Figure 1.
Smad4 knockdown and KRASG12V expression in the H6c7 cell line.
(A) A schema of introducing KRASG12V and Smad4 loss using the H6c7 cell line to produce the shSmad4, H6c7 KRAS shSmad4, TβR, TβR-KRAS , and TβR-KRAS -Smad4 cell lines. (B) Immunoblot of RAS-GTP, KRAS, and Smad4. GAPDH was used as a loading control. (C) PAI-1 mRNA expression after 48 hours of TGF-β stimulation (n=3). (D) Invasion assays through Matrigel coated membranes incubated with and without TGF-β (n=6). (* denotes significant differences between the test and control NS samples, and between treatment and no treatment groups two-way ANOVA and Bonferroni’s post hoc tests or paired student t-test; p<0.05; data are presented as mean ± SEM) .
Figure 2.
Characterization of the TβR cell line.
(A) Smad and TGF-β receptors expression were assessed by qPCR and compared to the control H6c7 cell line. (B) Methylation specific PCR was performed on bisulfite treated gDNA isolated from H6c7 and TβR cells. Where U and M represent unmethylated and methylated, respectively. H6c7 and TβR cells were treated with 5-azacytidine and Smad4 expression was assessed by (C) qPCR and (D) immunoblotting. (* denotes significant differences between H6c7 and TβR cell lines or treated compared to vehicle where appropriate, student t-tests, p<0.05, n=3; data are presented as mean ± SEM) .
Figure 3.
KRASG12V-mediated transformation of the TβR cell line.
(A) Immunoblots of activated RAS and Smad4. GAPDH was used as a loading control. (B) Immunoblots of phospho- and total Smad2/3, and Smad4. GAPDH was used as a loading control. (C) Growth curves of H6c7, TβR, TβR pBp, and TβR KRAS with TGF-β. (D) PAI-1 mRNA expression was assessed after 48 hours of TGF-β in the H6c7 and TβR cell lines (n=3). (E) Invasion assays through Matrigel coated membranes incubated with and without TGF-β (n=6). (F) Tumor growth curve after subcutaneous implantation of the TβR-pBp and TβR-KRAS cell lines into NOD-SCID mice (n=20). Representative H&E sections of orthotopic TβR-KRAS (G) xenografts and (H and I) metastases found in the liver and spleen. (* denotes significant differences between H6c7 and TβR cell lines or treated compared to vehicle where appropriate, one-way or two-way ANOVA, or linear regression where appropriate, p<0.05; data are presented as mean ± SEM).
Figure 4.
Smad4 expression restores TGF-β sensitivity and represses tumorigenicity in the TβR-KRAS cell line.
(A) Immunoblots of phospho- and total Smad2/3, and Smad4. GAPDH was used as a loading control. (B) Growth curves of H6c7, TβR, TβR pBp, and TβR KRAS after restoration of Smad4 with TGF-β. (C) PAI-1 mRNA expression after 48 hours of TGF-β stimulation after forced Smad4 expression (n=3). (D) Invasion assays through Matrigel coated membranes incubated with and without TGF-β (n=6). (E) Tumor growth curves of TβR-KRAS- EV and TβR-KRAS-Smad4 (n=10). (F) The liver and spleens after orthotopic implantation of the TβR-KRAS and TβR-KRAS-Smad4 cell line. Scale bars represent 5 mm. (G) Total metastatic area of each TβR-KRAS-EV and TβR-KRAS-Smad4 orthotopic model. (H) Representative histological images of xenografts formed by TβR-KRAS-EV (n=16) and – Smad4 (n=19) cells after H&E, and immunostaining for Smad4, cleaved caspase-3, and Ki67. Scale bars represent 50 μm. (I) Quantification of Ki67, cleaved caspase-3, and cleaved PARP positive pixels of the TβR KRAS EV and – Smad4 xenografts. (* denotes significant differences between H6c7 and TβR cell lines or treated compared to vehicle where appropriate, two-way ANOVA, student t-test, or linear regression where appropriate, p<0.05; data are presented as mean ± SEM) .
Figure 5.
The gene expression changes due to Smad4 loss and KRASG12V expression.
(A) The evolution from normal pancreatic duct epithelial cell to tumor cell line. The pathway alterations found in the analyses of the genomic and expression arrays between each transition are listed above the arrows. The red and blue alterations represent gains or upregulation, and losses or downregulation, respectively. (B) The pathways that were altered in the pancreatic duct cell carcinogenesis model. Yellow and purple corresponds to changes seen in the TβR and TβR-KRAS cell lines, respectively.