gRNA design and lentivirus infection
CRSIPR guides targeting Thrombin and PAR-1 of human and mouse sequences were generated and cloned into LentiCRISPRv2 at BsmBI restriction sites. LentiCRISPRv2 and packing constructs were transfected into 293T cells. Virus supernatants were collected 48 h after transfection. A549 and LLC cells were infected with viral supernatants in the presence of polybrene and were then selected in growth media containing puromycin. All the cell lines used have been tested and authenticated by karyotyping. Transfection with the empty plasmid was performed which was used as negative control (NC). A549THR-/- , LLCThr-/- , A549PAR-1-/- and LLCPar-1-/- cell lines were checked by PCR and sequencing.
Scoring ofimmunostained specimens
Immunostained specimens were reviewed by 2 investigators blinded to the patients’ clinical status using a multihead microscope. To evaluate expression of proteins using immunohistochemistry, the intensity of proteins staining was graded by consensus on a scale from 0 to 3 (0 = negative staining; 1 = weakly positive; 2 = moderately positive; 3 = strongly positive). The frequency of positive cells was graded by consensus on a scale from 0 to 4 (0 = less than 1%; 1 = 1% to 10%; 2 = 10% to 50%; 3 = 50% to 80%, 4 = greater than 80%). The immunohistochemical scores (HIS) were determined by staining intensity and positive cells. Based on the score of thrombin or PAR-1 in the central positive staining area in tumor section as a cutoff value, the score of thrombin or PAR-1 was classified positive (thrombin+ >2, and PAR-1 high expression >6) and negative (thrombin- ≤2, and PAR-1 low expression ≤6).