Additional file 4 of c-Rel drives pancreatic cancer metastasis through fibronectin-integrin signaling-induced isolation stress resistance and EMT

Supplementary Material 4. Supplementary Figure 4. A) REL mRNA fpkm values were compared with paired t tests from an RNA-seq experiment performed with human PDAC cell lines cultured under adherent and nonadherent conditions. B) Western blot analyses of patient cell lines cultured under adherent and nonadherent conditions. The data were normalized to the total protein signal. C) Surface expression of multiple CSC markers was assessed via flow cytometry. For each genotype, 6 separate lines were used. The cells were cultured under standard adherent conditions. One-way ANOVA with multiple comparison test results is given in the figure. D) A schematic demonstrating the auxin-inducible degree 2 (AID2) system. A degron (mAID, miniAID)-tagged c-Rel recruits OsTIR (F74G mutant), which facilitates E3 ligase complex formation in the vicinity after auxin analog (5phIAA) treatment. Subsequent protein ubiquitination leads to proteasome-mediated protein degradation in a short time. E) Green fluorescence images of stably transfected CCKP cells. While the empty vector produced GFP mostly in the nucleus, c-Rel tagged with GFP was present mostly in the cytoplasm. F) Flow cytometry histogram graphs of the GFP signal in degron cells indicating that 5phIAA induced a reduction in the GFP signal after 2 hours. Notably, CCKP cells already have a basal EGFP signal due to the recombined c-Rel flox allele. A time-dependent reduction in GFP-tagged c-Rel expression upon 5phIAA treatment is shown in the immunoblot below. G) Representative microscopy images of CCKP cells stably transfected with either empty vector (pAY15) or c-Rel (Rel-pAY15) and treated with 5 ng/mL TGFβ for two days. The relative surface expression of E-cadherin was analyzed via flow cytometry (c-Rel factor p=0.0078). H) Representative microscopy images of degron cell spheroids. The left and right panels show A2929 and A2770 CCKP cells, respectively, which were stably transfected. To rescue c-Rel expression from high to low, the auxin analog 5phIAA is given at multiple doses. The average spheroid diameter was quantified and analyzed (genotype X auxin factors A2929 p= 0.0009 and A2770 p=0.0007). I) Surface expression of CD61 was assessed via flow cytometry analysis of spheroids (genotype X auxin factors, A2929 p=0.0017 and A2770 p=0.0008). J) Flow cytometry analysis of the surface expression of EpCAM and CD133 in degron cells with or without 5phIAA treatment. Each dot represents one of the CCKP cell lines. Two-way ANOVA indicated that the variation factors were not significant. K) A multiplex IF image from CCKP, CKP and GCKP tumors are split to channels.