Knockdown of gene expression by shRNA

For gene knockdown, two pairs of shRNA-coding DNA oligos were synthesized and subsequently cloned into the shRNA expression vector pGreenPuro (System Biosciences) according to the manufacturer’s instruction. The control shRNA is a scrambled sequence that does not target any mammalian mRNA. The sequences of the shRNA target sites are indicated below.

GPC3sh1: 5′-GGAGCTCAAGTTCTTAATTAT-3′; GPC3sh2: 5′-ACTGCAAGTCAC

TAGGATCTT-3′; FAT1sh1: 5′-GGACCAGTATCGCAAGAGTCA-3′; FAT1sh2: 5′-G

AAGACAAGGAGGTACATAGT-3′; HIF1αsh1: 5′-GAATGAAGTGTACCCTAACT

A-3′; HIF1αsh2: 5′-GACGATCATGCAGCTACTACA-3′; shCtrl: 5′- GCGTAATAAC

GATGTCTCTAC-3′

For lentivirus packaging, the packing plasmids p-Mission-Gag/Pol and p-Mission-VSV-G were mixed with expression plasmid pGreenPuro in 1 ml of Opti-Mem medium at a ratio of 3:1:1, then threefold excess of PEI solution was mixed with the plasmid solution. After the plasmids and PEI formed complex at room temperature for 20 min, the plasmid-PEI mixture were transferred to a T-75 flask of 293 T cells, gently mixed and placed in the CO₂ incubator for 3 days. The lentivirus-containing cell culture medium was collected and used to transduce target cells. Transduced cells were selected with 2 μg/ml of puromycin for 7 days to get stable knockdown cells.