Puromycin

NONHSAT076754 was cloned into a pLVX-IRES-Puro expression vector (BD Biosciences, Franklin Lakes, NJ, USA). TPC1 cells were infected with the viral suspension 24 h before the start of the assay. We obtained stably transfected clones by Puromycin (Promega, 2 μg/mL) selection. A stable transfectant of the pLVX-IRES-Puro empty vector was used as a control. The expression of NONHSAT076754 was silenced by an antisense oligonucleotide (the antisense oligonucleotide sequence is 5’-TGATGTGGTGGTCTGGTCTC-3’ while the sequence of the negative control is 5’-TCTGCTCACTTGCATGCCTT-3’). The oligonucleotides were transfected into the cells at a concentration of 100 nM using Lipofectamine™2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

A549 cells were trasfected using TOPflash TCF reporter plasmid (wild type TCF binding site, Millipore), FOPflash plasmid (mutant TCF binding site, Millipore) and Lipofectamine²⁰⁰⁰ (Invitrogen) in an antibiotics-free medium. FOPflash-normalized TOPflash luciferase activity was represented to the relative TCF/LEF luciferase activity. A549 cells were also transfected using the NF-κB reporter (5×10⁵ TU; SABioscience), in an antibiotic-free medium. After 48 h, the medium was changed to culture medium containing 10% FBS, and transduced cells were selected in culture medium with 30 µg/mL of puromycin (Sigma, MO). After puromycin selection, cells (1×10⁴ cells/well) were incubated in a 96-well plate for 24 h. After TOP/FOPflash or NF-κB reporter transfection, serum deprived cells were treated with TGF-β1 and KY-05009 for 48 h. Cells were washed with PBS, then lysed using passive lysis buffer (Promega). Luciferase activity was evaluated using the luciferase reporter assay (Promega). All experiments were performed in triplicate.

Mouse Smg-1 and Smg-7 siRNAs have been described previously^{34 (link)}. Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′), Lat1 (target sequence: 5′-AGUGAAAGAGCAAGCCCAA-3′), Lat3 (target sequence: 5′-UGAAAAAGACCAAACUCAU-3′), and Snat2 (target sequence 5′-UGUUAGCGUCGGCAUUCAA-3′) siRNAs were designed using i-Score Designer^{40 (link)} and asymmetric siRNA^{41 (link)} were synthesized (GeneDesign, Inc.). Other synthetic siRNAs were purchased from Qiagen: mouse Abcc4, FlexiTube siRNA SI02833019; mouse Atf4, FlexiTube siRNA SI00905905; mouse Perk, FlexiTube siRNA SI01319269; and the All Star Negative Control siRNA. Synthetic siRNA transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and the cells were analyzed 48−64 h after transfection. The results were confirmed in more than 3 independent experiments.
The plasmid-expressed human eIF2α-S52A mutant was constructed by site-directed mutagenesis of pSR-strep-HA-eIF2α^{38 (link)}. Subsequently, a puromycin resistance gene expression cassette derived from silentGene-puro (Promega) was inserted into wild-type (WT) and S52A mutant eIF2α expression (SA) plasmids to generate pSR-strep-HA- eIF2α_puro and pSR-strep-HA-eIF2α-S52A_puro. The transfections of plasmid siRNAs were carried out using Lipofectamine 3000 (Thermo Fisher Scientific) and stable cell lines were selected by puromycin (Sigma-Aldrich).

BV2 cells (1 × 10⁵ cells per well) were transfected with shRNA plasmids targeting murine AnxA1 from the MISSION TRC shRNA collection (Sigma-Aldrich, St. Louis, MO, USA), or with an empty plasmid control (termed pKCON). Cells were transfected for 48 h using FuGENE HD (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions, followed by selection for stable clones using puromycin (Promega, Madison, Wisconsin, USA). Transfection was confirmed by western blot analysis.

HeLa MLH1, MSH2, HLTF, and SHPRH knockout cell lines and the HLTF and SHPRH double knockout cell line were generated by CRISPR-Cas9 technologies, using single guide RNA (sgRNA) sequences (Table 1) for each of the genes listed. The LentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid #52961). The plasmid was digested with BsmBI and gel purified using the QIAquick PCR purification kit according to the manufacturer’s instructions. Complementary oligonucleotides (synthesized by Integrated DNA Technologies) encoding the sgRNA were then annealed and cloned into LentiCRISPRv2. Cells were then transfected with Lipofectamine 3000 (Thermo Scientific L3000008) and the cells were selected with puromycin (Promega). Single cell clones were allowed to grow up under puromycin selection and expanded. Loss of protein expression was confirmed for each clone using SDS-PAGE and western blot analysis.

Five sgRNA sequences near the N-terminus of the RBM45 genomic locus were selected using the CRISPR Design Tool (http://crispr.mit.edu) [38 (link)]. Oligos encoding the five sgRNAs were individually cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (RRID:Addgene_62988) and transfected into HEK293 cells. Cells were selected in the presence of 5 μg/ml puromycin (InvivoGen) for 48 h before functional validation of sgRNAs. The cleavage efficiency of each sgRNA was evaluated by the TIDE (Tracking of Indels by DEcomposition) assay [39 (link)]. Briefly, genomic DNA from puromycin selected cells and wild-type HEK293 cells was extracted (Promega Wizard Genomic DNA Purification Kit #A1120; Promega, Madison, WI, USA) for PCR amplification of a 535 bp region flanking the sgRNA cleavage site. The PCR DNAs were gel purified (Promega Wizard SV Gel and PCR Clean-up System #A9282) and 30 ng of each PCR DNA was Sanger sequenced. The sequencing result for each sgRNA was aligned to the wild-type control by TIDE software (https://tide.nki.nl/) and the resulting traces were used to calculate cleavage efficiencies [39 (link)]. The two sgRNAs with the highest cleavage efficiency (both 85%) were chosen for co-transfection with a homology-directed repair (HDR) template for CRISPR-Cas9 genome editing.