGACAAAC-3′ (EIF4G3-Mut2, forward), and 5′-GTTTGTCTTTAAAACAAGTTTATATTTTCAAAGTG-3′ (EIF4G3-Mut2, reverse).

The circPDHK1 siRNA (si-circPDHK1#1) and PPP1CA siRNA (si-PPP1CA) utilized in the experimental procedures were synthesized by GenePharma (Shanghai, China). Additionally, circPDHK1 siRNA (si-circPDHK1#2) was synthesized by Tsingke (Beijing, China). The specific siRNA sequences are listed in Supplementary Table S3. The IRES and its truncated mutants were synthesized and cloned and inserted into the Luc2-Report plasmid (Geneseed, Guangdong, China) using EcoRI and BamHI sites. Full-length circPDHK1 and circPDHK1-3 × flag was synthesized and cloned and inserted into the pLC5-ciR vector (Geneseed) using EcoRI and BglII sites. The 3 × flag sequence is located after the start codon ATG in the predicted coding sequence (CDS) of circPDHK1. The circPDHK1-3 × flag ATG mutation overexpression vector was constructed by QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. The human PPP1CA overexpression vector pCDH-PPP1CA was synthesized by Tsingke, whereas the human HIF-2A overexpression vector pCDNA3.1-HIF-2A was obtained from Unibio (Chongqing, China). The plasmids were transfected using a commercial DNA transfection reagent (Neofect, Beijing, China) following the manufacturer’s instructions. siRNAs were transfected using Lipo8000 transfection reagent (Beyotime, Shanghai, China) following the manufacturer’s instructions.

The 3′ UTR of GPRIN1 was amplified from the full-length cDNA by PCR. After digestion with XhoI and NotI, the 3′-UTR fragment was cloned into the XhoI/NotI sites of the psiCHECK-2 vector (Promega) to obtain a wild-type (Wt) GPRIN1 3′ UTR-luciferase reporter plasmid. A mutant (Mut) GPRIN1 3′ UTR-luciferase reporter plasmid was constructed using a QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime). Luciferase reporter assay was performed by cotransfection with 250 ng of luciferase reporter vectors and miR-654-5p inhibitor or NC inhibitor (10 nM) using Lipofectamine 2000 (Invitrogen). After 48 h, the GC cells were assayed for luciferase activities on a Dual Luciferase Reporter Assay System (Promega). The firefly luciferase activity values were normalized to Renilla luciferase values to exclude the effects of difference in transfection efficiency.

All siRNAs, Flag-tagged OTUB1, Flag-tagged OTUD5, Myc-tagged CST1, and HA-tagged GPX4 plasmids were purchased from Vigene Biosciences (Shandong, China). All plasmids were generated by cloning the corresponding cDNA into the expression vector pCMV-MCS at the AsisI and M1uI restriction sites. D88A mutant plasmid of OTUB1 and K11A mutant plasmid of GPX4 were constructed in our laboratory by using QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime Biotechnology, Shanghai, China). HEK293T or GC cells were transfected with siRNAs and/or plasmids using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. To establish a stable cell line, the pLenti6.3/IRES/GFP lentiviral plasmid carrying green fluorescent protein (GFP) and puromycin resistance genes were used for the preparation of CST1 overexpression recombinant lentivirus. pLKO.1-puro lentiviral plasmid was used to prepare CST1 interference lentivirus. After 72 h of infection, 10 μg/ml puromycin was used for screening cells overexpressing CST1 and those with CST1 knockdown. Fluorescence microscope and flow cytometry were used to detect GFP and analyze the efficiency of lentivirus-mediated transgenesis. RT-qPCR and Western blot were used to detect overexpression and silencing efficiency of lentivirus-mediated CST1 in GC cells.

Full-length AppA_1-450 and OaPAC_1-366 contain additional downstream domains. In light of the potential influence of downstream domains on the conformational distribution of Trp (Supplementary Fig. 15), which can render fluorinated Trp less suitable as a readout, we have constructed AppA_1-124 and OaPAC_1-102, with a focus on the BLUF domain. The wild-type plasmids were constructed by incorporation of the protein sequence from GenBank, i.e., ABA77707.1 (AppA), AFY83176.1 (OaPAC), BAA18389.1 (SyPixD) into a pET28a vector at the NdeI-SacI, NdeI-HindIII, NdeI-BamHI restriction sites, respectively. Also, we constructed mutants plasmids (i.e., AppA_1-124 W64F, Y21F/W64F and W104F/W64F, OaPAC_1-102 Y6F and W90F, SyPixD_1-150 Y8F and W91F) based on the wild type plasmid by site-specific mutagenesis using QuickMutation site-directed mutagenesis kit (D0206S, Beyotime). The primers used in this work are listed in Supplementary Table 6.

The human DUB plasmid library and the HA-Ub WT, K11R, K27R, K29R, K33R, K48R and K63R plasmids were provided by Pro. CJ Gao [23 (link)]. The pCGN.CSH.FL42 plasmid encoding HA-tagged CDC6 was a gift from Pro. L. Drury (Clare Hall Laboratories, Cancer Research UK, London, England). The plasmids encoding Flag-YFP-N terminus (Flag-YN), HA-YFP-C terminus (HA-YC), Flag-YFP-N terminus-OTUD6A (YN-OTUD6A) or HA-YFP-C terminus-CDC6 (YC-CDC6) were purchase from GeneChem Inc. (Shanghai, China). The plasmids encoding Flag-tagged OTUD6A-N-terminal (1-145 aa) and OTUD6A-C-terminal (129–288 aa) and the plasmids encoding GST-tagged full-length OTUD6A, OTUD6A-N-terminal (1-145 aa) and OTUD6A-C-terminal (129–288 aa) were gifts from Pro. LY Huang [24 (link)]. The plasmids encoding the HA-tagged CDC6 AAA (S54A, S74A and S106A) and CDC6 DDD (S54D, S74D and S106D) mutants were gifts from Pro. JF Diffley [25 (link)]. The Myc-CDC6, Myc-CDH1 and Myc-Cyclin F plasmids were purchased from GeneCopoeia (Guangzhou, China). The OTUD6A catalytic site mutation (C152A) was generated by site-directed mutagenesis (QuickMutation™ Site-Directed Mutagenesis Kit, Beyotime) according to the manufacturer’s protocol. The primers used for the construction of mutant vectors are shown in Supplementary Table 1.