p her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2
    Assessment of <t>HER2</t> expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )
    P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines"

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-024-02600-7

    Assessment of HER2 expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )
    Figure Legend Snippet: Assessment of HER2 expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )

    Techniques Used: Expressing, Western Blot, Derivative Assay, Comparison, Staining, Flow Cytometry, Fluorescence

    Brightfield imaged and SERS intensity maps of a selected cell for the Raman peaks at 1080 and 1580 cm − 1 for the cell lines: ( A ) MCF10A ( B ) MDA-MB-468 ( C ) SKBR3. Scale bar = 10 μm. ( D ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines based on the SERS analysis
    Figure Legend Snippet: Brightfield imaged and SERS intensity maps of a selected cell for the Raman peaks at 1080 and 1580 cm − 1 for the cell lines: ( A ) MCF10A ( B ) MDA-MB-468 ( C ) SKBR3. Scale bar = 10 μm. ( D ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines based on the SERS analysis

    Techniques Used: Biomarker Assay, Membrane

    Raman classification of breast derived cell lines expressing different HER2 levels. ( A ) Median + IQR spectra of SKBR3 (lime), MCF10A (green) and MDA-MB-468 (blue), with PCA loading expressing high variance, black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) 3D score plot of PC2-4 scores demonstrating the classification achieved for SKBR3, MCF10A and MDA-MB-468 cells using 3 components. ( C ) Box-plot of the distribution for each cell line of PC4 scores. The asterisks represent Wilcoxon nonparametric test results with p < 0.0001 when ****. ( D - E ) LDA supervised classification with the separation achieved using the latent variables 1 and 2, and the misclassification rate, respectively
    Figure Legend Snippet: Raman classification of breast derived cell lines expressing different HER2 levels. ( A ) Median + IQR spectra of SKBR3 (lime), MCF10A (green) and MDA-MB-468 (blue), with PCA loading expressing high variance, black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) 3D score plot of PC2-4 scores demonstrating the classification achieved for SKBR3, MCF10A and MDA-MB-468 cells using 3 components. ( C ) Box-plot of the distribution for each cell line of PC4 scores. The asterisks represent Wilcoxon nonparametric test results with p < 0.0001 when ****. ( D - E ) LDA supervised classification with the separation achieved using the latent variables 1 and 2, and the misclassification rate, respectively

    Techniques Used: Derivative Assay, Expressing

    HER2 assessment in scrambled- and HER2-silenced SKBR3 cells. ( A ) Western blot analysis of HER2 on extracts from single clones isolated upon selection of SKBR3 stably transfected with a scrambled control (SCR) or with an shRNA directed against HER2 (HER2 shRNA Clones 1–3). α-Tubulin was used as loading control for each sample. ( B ) Flow cytometry analysis of HER2 cell surface exposure in the scrambled transfected SKBR3 (SCR) or in clone 3 (Clone 3). The cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The histogram shows the relative quantification expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( t -test). ( C ) Western blot analysis of HER2, AKT and the phosphorylated form levels in extracts from SKBR3 transfected with a scrambled control and from the HER2-silenced clone 3. The first histogram illustrates the quantification of HER2 relative to α-Tubulin and the second the AKT phosphorylation level to total AKT compared to the SCR control. Statistical significance is considered when *** p < 0.001 and **** p < 0.0001 ( t -test). ( D ) Brightfield images and SERS intensity maps of a selected cell for the peaks at 1080 and 1580 cm − 1 for scrambled- and HER2-silenced SKBR3 cells. Scale bar = 10 μm. ( E ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines
    Figure Legend Snippet: HER2 assessment in scrambled- and HER2-silenced SKBR3 cells. ( A ) Western blot analysis of HER2 on extracts from single clones isolated upon selection of SKBR3 stably transfected with a scrambled control (SCR) or with an shRNA directed against HER2 (HER2 shRNA Clones 1–3). α-Tubulin was used as loading control for each sample. ( B ) Flow cytometry analysis of HER2 cell surface exposure in the scrambled transfected SKBR3 (SCR) or in clone 3 (Clone 3). The cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The histogram shows the relative quantification expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( t -test). ( C ) Western blot analysis of HER2, AKT and the phosphorylated form levels in extracts from SKBR3 transfected with a scrambled control and from the HER2-silenced clone 3. The first histogram illustrates the quantification of HER2 relative to α-Tubulin and the second the AKT phosphorylation level to total AKT compared to the SCR control. Statistical significance is considered when *** p < 0.001 and **** p < 0.0001 ( t -test). ( D ) Brightfield images and SERS intensity maps of a selected cell for the peaks at 1080 and 1580 cm − 1 for scrambled- and HER2-silenced SKBR3 cells. Scale bar = 10 μm. ( E ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines

    Techniques Used: Western Blot, Clone Assay, Isolation, Selection, Stable Transfection, Transfection, shRNA, Flow Cytometry, Fluorescence, Biomarker Assay, Membrane

    Raman spectra of the scrambled- and HER2-silenced SKBR3 cells (clone 3). ( A ): Median IQR spectra of scrambled (lime) and HER2-shRNA SKBR3 (red) with PCA loadings expressing high variance, as black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) The box plot of distribution of PCA scores for PC4 for the scrambled SKBR3 and silenced counterpart is shown, with a Wilcoxon statistic. ( C ) The score plot of PC1-PC4, evidencing the best spatial separation among scrambled and silenced SKBR3, is shown
    Figure Legend Snippet: Raman spectra of the scrambled- and HER2-silenced SKBR3 cells (clone 3). ( A ): Median IQR spectra of scrambled (lime) and HER2-shRNA SKBR3 (red) with PCA loadings expressing high variance, as black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) The box plot of distribution of PCA scores for PC4 for the scrambled SKBR3 and silenced counterpart is shown, with a Wilcoxon statistic. ( C ) The score plot of PC1-PC4, evidencing the best spatial separation among scrambled and silenced SKBR3, is shown

    Techniques Used: shRNA, Expressing

    p-her2-tyr 122  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p-her2-tyr 122
    P Her2 Tyr 122, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2
    P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2 erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    P Her2 Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition"

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    Journal: bioRxiv

    doi: 10.1101/2024.05.09.593450

    ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    Figure Legend Snippet: ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Techniques Used: Western Blot, Expressing

    ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).
    Figure Legend Snippet: ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Techniques Used: Western Blot, Expressing, Fluorescence, Incubation

    (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.
    Figure Legend Snippet: (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Techniques Used: Inhibition, Activity Assay, Expressing

    phosphorylated p her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated p her2
    Pyrotinib inhibits proliferation, migration, invasion, and <t>HER2</t> downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
    Phosphorylated P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors"

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    Journal: Chinese Journal of Cancer Research

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Techniques Used: Migration, Expressing, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Techniques Used: Western Blot

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.
    Figure Legend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Techniques Used: Migration, Wound Healing Assay, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.
    Figure Legend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Techniques Used: Immunohistochemistry

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    Cell Signaling Technology Inc phosphorylated p her2
    Pyrotinib inhibits proliferation, migration, invasion, and <t>HER2</t> downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
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    1) Product Images from "Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors"

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    Journal: Chinese Journal of Cancer Research

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Techniques Used: Migration, Expressing, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Techniques Used: Western Blot

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.
    Figure Legend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Techniques Used: Migration, Wound Healing Assay, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.
    Figure Legend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Techniques Used: Western Blot

    Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.
    Figure Legend Snippet: Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Techniques Used: Immunohistochemistry

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    Structured Review

    Cell Signaling Technology Inc p her2
    P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2 erbb2 tyr877  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc p her2 erbb2 tyr877
    P Her2 Erbb2 Tyr877, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc p her2
    Structure and function of the <t>HER2</t> receptor and its inhibition by trastuzumab. ( A – C) show the structure of the receptor, its activation, internalization, recycling and shedding; ( D ) illustrates how the monoclonal antibody trastuzumab (TZ) interacts with HER2 at a site in the IV extracellular domain of the receptor, impairing its function.
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    1) Product Images from "Development of High-Loading Trastuzumab PLGA Nanoparticles: A Powerful Tool Against HER2 Positive Breast Cancer Cells"

    Article Title: Development of High-Loading Trastuzumab PLGA Nanoparticles: A Powerful Tool Against HER2 Positive Breast Cancer Cells

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S429898

    Structure and function of the HER2 receptor and its inhibition by trastuzumab. ( A – C) show the structure of the receptor, its activation, internalization, recycling and shedding; ( D ) illustrates how the monoclonal antibody trastuzumab (TZ) interacts with HER2 at a site in the IV extracellular domain of the receptor, impairing its function.
    Figure Legend Snippet: Structure and function of the HER2 receptor and its inhibition by trastuzumab. ( A – C) show the structure of the receptor, its activation, internalization, recycling and shedding; ( D ) illustrates how the monoclonal antibody trastuzumab (TZ) interacts with HER2 at a site in the IV extracellular domain of the receptor, impairing its function.

    Techniques Used: Inhibition, Activation Assay

    Biological effects of encapsulated or free trastuzumab on SKBR3 cells. ( A ) Western blot assessment of HER2 Y1248 and AKT S473 phosphorylation on extracts from SKBR3 cells exposed to NP or two increasing concentrations of TZPs or free TZ for the indicated time points. The histograms report the quantification of the phosphorylation levels normalized to total HER2 or AKT, respectively; FC indicates Fold-Change. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others using the same colour code as in the panel on the right side of the figure. ( B ) Viability of SKBR3 cells treated with NPs or two increasing concentrations of TZPs or free TZ for the indicated time points reported as percentage vs untreated CTR control cells. Data are reported as mean± SEM of two independent experiments performed at least in triplicate; the mean values from untreated or treated cells were compared by applying the t -test. Also, in this case, the tables below illustrate the results obtained by comparing the single dose of each formulation to all others using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p <0.05, ** p < 0.01, ***p < 0.001. **** p < 0.0001, ns= not significant.
    Figure Legend Snippet: Biological effects of encapsulated or free trastuzumab on SKBR3 cells. ( A ) Western blot assessment of HER2 Y1248 and AKT S473 phosphorylation on extracts from SKBR3 cells exposed to NP or two increasing concentrations of TZPs or free TZ for the indicated time points. The histograms report the quantification of the phosphorylation levels normalized to total HER2 or AKT, respectively; FC indicates Fold-Change. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others using the same colour code as in the panel on the right side of the figure. ( B ) Viability of SKBR3 cells treated with NPs or two increasing concentrations of TZPs or free TZ for the indicated time points reported as percentage vs untreated CTR control cells. Data are reported as mean± SEM of two independent experiments performed at least in triplicate; the mean values from untreated or treated cells were compared by applying the t -test. Also, in this case, the tables below illustrate the results obtained by comparing the single dose of each formulation to all others using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p <0.05, ** p < 0.01, ***p < 0.001. **** p < 0.0001, ns= not significant.

    Techniques Used: Western Blot, Formulation

    TZPs and TZ promote HER2 endocytosis and reduce shedding. ( A ) Flow cytometry analysis of cell surface HER2 in SKBR3 cells untreated (black line) or treated with NPs (grey line) or two increasing concentrations of TZPs (heavy and light green line) or free TZ (heavy and light orange line) for 24 h. The percentage of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The right-most panel is the merge of the single panels. ( B ) The histograms report the quantification of HER2 levels, expressed as percentage vs untreated controls, after treatment with NPs (grey), TZPs (heavy and light green) or free TZ (heavy and light orange). The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant. ( C ) Western blot analysis of total HER2 protein in SKBR3 cells untreated or treated as above for the indicated time points. The histograms report the quantification after normalization to α-Tubulin used as protein loading control. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant. ( D ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated conditioned media of SKBR3 cells treated with NPs or TZPs as above for the indicated time points. The histograms show the relative quantification after normalization to protein loading through Ponceau S staining of the filter. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant.
    Figure Legend Snippet: TZPs and TZ promote HER2 endocytosis and reduce shedding. ( A ) Flow cytometry analysis of cell surface HER2 in SKBR3 cells untreated (black line) or treated with NPs (grey line) or two increasing concentrations of TZPs (heavy and light green line) or free TZ (heavy and light orange line) for 24 h. The percentage of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The right-most panel is the merge of the single panels. ( B ) The histograms report the quantification of HER2 levels, expressed as percentage vs untreated controls, after treatment with NPs (grey), TZPs (heavy and light green) or free TZ (heavy and light orange). The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant. ( C ) Western blot analysis of total HER2 protein in SKBR3 cells untreated or treated as above for the indicated time points. The histograms report the quantification after normalization to α-Tubulin used as protein loading control. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant. ( D ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated conditioned media of SKBR3 cells treated with NPs or TZPs as above for the indicated time points. The histograms show the relative quantification after normalization to protein loading through Ponceau S staining of the filter. The tables below illustrate the results obtained by comparing the single dose of each formulation to all others, using the same colour code as in the panel on the right side of the figure. Statistical significance was considered when * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001, ns= not significant.

    Techniques Used: Flow Cytometry, Fluorescence, Formulation, Western Blot, Staining

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    Cell Signaling Technology Inc p her2
    Assessment of <t>HER2</t> expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )
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    Cell Signaling Technology Inc p-her2-tyr 122
    Assessment of <t>HER2</t> expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )
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    Cell Signaling Technology Inc p her2 erbb2
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
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    Cell Signaling Technology Inc phosphorylated p her2
    Pyrotinib inhibits proliferation, migration, invasion, and <t>HER2</t> downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
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    Cell Signaling Technology Inc p her2 y1221 2
    Pyrotinib inhibits proliferation, migration, invasion, and <t>HER2</t> downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
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    Cell Signaling Technology Inc p her2 erbb2 tyr877
    Pyrotinib inhibits proliferation, migration, invasion, and <t>HER2</t> downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.
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    Image Search Results


    Assessment of HER2 expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )

    Journal: Journal of Nanobiotechnology

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    doi: 10.1186/s12951-024-02600-7

    Figure Lengend Snippet: Assessment of HER2 expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )

    Article Snippet: Antibodies to p-HER2 (Y1248 # 2247), HER2 (#3250), p-AKT (S473) (#9271), AKT (#9272) were from Cell Signaling Technology (Beverly, MA USA); α-Tubulin (T5168) was from Sigma-Aldrich Co. (Merck KGaA, St. Louis, MO, USA).

    Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Staining, Flow Cytometry, Fluorescence

    Brightfield imaged and SERS intensity maps of a selected cell for the Raman peaks at 1080 and 1580 cm − 1 for the cell lines: ( A ) MCF10A ( B ) MDA-MB-468 ( C ) SKBR3. Scale bar = 10 μm. ( D ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines based on the SERS analysis

    Journal: Journal of Nanobiotechnology

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    doi: 10.1186/s12951-024-02600-7

    Figure Lengend Snippet: Brightfield imaged and SERS intensity maps of a selected cell for the Raman peaks at 1080 and 1580 cm − 1 for the cell lines: ( A ) MCF10A ( B ) MDA-MB-468 ( C ) SKBR3. Scale bar = 10 μm. ( D ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines based on the SERS analysis

    Article Snippet: Antibodies to p-HER2 (Y1248 # 2247), HER2 (#3250), p-AKT (S473) (#9271), AKT (#9272) were from Cell Signaling Technology (Beverly, MA USA); α-Tubulin (T5168) was from Sigma-Aldrich Co. (Merck KGaA, St. Louis, MO, USA).

    Techniques: Biomarker Assay, Membrane

    Raman classification of breast derived cell lines expressing different HER2 levels. ( A ) Median + IQR spectra of SKBR3 (lime), MCF10A (green) and MDA-MB-468 (blue), with PCA loading expressing high variance, black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) 3D score plot of PC2-4 scores demonstrating the classification achieved for SKBR3, MCF10A and MDA-MB-468 cells using 3 components. ( C ) Box-plot of the distribution for each cell line of PC4 scores. The asterisks represent Wilcoxon nonparametric test results with p < 0.0001 when ****. ( D - E ) LDA supervised classification with the separation achieved using the latent variables 1 and 2, and the misclassification rate, respectively

    Journal: Journal of Nanobiotechnology

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    doi: 10.1186/s12951-024-02600-7

    Figure Lengend Snippet: Raman classification of breast derived cell lines expressing different HER2 levels. ( A ) Median + IQR spectra of SKBR3 (lime), MCF10A (green) and MDA-MB-468 (blue), with PCA loading expressing high variance, black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) 3D score plot of PC2-4 scores demonstrating the classification achieved for SKBR3, MCF10A and MDA-MB-468 cells using 3 components. ( C ) Box-plot of the distribution for each cell line of PC4 scores. The asterisks represent Wilcoxon nonparametric test results with p < 0.0001 when ****. ( D - E ) LDA supervised classification with the separation achieved using the latent variables 1 and 2, and the misclassification rate, respectively

    Article Snippet: Antibodies to p-HER2 (Y1248 # 2247), HER2 (#3250), p-AKT (S473) (#9271), AKT (#9272) were from Cell Signaling Technology (Beverly, MA USA); α-Tubulin (T5168) was from Sigma-Aldrich Co. (Merck KGaA, St. Louis, MO, USA).

    Techniques: Derivative Assay, Expressing

    HER2 assessment in scrambled- and HER2-silenced SKBR3 cells. ( A ) Western blot analysis of HER2 on extracts from single clones isolated upon selection of SKBR3 stably transfected with a scrambled control (SCR) or with an shRNA directed against HER2 (HER2 shRNA Clones 1–3). α-Tubulin was used as loading control for each sample. ( B ) Flow cytometry analysis of HER2 cell surface exposure in the scrambled transfected SKBR3 (SCR) or in clone 3 (Clone 3). The cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The histogram shows the relative quantification expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( t -test). ( C ) Western blot analysis of HER2, AKT and the phosphorylated form levels in extracts from SKBR3 transfected with a scrambled control and from the HER2-silenced clone 3. The first histogram illustrates the quantification of HER2 relative to α-Tubulin and the second the AKT phosphorylation level to total AKT compared to the SCR control. Statistical significance is considered when *** p < 0.001 and **** p < 0.0001 ( t -test). ( D ) Brightfield images and SERS intensity maps of a selected cell for the peaks at 1080 and 1580 cm − 1 for scrambled- and HER2-silenced SKBR3 cells. Scale bar = 10 μm. ( E ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines

    Journal: Journal of Nanobiotechnology

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    doi: 10.1186/s12951-024-02600-7

    Figure Lengend Snippet: HER2 assessment in scrambled- and HER2-silenced SKBR3 cells. ( A ) Western blot analysis of HER2 on extracts from single clones isolated upon selection of SKBR3 stably transfected with a scrambled control (SCR) or with an shRNA directed against HER2 (HER2 shRNA Clones 1–3). α-Tubulin was used as loading control for each sample. ( B ) Flow cytometry analysis of HER2 cell surface exposure in the scrambled transfected SKBR3 (SCR) or in clone 3 (Clone 3). The cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The histogram shows the relative quantification expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( t -test). ( C ) Western blot analysis of HER2, AKT and the phosphorylated form levels in extracts from SKBR3 transfected with a scrambled control and from the HER2-silenced clone 3. The first histogram illustrates the quantification of HER2 relative to α-Tubulin and the second the AKT phosphorylation level to total AKT compared to the SCR control. Statistical significance is considered when *** p < 0.001 and **** p < 0.0001 ( t -test). ( D ) Brightfield images and SERS intensity maps of a selected cell for the peaks at 1080 and 1580 cm − 1 for scrambled- and HER2-silenced SKBR3 cells. Scale bar = 10 μm. ( E ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines

    Article Snippet: Antibodies to p-HER2 (Y1248 # 2247), HER2 (#3250), p-AKT (S473) (#9271), AKT (#9272) were from Cell Signaling Technology (Beverly, MA USA); α-Tubulin (T5168) was from Sigma-Aldrich Co. (Merck KGaA, St. Louis, MO, USA).

    Techniques: Western Blot, Clone Assay, Isolation, Selection, Stable Transfection, Transfection, shRNA, Flow Cytometry, Fluorescence, Biomarker Assay, Membrane

    Raman spectra of the scrambled- and HER2-silenced SKBR3 cells (clone 3). ( A ): Median IQR spectra of scrambled (lime) and HER2-shRNA SKBR3 (red) with PCA loadings expressing high variance, as black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) The box plot of distribution of PCA scores for PC4 for the scrambled SKBR3 and silenced counterpart is shown, with a Wilcoxon statistic. ( C ) The score plot of PC1-PC4, evidencing the best spatial separation among scrambled and silenced SKBR3, is shown

    Journal: Journal of Nanobiotechnology

    Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

    doi: 10.1186/s12951-024-02600-7

    Figure Lengend Snippet: Raman spectra of the scrambled- and HER2-silenced SKBR3 cells (clone 3). ( A ): Median IQR spectra of scrambled (lime) and HER2-shRNA SKBR3 (red) with PCA loadings expressing high variance, as black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) The box plot of distribution of PCA scores for PC4 for the scrambled SKBR3 and silenced counterpart is shown, with a Wilcoxon statistic. ( C ) The score plot of PC1-PC4, evidencing the best spatial separation among scrambled and silenced SKBR3, is shown

    Article Snippet: Antibodies to p-HER2 (Y1248 # 2247), HER2 (#3250), p-AKT (S473) (#9271), AKT (#9272) were from Cell Signaling Technology (Beverly, MA USA); α-Tubulin (T5168) was from Sigma-Aldrich Co. (Merck KGaA, St. Louis, MO, USA).

    Techniques: shRNA, Expressing

    ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Western Blot, Expressing

    ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Western Blot, Expressing, Fluorescence, Incubation

    (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Inhibition, Activity Assay, Expressing

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Migration, Expressing, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Western Blot

    Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Migration, Wound Healing Assay, Western Blot

    Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Western Blot

    Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Western Blot

    Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Journal: Chinese Journal of Cancer Research

    Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

    doi: 10.21147/j.issn.1000-9604.2024.02.03

    Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

    Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

    Techniques: Immunohistochemistry