Scramble shrna

Lentiviral vectors encoding the mouse mPGES1 gene and a control lentiviral vector were provided by Keygen Biotech. Co. (Nanjing, China). Furthermore, the following short hairpin sequences were synthesized and cloned into the lentiviral vectors: mPGES1 shRNA, 5′-GATCCGCCAGCAGCTGAAGCCTCCTCACTCGAGTGAGGAGGCTTCAGCTGCTGGCTTTTTG-3′, and scramble shRNA, 5′-GATCCGCTGAAGGTCGCTTGGTTCAAGAGACCAAGCGACCTCCAGCATCTTTTTTG-3′. The lentiviral vectors were purified and then co-transfected with packaging vectors (psPAX₂ and PMD₂G) (Invitrogen, Carlsbad, CA, United States) into HEK293T cells. After 48 h, the lentiviral particles in the supernatant were concentrated by ultracentrifugation and resuspended in PBS(-). For mPGES1 knockdown, the lentiviral particles containing mPGES1 shRNA or scramble shRNA (Santa Cruz, Delaware, CA, United States) were adjusted to 10⁶–10⁷ titers before injection into the ventricles and hippocampus of mice.

Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol. For the experiments involving spheroid cells transduced with shRNA, spheroids were grown from 5 to 7 days and then dissociated into single cells using Accutase. Cells were then transduced with shRNA and placed back into spheroid culture. Experiments were performed after 5 days expect where otherwise noted.
Overexpression of PIK3R3 was achieved via Nanog lentiviral activation particles (sc-402964-LAC; Santa Cruz) per the manufacturer’s protocol. Controls were scramble shRNA (sc-108080; Santa Cruz) or empty lentiviral activation particles (sc-437282, Santa Cruz). Maximal knockdown of genes occurred 72–96 h after transduction.

Mouse PSCs were seeded in a six-well plate in the growth medium. The lentiviral particles containing shRNA of the targeted gene or scramble-shRNA (Santa Cruz Biotech, Santa Cruz, CA, USA) were added to the cells (15 μL/mL medium). After 12 h, the lentiviral particles containing medium were replaced by fresh growth medium, and cells were further cultured for another 48 h. The expression of target protein and the equal loading in the infected cells was detected by western blotting. The TGFRII-shRNA lentiviral particles were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA); two mouse β-catenin-shRNAs (targeting non-overlapping β-catenin sequence) lentiviral particles were designed and synthesized by Kaiji Biotech (Shanghai, China).