Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Molecular characterization of Sting knockout cancer cell lines. ( A ) Analysis of human HER2 display on cell surface of LLC1-HER2 (left) and CT26_HER2 (right) by FACS analysis; an unrelated antibody was used as negative control. ( B ) The graphic shows Tmem173 (transcript ID ENSMUST00000115728.4) gene organization. Full and empty boxes represent, respectively, coding and untranslated exons. The positions of guide RNAs used for CRISPR/Cas9 genome editing to generate Sting knockout cancer cell lines are indicated by arrows. ( C ) Western blot analysis of Sting protein in CT26-HER2, LLC1_HER2 and their Sting knockout cell lines counterparts. Gamma tubulin was used as standard. ( D ) PCR screening of CT26-HER2_SKO and LLC1_HER2_SKO cell lines to assess the absence of eGFP and Cas9 residues in genomic DNA. Cas9/eGFP-encoding vector was used as positive control (C+). Genomic DNA from parental CT26-HER2 and LLC-HER2 cell lines was used as negative control (C−). ( E ) Cell doubling per day were assessed for Sting wild-type (grey lines) and Sting knockout (black lines) LLC1 (left) and CT26 (right) cell lines. The differences in cell doubling were calculated by Student’s t -test and were not statistically significant (Ns) to each passage.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Negative Control, CRISPR, Western Blot, Plasmid Preparation, Positive Control

Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Comparison of viral effectiveness in Sting knockout vs. parental wild-type cancer cell lines. ( A , B ) Spread of eGFP-encoding R-LM113 was evaluated by fluorescence microscopy in STING wild-type and knockout LLC1 (5×) ( A ) and CT26 (10×) ( B ) cell lines. ( C ) The lytic activity of R-LM113 was evaluated by extracellular LDH (lactate dehydrogenase) release in cell supernatants over the time course of infection (72, 96 and 120 h) in LLC1-HER2 (grey lines) and LLC1-HER2_SKO (black lines) at two different concentrations of viral particles (1 multiplicity of infection (MOI) continuous lines and 0.5 MOI dashed lines). ( D ) The same experiments performed in panel C were recapitulated in CT26-HER2 and CT26-HER2_SKO. All the infections were performed as biological replicates. The statistical significances for experiments described in panel c and d were calculated by Student’s t -test comparing MOI-matched Sting wild-type vs. knockout cell lines. The p -values were 0.00115 and 0.000219, respectively, for 1 and 0.5 MOI in panel C; 0.01583, 0.008543, respectively, for 1 and 0.5 MOI in panel D. ( E , F ) Evaluation of viral replication of R-LM113 in Sting wild-type and knockout LLC1 ( E ) and CT26 ( F ) infected with 0.3 PFU/cell. The qPCR-TaqMan analysis revealed the genome copies per mL (gc/mL) produced by the virus over time (24, 48, 72 h for LLC1 and 72, 96, 20 h for CT26). The statistical significances for experiments described in panel e and f were calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values calculated on biological replicates were 0.0013 for LLC1 cell line and 0.0005 for CT26 cell line. ( G , H ) Analysis of the R-LM113 viral titers obtained in Sting wild-type and knockout LLC1 ( G ) and CT26 ( H ) cells infected with 0.3 PFU/cell. Plaque assay was performed as biological replicate. The statistical significance for experiments described in panel g and h was calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values were 0.038 for LLC1 cell line and 0.02 for CT26 cell line. p < 0.05 *; p < 0.005 **; p < 0.0005 ***.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Fluorescence, Microscopy, Activity Assay, Infection, Produced, Plaque Assay

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: HER2 overexpression is a driver for invasive progression of DCIS (A) Representative IHC images of p-ERK and p-AKT. (B) Plotted H scores for p-ERK and p-AKT expression in HER2 + DCIS and HER2 – DCIS controls. (C) Tumor-free survival curves of responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (D) Tumor-free survival curves of non-responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (E) Representative whole-mount images and Ku80-stained sections of DCIS-injected mammary glands from DCIS063 mice treated with vehicle (left panels) or herceptin (right panels). (F) Schematic representation of the experimental setup for lentiviral overexpression of HER2 in HER2 – DCIS cells. (G) Western blot showing expression of HER2 and phospho-HER2 in parental 293T cells and 293T cells transduced with the HER2-GFP lentivirus. (H) Representative whole-mount images of intraductally injected mammary glands, showing non-invasive replacement growth of non-transduced HER2 – DCIS cells (left panel) and invasive expansive growth of the same DCIS cells transduced with the HER2-GFP lentivirus (right panel). Inset shows lesion without αSMA marker indicating GFP expression (green). Cells of human origin are marked with Ku80 (gray), myoepithelial cells are marked with αSMA (magenta). Inset shows lesion without αSMA marker indicating HER2-GFP expression (green). See also Figure S7 .

Article Snippet: Primary or PDX DCIS tissue was digested to a single-cell solution as described before and was transfected with a lentivirus (ERBB2-GFP lentivirus, Origene, RC222909L4, or Akaluciferase, courtesy of the lab of Shinae Kizaka-Kondo cloned into the pRRL vector backbone, Addgene plasmid #31485) or vehicle by putting the cells and the virus in a falcon tube and spin for 2 hours at 2500 rpm at RT.

Techniques: Over Expression, Expressing, Staining, Injection, Western Blot, Transduction, Marker

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 1. ERBB2 expression is upregulated in patient‑derived cervical cancer tissues and is associated with a poor prognosis. (A) RT‑qPCR and (B) WB analysis of ERBB2 transcript and protein expression, respectively, in patient‑derived cervical cancer tissues (n=65) vs. matched healthy cervical tissues (n=65). Data were analyzed via Wilcoxon signed‑rank test. (C) RT‑qPCR and (D) WB analysis of ERBB2 transcript and protein expression, respectively, in stage I/II vs. stage III/IV patient‑derived cervical cancer tissues (n=43 stage I/II; n=22 Stage III/IV). Data were analyzed via Mann‑Whitney U test. (E) RT‑qPCR and (F) WB analysis of ERBB2 transcript and protein expression, respectively, in lymph node metastatic and non‑metastatic patient‑derived cervical cancer biopsies [n=46 lymph node (‑); n=19 lymph node (+)]. Data were analyzed via Mann‑Whitney U test. (G) Survival analysis using the Kaplan‑Meier method according to high (above the median) or low (below the median) ERBB2 mRNA expression (n=32 in each cohort). The P‑value was calculated using the log‑rank test. For purposes of comparison across cohorts, the median ERBB2 mRNA and protein expression levels (normalized to the RT‑qPCR housekeeping control and WB loading control GAPDH) in the normal cohort have been set to 1.0. Data in box plots are expressed as the median ± IQRs (boxes) and absolute ranges (whiskers). n=3. **P<0.01. RT‑qPCR, reverse transcription‑quantitative PCR; WB, western blotting; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; Pt, patient.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Mann-Whitney U-Test, Comparison, Control, Western Blot

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 2. ERBB2 overexpression in cervical cancer cell lines stimulates viability, invasion and sphere‑formation. (A) Confirmation of ERBB2 KD in siERBB2 cells and OE in ERBB2 vec cells via WB. GAPDH was used as the loading control. (B) Invasion of siERBB2 vs. siCtrl cells via Transwell assay. (C) Invasion of ERBB2 vec vs. Ctrl vec cells via Transwell assay. (D) Cellular viability of siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells quantified using a Cell Counting Kit‑8. (E) Sphere‑formation of siERBB2 vs. siCtrl cells. (F) Sphere‑formation of ERBB2 vec vs. Ctrl vec cells. (G) Analysis of metastasis‑associated and cancer stem cell biomarkers mRNA and protein expression in siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells via RT‑qPCR and WB, respectively. GAPDH was used as the RT‑qPCR housekeeping control and WB loading control. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. siCtrl or Ctrl vec analyzed via unpaired Student's t‑test. KD, knockdown; OE, overexpression; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; Ctrl, control; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Over Expression, Control, Transwell Assay, CCK-8 Assay, Expressing, Knockdown, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 3. ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2‑ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT‑qPCR. GAPDH was used as the housekeeping control. PIK3CA, p‑AKT/AKT and p‑mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; †P<0.05 and ††P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one‑way ANOVA. IP, immunoprecipitation; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Phospho-proteomics, Control, Transfection, Transwell Assay, CCK-8 Assay, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 4. miR‑3184‑5p attenuates cervical cancer cell viability and invasion by targeting ERBB2. (A) Putative binding location for miR‑3184‑5p on ERBB2 3'‑UTR via TargetScan analysis. (B) miR‑3184‑5p expression in HeLa and SiHa cervical cancer cell lines compared with in the non‑cancerous human H8 cervical epithelial cell line assessed via RT‑qPCR. U6 was used as the housekeeping control. **P<0.01 vs. H8; ††P<0.01 vs. SiHa. Luciferase reporter assay of ERBB2‑3'‑UTRWT or ERBB2‑3'‑UTRMU in (C) HeLa or (D) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. **P<0.01 vs. Ctrl mimic or Ctrl inhib. WB of (E) HeLa and (F) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. Ctrl vec; ††P<0.01 vs. miR‑3184‑5p mimic. Data were analyzed via one‑way ANOVA. UTR, untranslated region; WT, wild‑type; MU, mutant; Ctrl, control; inhib, inhibitor; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Binding Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, CCK-8 Assay, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 5. p53‑activating Mithramycin A boosts miR‑3184‑5p expression, which lowers ERBB2 expression and attenuates viability and invasion of cervical cancer cell lines. (A) p53, p21 and ERBB2 protein expression in cervical cancer cultures incubated with MM or vehicle (DMSO) assessed via WB. GAPDH was used as the loading control. (B) miR‑3184‑5p expression in cervical cancer cultures incubated with MM or vehicle assessed via RT‑qPCR. U6 was used as the housekeeping control. (C) p53 and ERBB2 protein expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via WB. GAPDH was used as the loading control. (D) miR‑3184‑5p expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via RT‑qPCR. U6 was used as the housekeeping control. (E) Representative images of Transwell and sphere‑formation assays in (E) HeLa and (F) SiHa cells, and quantitative analysis of viability, invasion and sphere‑formation of cells treated with MM or vehicle. (G) Schematic diagram of the p53 activator MM rescuing miR‑3184‑5p expression, thereby suppressing ERBB2 transcription. This attenuates PIK3CA activity, which stimulates cervical cancer cell viability, invasion and sphere‑formation. Data are expressed as the mean ± SEM (n=3). **P<0.01 analyzed via unpaired Student's t‑test. MM, Mithramycin A; Ctrl, control; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Incubation, Control, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot

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.

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, 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.

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: 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.

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, Inhibition, Activity Assay