Journal: Gastroenterology

Article Title: Systems Biology Analyses Reveal Hyperactivation of Transforming Growth Factor beta and JNK Signaling pathways in Esophageal Cancer

doi: 10.1053/j.gastro.2019.01.263

Figure Lengend Snippet: (A–B) IncuCyte-based growth assessments of SMAD4-null EsoAd1 cells treated with varying concentrations of TGFβRI inhibitors, SB431542 or LY2157299. Y-axes represent the average fold-change in cell confluence values normalized to time zero (X-axes) in respective treatment versus DMSO vehicle control groups. (C) IncuCyte-based cell (line graphs, Top Left) and colony growth (bar graphs, Bottom Left) assessments of EsoAd1 cells treated with siRNAs targeting TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, SMAD2, SMAD3, or SMAD4. Y-axes in line graphs represent the average fold-change in cell confluence values normalized to time zero (X-axes) in respective treatment versus non-targeting control siRNA groups. Y-axes in bar graphs represent the colony numbers for each treatment siRNA groups, expressed as a fraction of colony counts observed in the non-targeting control siRNA. Protein levels of ligands (bar graph) were quantified by ELISA and receptor/SMAD components by Western Blots, at 48hrs following siRNA treatments. For growth assays, cells were plated in individual wells in replicates and inhibitor/siRNA treatments were performed in each of these wells independently for subsequent analyses. All data are plotted as mean ± SEM, obtained from atleast six biologic replicate measurements. * (P < 0.05) and *** (P < 0.0005) indicate significant differences between the respective test versus control groups, estimated using a Student’s t-test assuming unequal variances.

Article Snippet: In vitro phenotypic characterization using EAC cell line models Cell growth assessments were quantified following seeding using the IncuCyte ZOOM automated live cell kinetic imaging system (Essen BioScience), as previously described by our group 5 .

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Gastroenterology

Article Title: Systems Biology Analyses Reveal Hyperactivation of Transforming Growth Factor beta and JNK Signaling pathways in Esophageal Cancer

doi: 10.1053/j.gastro.2019.01.263

Figure Lengend Snippet: (A) Assessment of FLO-1 colony growth in soft agar. Y-axis in the bar graph shows FLO-1 colony numbers following TGFβ1 (10ng/ml) treatment, expressed as a fraction of colony counts observed in the untreated Parental (P) cells. All data are plotted as mean ± SEM, obtained from atleast three independent experiments, each performed in triplicates. Significant differences in colony growth were estimated using a Student’s t-test assuming unequal variances. Shown below the bar graph are representative images of colony growth in Parental and TGFβ1 treated FLO-1 cells. Also shown below are Western blot images depicting phospho-SMAD induction at 48hrs following TGFβ1 treatment in FLO-1 cells. (B–D) IncuCyte-based growth assessments of FLO-1 cells treated with varying concentrations of TGFβRI inhibitors, SB431542 or LY2157299 (B,C), or with a JNK inhibitor, SP600125 (D). Y-axes represent the average fold-change in cell confluence values normalized to time zero (X-axes) in respective treatment versus DMSO vehicle controls. Also shown are representative Western blot images depicting the protein levels of total and phospho-SMAD/c-JUN, with β-actin as a loading control, at 48hrs following treatment. (E) IncuCyte-based cell (line graphs, Top Left) and colony growth (bar graphs, Bottom Left) assessments of FLO-1 cells treated with siRNAs targeting TGFβ1, TGFβ2, TGFβ3, TGFβRI, TGFβR2, SMAD2, SMAD3, or SMAD4. Y-axes in line graphs represent the average fold-change in cell confluence values normalized to time zero (X-axes) in respective treatment versus non-targeting control siRNA groups. Y-axes in bar graphs represent the colony numbers for each treatment siRNA groups, expressed as a fraction of colony counts observed in the non-targeting control siRNA. Protein levels of ligands (bar graph) were quantified by ELISA and receptor/SMAD components by Western Blots, at 48hrs following siRNA treatments. (F) IncuCyte-based cell (line graphs, Top) and colony growth (bar graphs, Bottom) assessments of FLO-1 cells treated with siRNAs targeting c-JUN, along with representative Western blot images depicting the protein levels of total and phospho-c-JUN at 48hrs post-treatment. For growth assays, cells were plated in individual wells in replicates and inhibitor/siRNA treatments were performed in each of these wells independently for subsequent analyses. All data are plotted as mean ± SEM, obtained from atleast six biologic replicate measurements. * (P < 0.05), ** (P<0.005), and *** (P < 0.0005) indicate significant differences between the respective test versus control groups, estimated using a Student’s t-test assuming unequal variances.

Article Snippet: In vitro phenotypic characterization using EAC cell line models Cell growth assessments were quantified following seeding using the IncuCyte ZOOM automated live cell kinetic imaging system (Essen BioScience), as previously described by our group 5 .

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Gastroenterology

Article Title: Systems Biology Analyses Reveal Hyperactivation of Transforming Growth Factor beta and JNK Signaling pathways in Esophageal Cancer

doi: 10.1053/j.gastro.2019.01.263

Figure Lengend Snippet: (A,B) IncuCyte-based cell growth assessments in non-dysplastic (CP-A) and dysplastic (CP-B, CP-C, CP-D) Barrett’s esophagus cells treated with (A) TGFβ1 ligand or untreated (Parental/P), or (B) with varying concentrations of TGFβRI kinase inhibitors, SB431542 (SB) and LY2157299 (LY), or with DMSO vehicle (V). Y-axes in line graphs represent the average fold-change in cell confluence values normalized to time zero (X-axes) in respective treatment versus control groups. Also shown are protein levels of SMAD components quantified by Western Blots at 48hrs post-treatment. For growth assays, cells were plated in individual wells in replicates and inhibitors treatments were performed in each of these wells independently for subsequent analyses. All data are plotted as mean ± SEM, obtained from atleast six biologic replicate measurements. * (P < 0.05) and *** (P < 0.0005) indicate significant differences in cell growth at the final time-point between the respective treatment versus control groups, estimated using a Student’s t-test assuming unequal variances.

Article Snippet: In vitro phenotypic characterization using EAC cell line models Cell growth assessments were quantified following seeding using the IncuCyte ZOOM automated live cell kinetic imaging system (Essen BioScience), as previously described by our group 5 .

Techniques: Western Blot