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).