Ab181496

The pathological sections of NSCLC patients’ tumor tissues were retrieved by microwave antigen retrieval (Citrate-EDTA Antigen Retrieval Solution, Beyotime, Beijing, China). The sections were blocked with 5% BSA and incubated with rabbit polyclonal FN antibody (1:200, ab2431, Abcam, Cambridge, UK) and mouse monoclonal CD90 (1:100, ab181496, Abcam, Cambridge, UK) overnight at 4 °C. Signal amplification staining was performed using the ABC HRP Kit (Thermo, MA, USA) and counterstained with hematoxylin. Next, samples were treated with dehydration, cleaned with xylene and covered with neutral resin. Sections were then captured with microscope (Leica, Barnack, Germany). To further analyze the number of CD90 positive CAFs in tumor tissues, we randomly selected 25 views of the IHC sections. The number of CD90 positive cells were counted, and the average of 25 views were calculated. The low malignant group were set as control (value = 1), and the high malignant group were calculated relative to the control (Fig. S1A). The relative expression of FN was analyzed by using the Image-Pro Plus 2.0 software.

The pathological sections of NSCLC patients’ tumor tissues were retrieved by microwave antigen retrieval (Citrate-EDTA Antigen Retrieval Solution, Beyotime). The sections were then blocked with 5% BSA and incubated with mouse monoclonal anti-integrin αvβ3 (1:100, ab7166, Abcam, UK), rabbit monoclonal SOX2 (1:200, ab92494, Abcam, Cambridge, UK), rabbit polyclonal p-AKT (1:200, ab38449, Abcam, Cambridge, UK), rabbit monoclonal KLF (1:200, ab215036, Abcam, Cambridge, UK), rabbit monoclonal total-PI3K (1:500, ab302958, Abcam, Cambridge, UK), mouse monoclonal CD90 (1:100, ab181496, Abcam, Cambridge, UK) for 4 °C overnight, and followed by horse radish peroxidase (HRP) conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI. In addition, the A594 and Lewis cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% triton-X100, and blocked by 5%BSA, followed by incubating with anti-SOX2 antibody (1:200, Abcam, Cambridge, UK) for 4 °C overnight, and followed by HRP conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI. All immunofluorescence images were captured from FV1000 laser scanning confocal microscope (Leica, Barnack, Germany) and analyzed by IPP software.