Recombinant adenoviruses expressing GFP alone (Ad-GFP), UCHL1 (Ad-UCHL1), siRNA-control, siRNA-UCHL1, or siRNA-EGFR were generated by Hanbio Biotechnology (Shanghai, China). To induce exogenous expression of UCHL1 in vivo, the pAV-CMV-mUCHL1(C-3 × Flag)-P2A-GFP vector (rAAV9-UCHL1) was used (ViGene Biosciences, Shandong, China). Mouse UCHL1 complementary DNA (cDNA) was first amplified by PCR from pCMV6-Entry-mUCHL1 plasmid, and a 3 × Flag-tag was added at the C terminus of UCHL1. The PCR products were digested with Asi SI and Mlu I and inserted into Asi SI Mlu I–digested pAAV9-p2A-GFP to construct the pAV-CMV-mUCHL1 (C-3 × Flag)-P2A-GFP vector (rAAV9-UCHL1). Both UCHL1-Flag and GFP could be expressed in the same cells and tissues and controlled by only one promoter, which was accomplished by using a “self-cleaving” 2A [porcine teschovirus-1 2A (P2A)] sequence (41, 42). AAV carrying GFP (rAAV-GFP) was simultaneously prepared as a control vector. For local knockdown of EGFR in vivo, rAAV9 vectors expressing short hairpin RNAs (shRNA) directed at EGFR (rAAV-siEGFR) or a control hairpin (rAAV-siControl) were used (ViGene Biosciences). The following primer sequences were used: EGFR-siRNA1, 5′-GGAAATTACCTATGTGCAA-3′; EGFR-siRNA2, 5′-AATGGACTTACAGA GCCATCC-3′. The shRNA expression was driven by a mouse U6 promoter (pol III) and used GFP as a reporter. The final virus in normal saline (saline) had a titer of 1 × 1013 to 3.5 × 1013 vg/ml.

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