Cisplatin

Rabbit polyclonal anti-CYLD antibody (SAB4200060) was purchased from Sigma-Aldrich, Inc (Saint Louis, MO, USA). Rabbit monoclonal anti-EGFR antibody (#4267), rabbit monoclonal anti-Phospho-EGFR antibody (#3777), rabbit monoclonal anti-Akt antibody (#4691S), rabbit monoclonal anti-Phospho-Akt antibody (#4060S), rabbit monoclonal anti-ERK antibody (#4695), rabbit monoclonal anti-Phospho-ERK antibody (#4370S), rabbit monoclonal anti-Smad2/3 antibody (#3102S), and rabbit monoclonal anti-Phospho-Smad3 antibody (#9520S) were purchased from Cell Signaling Technology (Beverly, MA, USA). PI3K inhibitor (LY294002, HY-10108), MEK inhibitor (PD98059, HY-12028), and ALK5 inhibitor (TGF-β RI Kinase Inhibitor II, #616452) were purchased from Calbiochem (San Diego, CA, USA). Cisplatin (Nippon Kayaku, Tokyo, Japan), 5-FU (Sigma-Aldrich, Inc), cetuximab (Merck BioPharma, Darmstadt, Germany), gefitinib (Tocris Bioscience, Bristol, UK), erlotinib (Wako, Tokyo, Japan), afatinib (abcam, Cambridge, UK), and osimertinib (Selleck Chemicals, Houston, USA) were also used in this study. For other reagents, commercially available special grades were used.

Plasmid pSpCas9n-(BB)-2A-GFP (PX461) was purchased from Addgene (Watertown, MA, US) [23 (link), 24 (link)]. To create convenient 5′ overhangs for single guide (sg)RNA cloning in our setting, we digested the plasmid with BbsI restriction enzyme (NEB, Ipswich, MA, US) to insert phosphorylated and annealed oligo sets: set 1, (5′-CACCGTCTGTGATCTTGACATGCTG-3′ and 5′-AAACCAGCATGTCAAGATCACAGAC-3′) and set 2 (5′-CACCGGCTGGCCAAACTGCTGGGTG-3′ and 5′-AAACCACCCAGCAGTTTGGCCAGCC-3′). Long ssODNs were purchased from Sigma-Aldrich. Gefitinib was also from Sigma-Aldrich, and afatinib (BIBW2992) was from Cayman Chemical Company (Ann Arbor, MI, US). Cisplatin and taxol were purchased from Nippon Kayaku Co., Ltd. (Tokyo, Japan).

Cisplatin (Nippon Kayaku, Tokyo, Japan) was reconstituted with sterile 0.9% saline in a laminar air-flow hood under sterile conditions. Our preliminary experiment showed that the 50% lethal dose (LD₅₀) of Cisplatin was 16 mg/kg, therefore animals received i.p. chemotherapy with Cisplatin at a dose of 2 mg/kg (1/8 of the LD₅₀). This dose was approximately equivalent to the clinical dose used for treatment of cervical cancer in humans (40 mg/m²) (20 (link)) based on the ratio of mass and body surface area between mice and adult human females (21 (link)).

TACE was conducted using the following procedure: angiography was performed through the celiac and common hepatic arteries using a 3 or 4 Fr catheter, and digital subtraction angiography was carried out employing a nonionic iodine contrast agent. After assessing the tumor-contained segment using imaging techniques such as cone-beam computed tomography, a 1.7 or 1.9 Fr microcatheter (Piolax Inc., Kanagawa, Japan) was inserted towards the tumor-feeding artery. TACE was performed using 20–50 mg of epirubicin (Sawai Pharmaceutical Co., Ltd., Osaka, Japan) or cisplatin (Nippon Kayaku Co., Ltd., Tokyo, Japan) with lipiodol (Guerbet Co., Ltd., Tokyo, Japan), based on the size and number of tumors, in addition to absorbable gelatin sponge particles (Nippon Kayaku Co., Ltd.) [45 (link)]. Subsequent TACE procedures were repeated, as determined by the investigators.

Cells were plated into 96-well plates at 2,000~4,000 cells per well. Twenty-four hours later, the culture medium was replaced with fresh medium containing various drug concentrations, including cisplatin (Nippon Kayaku, Kyoto, Japan) paclitaxel (Bristol-Myers Squibb, New York, USA), gemcitabine (Eli Lilly Japan, Kobe, Japan) and liposomal doxorubicin (Janssen Pharmaceutical K.K., Tokyo, Japan) for 72 hours. The percentage of viable cells was then examined using the WST-1 assay kit following the manufacturer’s instructions (Premix WAT-1®, Takara Bio). All cytotoxicity data shown are the means of at least three independent experiments.