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OriGene human erbb2 complementary dna cdna

Time-dependent expression of CDK4 and cyclin D1 in G1/S transition. A – D , dynamic changes in the expression or phosphorylation of individual proteins in MCF-7 cells stably expressing FUCCI, detected by western blotting up to 36 h after treatment with 10 nM HRG. A and B , <t>ErbB2</t> pathway proteins. C and D , proteins regulating G1/S transition. Protein and phosphorylation levels were normalized relative to those of GAPDH and divided by that of the 0 h sample (0 h = 1); n = 3. The data are displayed as the means ± SEM. FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin.

Human Erbb2 Complementary Dna Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 96/100, based on 1039 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1039 article reviews

human erbb2 complementary dna cdna - by Bioz Stars, 2026-09

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1) Product Images from "The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition"

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2025.110865

Figure Legend Snippet: Time-dependent expression of CDK4 and cyclin D1 in G1/S transition. A – D , dynamic changes in the expression or phosphorylation of individual proteins in MCF-7 cells stably expressing FUCCI, detected by western blotting up to 36 h after treatment with 10 nM HRG. A and B , ErbB2 pathway proteins. C and D , proteins regulating G1/S transition. Protein and phosphorylation levels were normalized relative to those of GAPDH and divided by that of the 0 h sample (0 h = 1); n = 3. The data are displayed as the means ± SEM. FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin.

Techniques Used: Expressing, Phospho-proteomics, Stable Transfection, Western Blot, Ubiquitin Proteomics

High ErbB2 expression induces cell cycle arrest by repressing cyclin D1 expression via c-Myc under CDK4 inhibition. A and B , WT MCF-7 and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and harvested 24 h after HRG stimulation. Western blotting of ( A ) p-ErbB2, p-RB (Ser807/811), p-RB (Thr373), RB, p-AKT (Thr308), AKT, p-ERK (Thr202/Thr204), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, with DMSO treatment) after normalization with GAPDH expression ( B ). The data are displayed as the means ± SD. C - F , ErbB2-overexpressing cells (moderate) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). Hsp90 and ErbB2 were coimmunostained. The scale bar represents 25 μm ( C ). Colocalization rate of Hsp90 and ErbB2 based on Mander’s tM1 and Pearson’s R values determined using Coloc 2 plugin of Image J; n = 10 ( D ). Hsp90 and PY100 were coimmunostained. The scale bar represents 25 μm ( E ). Colocalization rate of Hsp90 and PY100 based on Mander’s tM2 and Pearson’s R values; n = 10 ( F ). G and H , PLA signals detected in WT MCF-7 cells. Cells were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). PLA was performed using ErbB2 and Hsp90 antibodies. G , PLA signals detected in the presence of HER2 and Hsp90 antibodies ( left ). No signals detected after omitting Hsp90 antibody ( right ). The scale bar represents 100 μm. H , mean intensity of PLA signals per image was measured under each condition in ( G ) ( Left ). Number of nuclei per image was counted ( Right ). n = 59. I and J , FUCCI stably expressing MCF-7 cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG and collected 8 h later. I , ErbB2 and GAPDH expression determined by western blotting. ( J ) ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SD. K and L , ErbB2 cycloheximide pulse-chase assay was performed with FUCCI stably expressing MCF-7 cells. Cells were treated with CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, then 100 μg/ml cycloheximide was added 16 h after stimulation with 10 nM HRG. Cells were harvested at 0, 2, and 4 h after cycloheximide treatment; n = 3. K , ErbB2 and GAPDH expression determined by western blotting. L , ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SDs. M , coimmunoprecipitation assay in ErbB2-overexpressing MCF-7 cells (moderate). Cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG, and collected 8 h later. Immunoprecipitates and whole cell lysates were immunoblotted with anti-ubiquitin or anti-ErbB2, or GAPDH antibodies. N , schematic model: In CDK4i-unresponsive WT MCF-7 cells, the loss of ErbB2 protein stability impairs the regulation of c-Myc-mediated G1/S transition, leading to the delayed G1/S transition. O , WT MCF-7 and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after stimulation. The scale bar represents 100 μm. P , Images in ( J ) were analyzed to determine the proportion (%) of each cell cycle phase in the entire set of images; n = 3. In ( B ) and ( J ), ∗ p < 0.05 (Tukey’s test). In ( D ), ∗ p < 0.05 (with respect to control and CDK4i; Welch’s t test). In ( F ) and ( H ), ∗ p < 0.05 (with respect to control and CDK4i; two-tailed Student’s t test). In ( L ), the two-way ANOVA revealed significant main effects of both time ( p < 0.0001) and CDK4i treatment (∗ p = 0.02). FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin; PLA, proximity ligation assay; DMSO, dimethyl sulfoxide; RB, retinoblastoma protein.

Figure Legend Snippet: High ErbB2 expression induces cell cycle arrest by repressing cyclin D1 expression via c-Myc under CDK4 inhibition. A and B , WT MCF-7 and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and harvested 24 h after HRG stimulation. Western blotting of ( A ) p-ErbB2, p-RB (Ser807/811), p-RB (Thr373), RB, p-AKT (Thr308), AKT, p-ERK (Thr202/Thr204), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, with DMSO treatment) after normalization with GAPDH expression ( B ). The data are displayed as the means ± SD. C - F , ErbB2-overexpressing cells (moderate) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). Hsp90 and ErbB2 were coimmunostained. The scale bar represents 25 μm ( C ). Colocalization rate of Hsp90 and ErbB2 based on Mander’s tM1 and Pearson’s R values determined using Coloc 2 plugin of Image J; n = 10 ( D ). Hsp90 and PY100 were coimmunostained. The scale bar represents 25 μm ( E ). Colocalization rate of Hsp90 and PY100 based on Mander’s tM2 and Pearson’s R values; n = 10 ( F ). G and H , PLA signals detected in WT MCF-7 cells. Cells were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). PLA was performed using ErbB2 and Hsp90 antibodies. G , PLA signals detected in the presence of HER2 and Hsp90 antibodies ( left ). No signals detected after omitting Hsp90 antibody ( right ). The scale bar represents 100 μm. H , mean intensity of PLA signals per image was measured under each condition in ( G ) ( Left ). Number of nuclei per image was counted ( Right ). n = 59. I and J , FUCCI stably expressing MCF-7 cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG and collected 8 h later. I , ErbB2 and GAPDH expression determined by western blotting. ( J ) ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SD. K and L , ErbB2 cycloheximide pulse-chase assay was performed with FUCCI stably expressing MCF-7 cells. Cells were treated with CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, then 100 μg/ml cycloheximide was added 16 h after stimulation with 10 nM HRG. Cells were harvested at 0, 2, and 4 h after cycloheximide treatment; n = 3. K , ErbB2 and GAPDH expression determined by western blotting. L , ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SDs. M , coimmunoprecipitation assay in ErbB2-overexpressing MCF-7 cells (moderate). Cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG, and collected 8 h later. Immunoprecipitates and whole cell lysates were immunoblotted with anti-ubiquitin or anti-ErbB2, or GAPDH antibodies. N , schematic model: In CDK4i-unresponsive WT MCF-7 cells, the loss of ErbB2 protein stability impairs the regulation of c-Myc-mediated G1/S transition, leading to the delayed G1/S transition. O , WT MCF-7 and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after stimulation. The scale bar represents 100 μm. P , Images in ( J ) were analyzed to determine the proportion (%) of each cell cycle phase in the entire set of images; n = 3. In ( B ) and ( J ), ∗ p < 0.05 (Tukey’s test). In ( D ), ∗ p < 0.05 (with respect to control and CDK4i; Welch’s t test). In ( F ) and ( H ), ∗ p < 0.05 (with respect to control and CDK4i; two-tailed Student’s t test). In ( L ), the two-way ANOVA revealed significant main effects of both time ( p < 0.0001) and CDK4i treatment (∗ p = 0.02). FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin; PLA, proximity ligation assay; DMSO, dimethyl sulfoxide; RB, retinoblastoma protein.

Techniques Used: Expressing, Inhibition, Western Blot, Control, Stable Transfection, Pulse Chase, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Two Tailed Test, Proximity Ligation Assay

Reversible cell cycle arrest is maintained under c-Myc transcriptional activity. A - G , MCF-7 cells overexpressing ErbB2 (high levels). A and B , cells were treated with 250 nM CDK4i 12 h after HRG stimulation, subjected to a wash/no-wash treatment 4 h later, and harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, p-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. The data are displayed as the means ± SD. C , washing operations were performed under the same conditions as in ( A and B ); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3859. 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 circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. D and E , cells were pretreated 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 , percentage distributions of G1 and S/G2/M phases based on the images in ( D ); n = 4. The scale bar represents 100 μm. The data are displayed as the means ± SD. F , cells were treated with 250 nM CDK4i or DMSO (control) 12 h after HRG stimulation and CDKN1B mRNA levels up to 20 h after HRG stimulation were examined by qPCR; n = 3. 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 CDKN1B mRNA levels were examined by qPCR; n = 3. The data are displayed as the means ± SD. H , WT MCF-7, WT T47D, and ErbB2 ( high ) OE MCF-7 cells were pretreated for 30 min with/without 4 μM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation, incubated with 250 nM CDK4i for 6 days, and SA-β-Gal staining was assessed. I , percentage SA-β-Gal-stained cells based on the images in ( H ); n = 12. J , ErbB2 levels modulate the irreversibility of the G1/S transition under CDK4i treatment. In ( B ), ( E ), ( F ), and ( G ), ∗ p < 0.05 (with respect to control and each inhibitor; two-tailed Student’s t test). In ( I ), ∗ p < 0.05 (with respect to control and each inhibitor; Welch’s t test). DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; SA-β-Gal, senescence-associated β-galactosidase; qPCR, quantitative PCR; RB, retinoblastoma protein.

Figure Legend Snippet: Reversible cell cycle arrest is maintained under c-Myc transcriptional activity. A - G , MCF-7 cells overexpressing ErbB2 (high levels). A and B , cells were treated with 250 nM CDK4i 12 h after HRG stimulation, subjected to a wash/no-wash treatment 4 h later, and harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, p-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. The data are displayed as the means ± SD. C , washing operations were performed under the same conditions as in ( A and B ); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3859. 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 circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. D and E , cells were pretreated 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 , percentage distributions of G1 and S/G2/M phases based on the images in ( D ); n = 4. The scale bar represents 100 μm. The data are displayed as the means ± SD. F , cells were treated with 250 nM CDK4i or DMSO (control) 12 h after HRG stimulation and CDKN1B mRNA levels up to 20 h after HRG stimulation were examined by qPCR; n = 3. 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 CDKN1B mRNA levels were examined by qPCR; n = 3. The data are displayed as the means ± SD. H , WT MCF-7, WT T47D, and ErbB2 ( high ) OE MCF-7 cells were pretreated for 30 min with/without 4 μM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation, incubated with 250 nM CDK4i for 6 days, and SA-β-Gal staining was assessed. I , percentage SA-β-Gal-stained cells based on the images in ( H ); n = 12. J , ErbB2 levels modulate the irreversibility of the G1/S transition under CDK4i treatment. In ( B ), ( E ), ( F ), and ( G ), ∗ p < 0.05 (with respect to control and each inhibitor; two-tailed Student’s t test). In ( I ), ∗ p < 0.05 (with respect to control and each inhibitor; Welch’s t test). DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; SA-β-Gal, senescence-associated β-galactosidase; qPCR, quantitative PCR; RB, retinoblastoma protein.

Techniques Used: Activity Assay, Western Blot, Expressing, Incubation, Control, Staining, Two Tailed Test, Real-time Polymerase Chain Reaction

Proposed model the ErbB2 receptor regulation during G1/S transition via cyclin D1 and c-Myc in CDK4i nonresponsive cells. A , in low ErbB2-expressing cells, CDK4 inhibition leads to reduced pathway activity due to ErbB2 degradation via Hsp90 dis-colocalization. This attenuates the transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells unresponsive to CDK4i exhibits a delayed G1/S transition. B , in high ErbB2-expressing cells, 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 cell cycle arrest after CDK4 inhibition. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Figure Legend Snippet: Proposed model the ErbB2 receptor regulation during G1/S transition via cyclin D1 and c-Myc in CDK4i nonresponsive cells. A , in low ErbB2-expressing cells, CDK4 inhibition leads to reduced pathway activity due to ErbB2 degradation via Hsp90 dis-colocalization. This attenuates the transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells unresponsive to CDK4i exhibits a delayed G1/S transition. B , in high ErbB2-expressing cells, 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 cell cycle arrest after CDK4 inhibition. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Techniques Used: Expressing, Inhibition, Activity Assay

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Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Time-dependent expression of CDK4 and cyclin D1 in G1/S transition. A – D , dynamic changes in the expression or phosphorylation of individual proteins in MCF-7 cells stably expressing FUCCI, detected by western blotting up to 36 h after treatment with 10 nM HRG. A and B , ErbB2 pathway proteins. C and D , proteins regulating G1/S transition. Protein and phosphorylation levels were normalized relative to those of GAPDH and divided by that of the 0 h sample (0 h = 1); n = 3. The data are displayed as the means ± SEM. FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin.

Article Snippet: MCF-7 (American Type Culture Collection, ATCC, HTB-22) cells overexpressing full-length human ErbB2 (HER2) were established as follows: Human ErbB2 complementary DNA (cDNA) in the pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161).

Techniques: Expressing, Phospho-proteomics, Stable Transfection, Western Blot, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: High ErbB2 expression induces cell cycle arrest by repressing cyclin D1 expression via c-Myc under CDK4 inhibition. A and B , WT MCF-7 and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and harvested 24 h after HRG stimulation. Western blotting of ( A ) p-ErbB2, p-RB (Ser807/811), p-RB (Thr373), RB, p-AKT (Thr308), AKT, p-ERK (Thr202/Thr204), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, with DMSO treatment) after normalization with GAPDH expression ( B ). The data are displayed as the means ± SD. C - F , ErbB2-overexpressing cells (moderate) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). Hsp90 and ErbB2 were coimmunostained. The scale bar represents 25 μm ( C ). Colocalization rate of Hsp90 and ErbB2 based on Mander’s tM1 and Pearson’s R values determined using Coloc 2 plugin of Image J; n = 10 ( D ). Hsp90 and PY100 were coimmunostained. The scale bar represents 25 μm ( E ). Colocalization rate of Hsp90 and PY100 based on Mander’s tM2 and Pearson’s R values; n = 10 ( F ). G and H , PLA signals detected in WT MCF-7 cells. Cells were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG and fixed 16 h after stimulation with 10 nM HRG (CDK4i or DMSO addition 4 h later). PLA was performed using ErbB2 and Hsp90 antibodies. G , PLA signals detected in the presence of HER2 and Hsp90 antibodies ( left ). No signals detected after omitting Hsp90 antibody ( right ). The scale bar represents 100 μm. H , mean intensity of PLA signals per image was measured under each condition in ( G ) ( Left ). Number of nuclei per image was counted ( Right ). n = 59. I and J , FUCCI stably expressing MCF-7 cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG and collected 8 h later. I , ErbB2 and GAPDH expression determined by western blotting. ( J ) ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SD. K and L , ErbB2 cycloheximide pulse-chase assay was performed with FUCCI stably expressing MCF-7 cells. Cells were treated with CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, then 100 μg/ml cycloheximide was added 16 h after stimulation with 10 nM HRG. Cells were harvested at 0, 2, and 4 h after cycloheximide treatment; n = 3. K , ErbB2 and GAPDH expression determined by western blotting. L , ErbB2 expression normalized to GAPDH expression; n = 3. The data are displayed as the means ± SDs. M , coimmunoprecipitation assay in ErbB2-overexpressing MCF-7 cells (moderate). Cells were treated with CDK4i or DMSO (control) as well as 10 μM MG132, 12 h after stimulation with 10 nM HRG, and collected 8 h later. Immunoprecipitates and whole cell lysates were immunoblotted with anti-ubiquitin or anti-ErbB2, or GAPDH antibodies. N , schematic model: In CDK4i-unresponsive WT MCF-7 cells, the loss of ErbB2 protein stability impairs the regulation of c-Myc-mediated G1/S transition, leading to the delayed G1/S transition. O , WT MCF-7 and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (control) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after stimulation. The scale bar represents 100 μm. P , Images in ( J ) were analyzed to determine the proportion (%) of each cell cycle phase in the entire set of images; n = 3. In ( B ) and ( J ), ∗ p < 0.05 (Tukey’s test). In ( D ), ∗ p < 0.05 (with respect to control and CDK4i; Welch’s t test). In ( F ) and ( H ), ∗ p < 0.05 (with respect to control and CDK4i; two-tailed Student’s t test). In ( L ), the two-way ANOVA revealed significant main effects of both time ( p < 0.0001) and CDK4i treatment (∗ p = 0.02). FUCCI, fluorescent ubiquitination-based cell cycle indicator; HRG, heregulin; PLA, proximity ligation assay; DMSO, dimethyl sulfoxide; RB, retinoblastoma protein.

Techniques: Expressing, Inhibition, Western Blot, Control, Stable Transfection, Pulse Chase, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Two Tailed Test, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Reversible cell cycle arrest is maintained under c-Myc transcriptional activity. A - G , MCF-7 cells overexpressing ErbB2 (high levels). A and B , cells were treated with 250 nM CDK4i 12 h after HRG stimulation, subjected to a wash/no-wash treatment 4 h later, and harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, p-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. The data are displayed as the means ± SD. C , washing operations were performed under the same conditions as in ( A and B ); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3859. 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 circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. D and E , cells were pretreated 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 , percentage distributions of G1 and S/G2/M phases based on the images in ( D ); n = 4. The scale bar represents 100 μm. The data are displayed as the means ± SD. F , cells were treated with 250 nM CDK4i or DMSO (control) 12 h after HRG stimulation and CDKN1B mRNA levels up to 20 h after HRG stimulation were examined by qPCR; n = 3. 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 CDKN1B mRNA levels were examined by qPCR; n = 3. The data are displayed as the means ± SD. H , WT MCF-7, WT T47D, and ErbB2 ( high ) OE MCF-7 cells were pretreated for 30 min with/without 4 μM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation, incubated with 250 nM CDK4i for 6 days, and SA-β-Gal staining was assessed. I , percentage SA-β-Gal-stained cells based on the images in ( H ); n = 12. J , ErbB2 levels modulate the irreversibility of the G1/S transition under CDK4i treatment. In ( B ), ( E ), ( F ), and ( G ), ∗ p < 0.05 (with respect to control and each inhibitor; two-tailed Student’s t test). In ( I ), ∗ p < 0.05 (with respect to control and each inhibitor; Welch’s t test). DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; SA-β-Gal, senescence-associated β-galactosidase; qPCR, quantitative PCR; RB, retinoblastoma protein.

Techniques: Activity Assay, Western Blot, Expressing, Incubation, Control, Staining, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The receptor tyrosine kinase ErbB2/HER2 governs CDK4 inhibitor sensitivity, timing, and irreversibility of the G1/S transition

doi: 10.1016/j.jbc.2025.110865

Figure Lengend Snippet: Proposed model the ErbB2 receptor regulation during G1/S transition via cyclin D1 and c-Myc in CDK4i nonresponsive cells. A , in low ErbB2-expressing cells, CDK4 inhibition leads to reduced pathway activity due to ErbB2 degradation via Hsp90 dis-colocalization. This attenuates the transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells unresponsive to CDK4i exhibits a delayed G1/S transition. B , in high ErbB2-expressing cells, 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 cell cycle arrest after CDK4 inhibition. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

Techniques: Expressing, Inhibition, Activity Assay

( 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: A method for constructing MCF-7 (ATCC, HTB-22) overexpressing full-length human ErbB2 (HER2) is described as follows: A human ErbB2 cDNA in pCMV6-XL5 vector was purchased from OriGene (Catalog No: TC128161, Rockville, MD).

Techniques: Control, 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).

Techniques: Western Blot, Expressing, Fluorescence, Incubation, Control

(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.

Techniques: Inhibition, Activity Assay, Expressing

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