Fig 2: Immunogold-TEM characterization of HT-22 exosomes. (A) IgG control for single immunogold labeling. (B) Single immunogold labeling shows that exosome-like vesicles secreted by HT-22 cells express Wnt3a, Wnt5a, Wnt7a, and GPR177. (C) IgG control for double immunogold. (D) Doubled immunogold analysis detected co-expression of Wnt5a and GPR177. Red and black arrows show the presence of Wnt5 and GPR177, respectively.

Fig 3: HDACi-resistant HCT-R cells exhibit high expression levels of pAKT, a downstream effector of WNT5A and ROR2.(A) HDACi-resistant HCT-R cells express higher levels of pAKT than HDACi-sensitive HCT-116 cells. Cells were plated in 6-well plates at 450,000 cells per well, and at 24 hrs were exposed to mock (M) or 5 mM butyrate (B) treatment for 17 hrs. Total protein lysates were analyzed by western blot analyses, and levels of Ser473-phosphorylated AKT (pAKT), total AKT(AKT), and ACTIN were detected as described in Materials and Methods. (B) HCT-R cells are more resistant to 5-fluorouracil (5-FU) induced apoptosis than parental HDACi-sensitive HCT-116 cells. Cells were exposed to 1.5 mM 5-FU for 24 hrs, and apoptotic assays were performed as described in Materials and Methods. Percentage of live cells was calculated by dividing the number of live cells by the total number of analyzed cells. (C) Silencing of ROR2 expression in HCT-R cells decreases the levels of Ser473-phosphorylated AKT. One-million cells were nucleofected with control or ROR2 siRNAs (175 pmol, Invitrogen), and plated at 500,000 per well in 6-well plates. At 24 hrs post-transfection, the cells were exposed to mock (M) or 5 mM butyrate (B) for 19 hrs. Total cell lysates were analyzed by western blot; a representative western blot is shown. The bar graph under the western blot image represents the quantification of pAKT levels in three independent experiments by densitometry, the difference between butyrate-treated cells are statistically significant (P<0.05), (D).Overexpression of recombinant WNT5A in HCT-116 cells results in increased pAKT levels. HCT-116 cells were transfected with an empty vector or a WNT5A expression vector, and selected for stable expression of these constructs. Duplicate samples were analyzed for expression levels of WNT5A and pAKT as described above; a representative western blot is shown. The bar graph under the western blot represents the quantification of pAKT in three independent experiments by densitometry, P<0.05. (E) Representative western blot analysis of SW620 human CC cells and SW620R cells that grow at 3 mM butyrate. The two cell lines were exposed to mock (m) or 5 mM butyrate (b) treatment for 17 hrs and analyzed as in Fig.2A. (F). SW620 cells expressing exogenous WNT5A exhibit suppressed induction of Wnt/beta-catenin transcription by butyrate, P<0.05. Transfection and analyses were performed as described in Fig. 1D.

Fig 4: HDACi-resistant HCT-R cells suppress Wnt/beta-catenin activity by overexpressing mediators of noncanonical Wnt signaling.(A) Representative western blot analyses of CC cells and their conditioned media after exposure to mock treatment (M) or 5 mM butyrate (B) for 19 hrs. (B and C). Silencing of WNT5A and ROR2 expression increases Wnt/catenin transcriptional activity of HCT-116 (B) and HCT-R (C) cells. Cells (50,000/well) were co-transfected with reporters for Wnt/catenin transcriptional activity (TopFlash or FopFlash, 50 ng/well), pRL-TK as a control for transfection efficiency, and control, ROR2, or WNT5A siRNAs (10 pmol/well). Nucleic acids were introduced in the cells via a reverse transfection protocol with Lipofectamine 2000 in 96-well plates. At 31 hrs post-transfection, cells were exposed for 20 hrs to mock (m) or 5 mM butyrate (b) treatment. Wnt transcriptional activity was calculated as the ratio of the activity of TopFlash (with wild type Lef/Tcf binding sites) and FopFlash (with mutant Lef/Tcf binding sites). Each transfection was performed in duplicate wells; data represent the mean from results of three experiments. (D). Overexpression of WNT5A suppresses canonical Wnt transcriptional activity of HCT-116 cells. The co-transfection of HCT-116 cells (50,000/well) with the Wnt activity reporters TopFlash or FopFlash (50 ng per well), and pcDNANeo3.1 or WNT5A-expressing construct (150 ng per well) were carried out through a reverse transfection with Lipofectamine (0.7 µl/well) in 96-well plates. Post-transfection, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 20 hrs. Each transfection was performed in duplicate wells; data represent the mean from results of at least three experiments. (E) Suppression of ROR2 expression increases the sensitivity of the HDACi-resistant HCT-R cells to the growth-suppressive effect of butyrate. HCT-R cells (106) were nucleofected with 175 pmol of ROR2 (Invitrogen) or control siRNA, and 500,000 cells were plated per well in a 6-well plate. At 24 hrs, cells were exposed to mock treatment (m) or 5 mM butyrate (b) for 17 hrs. At 48 hrs post-nucleofection, cells were plated at 100 or 200 per well in triplicates. Percent clonal growth was calculated as the number of colonies arising from a single cell suspension of 100 cells. The experiment was repeated three times and the results are the mean from the data of the three experiments. Statistically significant differences are noted by stars.

Fig 5: Western blot analysis for confirmation of differential expression at the protein level of 3 selected candidates—KRT19, WNT5a and SOX2. GAPDH occurred as normalization protein