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

Journal: International Journal of Molecular Sciences

Article Title: When Isolated at Full Receptivity, in Vitro Fertilized Wheat ( Triticum aestivum , L.) Egg Cells Reveal [Ca 2+ ] cyt Oscillation of Intracellular Origin

doi: 10.3390/ijms151223766

Figure Lengend Snippet: Calcium dynamics in reconstructions of temporal sections obtained with the Line Image function of the Lucida software. The line trace plot is represented as pixel intensities converted into pseudocolor values in the Line Image. Rows in the Line Image correspond to successive images ( x ) along the active dimension, time ( t ). Thus, the Line Image is an ( x , t ) plot showing [Ca 2+ ] cyt change along an axis through the “stack” image composed of the overlaid images taken successively during [Ca 2+ ] cyt measurement. The increase in [Ca 2+ ] cyt is represented by yellow-red bands. The bar represents a pseudocolor code of the pixel values digitized to 256 grey levels. ( a ) Line Image of an egg cell isolated three DAE, injected with fura-2 dextran and ratio-imaged following electrofusion with a sperm cell. The axis along which the [Ca 2+ ] cyt changes were measured passed through the sperm entry site; ( b ) [Ca 2+ ] cyt changes over time in a receptive egg cell (isolated six DAE) microinjected with fura-2 dextran and fertilized in vitro ; ( c ) Time-lapse series of an axis “drawn” through time, the active dimension, in an overmature (18 DAE) egg cell fertilized in vitro following fura-2 dextran injection.

Article Snippet: Fura-2 dextran-loaded egg cells were observed with a Zeiss Axiovert 35M inverted microscope equipped with a 75-W xenon epifluorescence burner (Osram Licht AG, Munich, Germany).

Techniques: Software, Isolation, Injection, Electrofusion, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: When Isolated at Full Receptivity, in Vitro Fertilized Wheat ( Triticum aestivum , L.) Egg Cells Reveal [Ca 2+ ] cyt Oscillation of Intracellular Origin

doi: 10.3390/ijms151223766

Figure Lengend Snippet: The 340/380 nm excitation ratios of fura-2 dextran-injected wheat egg cells showing [Ca 2+ ] cyt variations in response to the different maturational stages of the batch of the egg cells used for in vitro fertilization. ( a ) Typical, fertilization-associated [Ca 2+ ] cyt rise in an in vitro fertilized wheat egg cell developed in situ and isolated three DAE (the arrow denotes 10 min after in vitro fertilization (IVF)). The bright field image (inset) at the right upper corner shows the lines (axes) of pixels along which the pixel intensities ( i.e ., the changes in calcium concentrations) were measured through the active dimension, time; the arrow shows the site of sperm incorporation, and the bar represents: 15 µm; ( b ) Representative [Ca 2+ ] cyt changes occurring concomitantly upon in vitro fertilization of mature wheat egg cells (isolated at six DAE). This dynamics of the [Ca 2+ ] cyt change could be seen in 66 out of 80 (81.5%) egg cells fertilized with the sperm (the arrow indicates the time at which fusion between the sperm and the egg cell occurred). The [Ca 2+ ] cyt peak elicited by the sperm ensued 10 min after the in vitro fusion of the gametes of opposite sexes. The pseudo-colored images (insets) give a visual representation of the change in [Ca 2+ ] cyt , whereas the bright-field image shows the axis along which the pixel intensities ( i.e ., the changes in calcium concentrations) were measured. The arrow shows the site of sperm entry, and the bar represents: 20 µm; ( c ) A slow [Ca 2+ ] cyt rise induced by sperm cell fusion in an overmature egg cell isolated 11 DAE (the arrow shows the time lapse, 17 min, between sperm–egg fusion and the commencement of the slow [Ca 2+ ] cyt elevation). The bright-field image at the right upper corner shows the axis along which the pixel intensities ( i.e ., the changes in calcium concentrations) were measured. The arrow shows the site of sperm entry, and the bar represents: 25 µm.

Article Snippet: Fura-2 dextran-loaded egg cells were observed with a Zeiss Axiovert 35M inverted microscope equipped with a 75-W xenon epifluorescence burner (Osram Licht AG, Munich, Germany).

Techniques: Injection, In Vitro, In Situ, Isolation

Journal:

Article Title: The Glomerulosclerosis of Aging in Females

doi:

Figure Lengend Snippet: Increased NF-κB transcriptional activity in mesangial cells isolated from 28-month-old mice. Two separate mesangial cell lines were cultured from 5-month-old (5-month MC) or 28-month-old (28-month MC) female B6 mice. NF-κB transcriptional activity was measured by transfecting cells with a luciferase NF-κB reporter construct and a β-galactosidase plasmid. Basal NF-κB transcriptional activity in 5-month MC were arbitrarily defined as 1. Data shown are from six measurements of each of four independent experiments. A: Basal NF-κB transcriptional activity (base) was 2.6-fold higher in 28-month MC than in 5-month MC. The introduction of an exogenous IKK expression vector to 5-month MC or 28-month MC increased cell NF-κB transcriptional activity. In contrast, transfection of 5-month or 28-month MC with an IκB-α or dominant-negative IKK (dn) expression vector decreased cell NF-κB transcriptional activity. B: Increased sensitivity to TNF-α induced NF-κB activation in 28-month MC. Five ng/ml of TNF-α increased NF-κB transcriptional activity in 28-month MC but not in 5-month MC. An increase in TNF-α levels to 10 ng/ml was required to stimulate NF-κB activity in 5-month MC.

Article Snippet: Transfection and NF-κB Reporter Gene Assay Transient transfection was performed in mesangial cells from 5- and 28-month-old mice using Transfast according to the manufacturer’s protocol (Promega, Madison, WI).

Techniques: Activity Assay, Isolation, Cell Culture, Luciferase, Construct, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Activation Assay