wnt β catenin signaling activity  (BPS Bioscience)

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of HER2 G776S mutation on the HER2 signaling pathway and anchorage-independent growth in several cell lines. ( A ) Detection of APC proteins in each cell by Western blotting. Full-length APC was detected with APC antibody (#2504, Cell Signaling Technology), and truncated APC was detected with APC antibody (sc-9998, Santa Cruz Biotechnology) raised against the N-terminus of APC. ( B ) The activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. The assay was performed in triplicate and the results were standardized to the mean of the activity of HeLa cells. ( C ) Phosphorylation and expression of HER2 and the downstream signaling in WT HER2 - or HER2 G776S-transfected HeLa and colon cells (FHC, CACO-2 and COLO-320). ( D ) Soft agar colony-forming assays showing the effects of stable transfection with HER2 WT or HER2 G776S in FHC (WT APC ) and COLO-320 (mutant APC ) cells. The cells were seeded into six-well plates in triplicate, and the number of colonies per well was counted. ns, not significant. ** P < 0.01 HER2 WT vs HER2 G776S.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot, Activity Assay, Luciferase, Reporter Assay, Expressing, Transfection, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of APC KO on HER signaling in HeLa cells (with WT APC ). ( A ) Confirmation of APC KO and its effect on the HER2–ERK pathway in APC -KO HeLa cells using Western blotting. β-Actin served as a loading control. ( B ) Activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. Nontargeting control cells (NTC) incubated with Wnt3a (100 ng/ml) for 24 h were used as a positive control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.01, * P < 0.05, ** P < 0.01 vs NTC cells ( n = 3). ( C ) Measurement of activated RAS (RAS–GTP) using the G-LISA assay. NTC plus Wnt3a (100 ng/ml) was used as a control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.05, * P < 0.05 vs NTC cells ( n = 3). ( D ) Western blot results showing the effects of HER2 WT or HER2 G776S transfection on the HER2 signaling pathway in APC -KO cells. APC -KO cells or NTC cells were transfected with mock or HER2 expression vectors, and the experiments were performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of RAS-GTP using the G-LISA assay performed 48 h after HER2 expression vector transfection. The data were standardized to the mean of the activity of NTC cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, * P < 0.05, ** P < 0.01 ( n = 3). ( F ) Colony-forming assay results showing the effects of stable transfection with HER2 WT or HER2 G776S in APC -KO cells. The number of colonies was counted in six random low-power fields. Scale bar, 200 μm. Two-way ANOVA: interaction P < 0.01, ** P < 0.01.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Assay, Incubation, Positive Control, Transfection, Expressing, Plasmid Preparation, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of WT APC overexpression on the HER2 signaling pathway in COLO-320 cells (with mutant APC ). ( A ) Confirmation of APC overexpression in cells transfected with pCMV_APC vector using Western blotting. ( B ) Activity of the Wnt/β-catenin pathway in COLO-320 cells transfected with pCMV_APC measured using the TCF/LEF luciferase reporter assay. The reporter vectors were cotransfected into COLO-320 cells along with the pCMV empty vector (mock) or pCMV-APC. The data were standardized to the mean of the activity of mock cells. ** P < 0.01 vs mock ( n = 3). ( C ) Measurement of activated RAS using the G-LISA assay performed 48 h after the transfection with pCMV. The data were standardized to the mean of the activity of cells transfected with mock vectors. * P < 0 .05 vs mock ( n = 3) ( D ) Western blot results showing the effects of APC overexpression on the HER2 signaling pathway in COLO-320 cells. The cells were cotransfected with pcDNA_HER2-WT/G776S vectors and/or pCMV_APC vectors. Western blotting was performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of activated RAS using the G-LISA assay performed 48 h after transfection. The data were standardized to the mean of the activity of cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, ** P < 0.01 ( n = 3). ( F ) Activity of the Wnt/β-catenin pathway in COLO-320 cells measured 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate. ( G ) Measurement of activated RAS using the G-LISA assay in COLO-320 cells 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Luciferase, Reporter Assay

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Schema of the relationship between HER2 mutations and APC mutations in colorectal cancer cells. HER2 G776S is a mutation that increases HER2 kinase activity, although the activity is weak. APC loss-of-function increases Wnt/β-catenin pathway activation but also increases the amount of RAS-GTP, which helps the HER2 G776S mutation to activate the HER2-ERK pathway.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Activity Assay, Activation Assay