Lipo3000 4 μg

The shRNAs plasmids and overexpression plasmids were constructed by Genechem (Shanghai, China). The plasmids were introduced into 200 μL Opti-MEM medium (Gibco, Thermo Fisher, Waltham, MA, USA). Transfection reagents (Lipo3000 4 μg and P3000 4 μg) (Invitrogen, Thermo Fisher, Waltham, MA, USA) were combined with 200 μL Opti-MEM medium. Following a 20-minute incubation period, the mixture was added to 293T cells. After 6–8 h, the medium was replaced with 2 mL DMEM medium (10% FBS). The virus solution was harvested 72 h later, filtered using a 0.22 μm filter, and applied to GH3 or GH4 cells. Another 6–8 h later, the medium was once again replaced with 2 mL of DMEM (10% FBS). To establish a stable transgenic cell line, selection with 2.5 mg/mL Puromycin was carried out for 3 days, commencing 72 h after infection.

Lentiviruses of shRNAs were constructed by Genechem (Shanghai, China). The target sequences are as follows:
GJD2:
sh1: GCAGCACTCCACTATGATTGG; sh2: GCATTTGTGTGGTGCTCAATC;
sh3: GGAGCAAGCGAGAAGATAAGA.
SSTR2:
sh1: CCAGCCCTTAAAGGCATGTTT; sh2: CCCTATCCTATATGCCTTCTT;
sh3: GCAGTCCTCACATTCATCTAT.
SSTR5:
sh1: CGTCACCAACATCTACATTCT; sh2: CTTCACCGTCAACATCGTCAA;
sh3: CAACCAGTTCACCAGTGTCTT
Viral capsid plasmids (PSPAX 1μg and PMD2G 1μg) and plasmids (NC-shRNA 2μg or GJD2-shRNA 2μg) were added to 200μL opti-mem medium (Gibco, Thermo Fisher, Waltham, MA, USA); transfection reagents (Lipo3000 4μg and P3000 4μg) (Invitrogen, Thermo Fisher, Waltham, MA, USA) were added to 200μL opti-mem medium. The two solutions were mixed, and after standing for 20 minutes, the mixed solution was added to 293T cells; after 6-8 hours, the solution was changed with 2mL DMEM medium (10%FBS). The virus solution was collected 72 hours later, filtered with a 0.22μm filter, and added to GH3 or GH4 cells. The virus solution was changed with 2mL DMEM medium (10% FBS) after 6-8 hours. The stable transformation cell line was obtained by screening with medium containing 2.5mg/mL Puromycin 72 hours later for 3 days.