Lip2000

We confirmed the splicing effect of the synonymous variant c.774C > T using a pSPL3 minigene reporter vector as previously described (PMID: 29637721). In brief, genomic fragments containing the variants located in exon 6 were amplified by PCR from the patients' genomic DNA using primers (MBTPS1-EXON6: Forward accagaattctggagctcgagACGTCTAAGGGGATCGTAGA; Reverse 5′-atcaccagatatctgggatccTGCAGTATGAATGGCTCAGC-3′). Then, the PCR products were cloned into pSPL3 vector using the ClonExpressTM II One step Cloning Kit (Vazyme Biotech Co., Ltd). All constructs were sequenced by Sanger sequencing. HEK293 cells were cultured in 12-well plates and transfected with 1 µg purified pSPL3, wild-type and variant constructs plasmids using Lip2000 (Invitrogen). After overnight incubation, cells were transfected with ASO at the concentration of 0.5 μM. Forty-eight hours after ASO delivery, cells were harvested for transcriptional analysis by RT-PCR. Finally, aberrant splicing transcripts amplification by RT-PCR, PCR product separation by agarose gel and proven by Sanger sequencing were performed.

Notch1 shRNA, negative control, pCMV-Notch1 (H3176 pLenti-CMV-MCS-HA-3Flag-P2A-EGFPT2A-Puro), and control vectors were purchased from Oobio Corporation (Shanghai, China) and the vectors carry puromycin-resistance function. siRNA sequences of Notch1 or negative control (NC) were as follows: Notch1, GCAACAGCTCCTTCCACTT; NC, TTCTCCGAACGTGTCACGT. Lip2000 (Invitrogen, Carlsbad, USA) was used to transfect vectors into GC cells and then transfected cells were selected by treatment with puromycin. The effects of shRNA and pCMV-Notch1 were confirmed at protein level using western blot assay.

USP6NL promoter was amplified with the primers: 5′-CCCTCGAGTATCATTTAATGGGTTCTGGAGGTC-3′ (forward with XhoI site as indicated by the underline) and 5′-CCAAGCTTATAGAGTAACTTATCTATTCCAAGATTTC-3′ (reversed, HindIII site as indicated by the underline), and inserted into pGL3-Enhance to construct pGL3-Enhancer-p USP6NL. SW480 cells (5 × 10⁵ cells/well) on a 6-well plate were cultured at 37 °C overnight for adherent growth, and then transfected with plasmid pGL3-Enhancer-p USP6NL (1.5 μg) and internal control plasmid pRL-TK (0.05 μg) using Lip 2000 (11668-019, Invitrogen) according to a provided instruction. Besides, pGL3-Enhancer was used as a negative control.
6 h later, SW480 cells with USP6NL overexpression were maintain in fresh culture medium with or without 10058-F4 (100 μmol/l) at 37 °C for 24 h. After fully lysis, Dual-Luciferase^® Reporter Assay System (E1910, Promega) was used to detect the activity of firefly luciferase in plasmid pGL3-Enhancer and pGL3-Enhancer-p USP6NL, as well as the activity of renilla luciferase in internal control plasmid pRL-TK. The activity of USP6NL promoter was presented as the ratio of firefly/renilla. Each group set 3 wells, and the experiment was repeated 3 times.