HT3 cells were transfected with 30 n
M siRNA (SCCA1 (h) sc-40950 or
scramble siRNA sc-37007, Santa Cruz Biotechnology, Dallas, TX, USA) with
Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) per manufacturer’s protocol. Knockdown was evaluated by qRT-PCR (primers in
Supplementary Table 1) and WB after 24 and 48 h respectively. siRNA treated cells were seeded for radiation 24 h later. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated knockout of
SERPINB3 was achieved using the
LentiCRISPR V2 (Addgene 52961, Addgene, Cambridge, MA, USA) plasmid with either an empty guide RNA (gRNA) cassette or gRNA directed to the
SERPINB3 sequence inserted as previously described (Shalem
et al, 2014 (
link)). Briefly, an all-in-one lentiviral plasmid,
LentiCRISPR V2 (Addgene 52961), was engineered to house the
SERPINB3-specific single guide RNA (sgRNA), 5′-CACCGGCCTGTACATCCTCCAGCG-3′, identified using the publically available tool WU-CRISPR (available at
http://crispr.wustl.edu/). 293 T cells were transduced with the plasmid for viral packaging, and HT3 and SW756 parent cell lines were infected with lentivirus. Puromycin-selected single cell clones were established, and knockout confirmed with WB analysis and genomic sequencing for
SERPINB3 and potential off-target loci (
Supplementary Table 1).
Markovina S., Wang S., Henke L.E., Luke C.J., Pak S.C., DeWees T., Pfeifer J.D., Schwarz J.K., Liu W., Chen S., Mutch D., Wang X., Powell M.A., Siegel B.A., Dehdashti F., Silverman G.A, & Grigsby P.W. (2017). Serum squamous cell carcinoma antigen as an early indicator of response during therapy of cervical cancer. British Journal of Cancer, 118(1), 72-78.
