4.10. STC1 Gene Knockdown (KD) and Knockout (KO)

The KD small interference RNA (siRNA) for STC1 and negative control (scrambled) siRNA were purchased from Qiagen (Germantown, MD, USA). The target sequence for STC1 siRNA is 5′-CTGCTTAAACAAAGCAGTATA-3′. siRNA transfection was performed according to the manufacturer’s instruction and as previously described [29], using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific). The KD efficiency was determined by western blot using an anti-STC1 antibody (Abcam). The KO of STC1 gene was performed as described previously [26]. The CRISPR gRNA containing 20-nucleotide target sequences of STC1 (3′ACCCACGAGCTGACTTCAAC-5′) was designed and prepared by GenScript (Piscataway, NJ, USA). Homogeneous cell populations (double allele KO cells) were selected for subsequent studies.