Fig. 6

α-PD-1 therapy induces pSmad3 signaling in CCK168 tumors to enhance EMT and suppress gene expression of antigen presenting machinery. a-f CCK168 tumor-bearing mice were treated either with α-PD-1 monotherapy or IgG control antibodies on day 0 and 4. As indicated in (a), on day 8, the α-PD-1 treated group were randomly split into two further groups and treated with either control IgG or α-TGFβ monotherapy, and all tumors were harvested 2 h later for pSmad3 immunofluorescence staining. b and c quantification of the three arms of the experiment b as percentage of DAPI+ nuclei stained with pSmad3 and c intensity of pSmad3 staining per nucleus. d-f representative images of pSmad3 immunofluorescence staining. Note rim of tumor in α-TGFβ treated sample f shows dramatically reduced pSmad3 staining. g, h CCK168 tumor-bearing mice were treated with α-PD-1 or α-TGFβ on day 0 and day 4, and analyzed by pSmad3 immunofluorescence on day 8. i-n CCK168 cells grown in vitro were treated with TGFβ and/or α-TGFβ antibodies. I-m) phase contrast analysis shows reversible TGFβ-induced EMT. n RNA from cultures shown in (l-m) was extracted and subjected to qRT-PCR to quantify gene expression of components of the tumor cell antigen presentation machinery, Mhc1, B2M (β2-microglobulin), Tap1 and Tap2. * = P < 0.05, **** = P < 0.0001; Unpaired two-tailed Student’s T test