Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Differential regulation of human monocytes and NK cells by antibody-opsonized tumors

doi: 10.1007/s00262-018-2179-z

Figure Lengend Snippet: Monocyte phagocytosis is increased with HER2 gene-amplified targets. Antibody-dependent phagocytosis, degranulation, and tumor cell death were evaluated on HER2-amplified and -non-amplified tumor cell lines. a Titration curves of antibody-dependent phagocytosis, degranulation, and tumor cell death from SKBR3 (HER2 amplified) and T47D (HER2 non-amplified) tumor cell lines. All samples were performed in triplicate; means ± SEM and regression line are plotted on the graph. Two-way ANOVA with Sidak’s multiple comparisons test was used to determine significance. Asterisk represents significance between SKBR3 and T47D. b Comparison between antibody-dependent phagocytosis, degranulation, and tumor cell death of breast cancer cell lines that are amplified and non-amplified for HER2. Each point on the graph represents a different cell line and is the mean of a minimum of two experiments at the trastuzumab concentration of 316 ng/ml. Two tailed t tests were used to obtain P values for phagocytosis (P < 0.03), NK cell degranulation (P < 0.01), and tumor cell death (P > 0.8)

Article Snippet: The MCF-7 cell line was modified with pcDNA3.1 (V79020), purchased from Thermo Fisher Scientific (Waltham, MA), or pcDNA3 HER2 WT, which was a gift from Mien-Chie Hung (Addgene plasmid # 16257) [ 20 ].

Techniques: Amplification, Titration, Comparison, Concentration Assay, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Differential regulation of human monocytes and NK cells by antibody-opsonized tumors

doi: 10.1007/s00262-018-2179-z

Figure Lengend Snippet: Antigen density is important for phagocytosis. The MCF-7 breast cancer cell line was transfected with pcDNA HER2, and individual clones were selected by serial dilution. a Characterization of the protein expression by flow cytometry. Filled histogram is pcDNA HER2, bold line is empty vector, and normal line is unstained control. b Phagocytosis was performed as previously described in Fig. 1. Phagocytosis was performed in triplicate; means ± SEM and regression line are plotted on the graph. Two-way ANOVA with Sidak’s multiple comparisons test was used to determine significance. Asterisk represents significance between MCF7-HER2 and MCF7-Vector

Article Snippet: The MCF-7 cell line was modified with pcDNA3.1 (V79020), purchased from Thermo Fisher Scientific (Waltham, MA), or pcDNA3 HER2 WT, which was a gift from Mien-Chie Hung (Addgene plasmid # 16257) [ 20 ].

Techniques: Transfection, Clone Assay, Serial Dilution, Expressing, Flow Cytometry, Plasmid Preparation, Control

Journal: Autophagy

Article Title: Autophagy inhibition by TSSC4 (tumor suppressing subtransferable candidate 4) contributes to sustainable cancer cell growth.

doi: 10.1080/15548627.2021.1973338

Figure Lengend Snippet: Figure 8. ERBB2 increases TSSC4 expression which inhibits cell growth in breast cancer cells. (A–B) ERBB2 expression increases TSSC4 protein level and cell growth in MDA-MB-231 cells and TSSC4 knockdown in the ERBB2 expressing cells further increases cell growth. (A) Western blot showed expression of ERBB2 and TSSC4, and knockdown of TSSC4. (B) cell growth was measured by cell counting. (C–F) Effects of ERBB2 knockout and TSSC4 overexpression or knockout on cell growth in SKBR3 cells. (C) Western blot showed ERBB2 knockout and TSSC4 expression. (D) Western blot showed expression of Vector (HA), TSSC4 (TSSC4-HA) and TSSC4M (TSSC4M- HA) in SKBR3 ERBB2 knockout cells (NIC-ERBB2). (E) Effects of ERBB2 knockout and TSSC4/TSSC4M overexpression on cell growth that was measured by cell counting. (F) Effect of TSSC4 knockout on cell growth. Western blot showed TSSC4 knockout. NIC-Con, Control cells; NIC-TSSC4, cells with TSSC4 knockout (the same hereafter). ACTB was used as the loading control for western blot.

Article Snippet: Generation of stable cell lines expressing TSSC4 or ERBB2 plasmids Human ERBB2 plasmid (16,257) [83] was purchased from Addgene (deposited by Mien-Chie Hung).

Techniques: Expressing, Knockdown, Western Blot, Cell Counting, Knock-Out, Over Expression, Plasmid Preparation, Control

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: 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′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY, Real-time Polymerase Chain Reaction, 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: 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′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Over Expression, Transwell Assay, Cell Counting, Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, 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: 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′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Transfection, Quantitative RT-PCR, Transwell Assay, Cell Counting, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, 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: 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′-UTR WT or ERBB2-3′-UTR MU 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′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, Cell Counting, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, 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: 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′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Incubation, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Translational Lung Cancer Research

Article Title: Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study

doi: 10.21037/tlcr-21-216

Figure Lengend Snippet: Mutation spectrum of HER2 in NSCLC detected in the cBioPortal database. In the cBioPortal database, we identified three HER2 exon 20 insertions and four HER2 point mutations that are common in NSCLCs from eight studies that performed comprehensive genetic screening (2,15-21). NSCLC, non-small cell lung cancer.

Article Snippet: Briefly, a pBABE retrovirus vector subcloned cDNA of the human WT HER2 gene was purchased from Addgene (Cambridge, MA, USA).

Techniques: Mutagenesis

Journal: Translational Lung Cancer Research

Article Title: Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study

doi: 10.21037/tlcr-21-216

Figure Lengend Snippet: Tarloxotinib-E demonstrated high inhibition activity against HER2 mutations, and tarloxotinib showed low activity against WT HER2. (A) Growth inhibition curves of H1781 and Ba/F3 cells harboring WT HER2, HER2 with exon 20 insertions or HER2 with point mutations for each HER2 TKI. (B) A mosaic table that summarizes the measured IC50 values of (A) shows comparable efficacies of tarloxotinib-E and poziotinib and the minor activity of tarloxotinib. Each value indicates the IC50 calculated from the growth inhibition curve. Green indicates high sensitivity, corresponding to when the IC50 is less than 5 nM. Yellow indicates moderate sensitivity, corresponding to when the IC50 is higher than 5 nM but lower than 50 nM. Red indicates resistance, corresponding to when the IC50 is higher than 50 nM. (C) The plot table of SI of HER2 TKIs revealed the high SI of tarloxotinib/tarloxotinib-E. Each plot was calculated by [IC50 of Ba/F3 cells expressing WT HER2]/[IC50 of Ba/F3 cells expressing each HER2 mutant]. Regarding the plots of tarloxotinib-E, the SI was calculated by [IC50 of WT HER2 treated with tarloxotinib]/[IC50 of each HER2 mutant treated with tarloxotinib-E]. YVMA, A775_G776insYVMA; VC, G776_delinsVC; GSP, P780_Y781insGSP; WT, wild type; TKI, tyrosine kinase inhibitor; IC50, half maximal (50%) inhibitory concentration; SI, sensitivity index.

Article Snippet: Briefly, a pBABE retrovirus vector subcloned cDNA of the human WT HER2 gene was purchased from Addgene (Cambridge, MA, USA).

Techniques: Inhibition, Activity Assay, Expressing, Mutagenesis, Concentration Assay

Journal: Translational Lung Cancer Research

Article Title: Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study

doi: 10.21037/tlcr-21-216

Figure Lengend Snippet: The secondary C805S mutation was identified in TR clones of Ba/F3 cells harboring a HER2 mutation through ENU mutagenesis. (A) Number of TR clones established from ENU mutagenesis. The only secondary mutation identified in the HER2 TKD was C805S. (B) Chromatogram of the secondary C805S mutation identified in TR clones. (C) Ba/F3 cells harboring either HER2 exon 20 insertions or HER2 point mutations with the secondary C805S mutation acquired resistance to tarloxotinib-E and poziotinib. The killing curves of parental cells with HER2 exon 20 insertions or point mutations were extracted from Figure 2A as a control. (D) A mosaic table of the measured IC50 values of the acquired C805S clones shows that the secondary C805S mutation causes significant resistance to all HER2 TKIs. Yellow indicates moderate sensitivity, corresponding to when the IC50 is higher than 5 nM but lower than 50 nM. Red indicates resistance, corresponding to when the IC50 is higher than 50 nM. YVMA, A775_G776insYVMA; VC, G776_delinsVC; GSP, P780_Y781insGSP; TKD, tyrosine kinase domain; CS, C805S; TR, tarloxotinib-E-resistant; ENU, N-ethyl-N-nitrosourea; IC50, half maximal (50%) inhibitory concentration.

Article Snippet: Briefly, a pBABE retrovirus vector subcloned cDNA of the human WT HER2 gene was purchased from Addgene (Cambridge, MA, USA).

Techniques: Mutagenesis, Clone Assay, Control, Concentration Assay

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: The cytoplasmic tail of HER-2 is cleaved by multiple caspases in vitro. Small pool expression cloning was used to identify caspase substrates from a prostate adenocarcinoma cDNA library. A, cDNA pools were transcribed and translated in vitro in the presence of [35S]methionine and incubated with control (C) buffer or 25 ng of recombinant caspase-2 (C2), -3 (C3), or -8 (C8). Pool 158 contained a ∼76-kDa protein (indicated by an asterisk), which was cleaved by caspase-3 and -8. B, pool 158 was progressively subdivided and reanalyzed until a single cDNA (158-5E) encoding a ∼76-kDa protein, which was cleaved by caspase-3 into several fragments (indicated by arrows and labeled I-III), was isolated. 158-5E was identified as a partial human HER-2 cDNA encoding amino acids 762-1254. C, 35S-labeled 158-5E protein was incubated with 2.5 or 25 ng of caspase-1, -2, -3, -5, -6, -7, -8, or -9 or buffer (C) for 1 h at 37 °C, and the cleavage products (labeled I-III) were identified.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: In Vitro, Expressing, Clone Assay, cDNA Library Assay, Incubation, Recombinant, Labeling, Isolation

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: HER-2 is proteolyzed by caspases in HER-2-overexpressing breast cancer cells during the induction of apoptosis. A, HER-2-overexpressing SKBR3 human breast cancer cells were treated with 2 μg/ml TRAIL and 1 μg/ml cycloheximide for 0-16 h with or without pretreatment with 50 μm Z-VAD-fmk for 1 h. HER-2 was detected by immunoblotting using a HER-2 mAb that recognizes the carboxyl terminus. B, SKBR3 cells were preincubated with 100 nm epoxomicin and 50 μm Z-VAD-fmk for 1 h as indicated and then treated with 2 μg/ml TRAIL and 1 μg/ml cycloheximide for 0-16 h. C, SKBR3 cells were preincubated with 100 nm epoxomicin and 50 μm Z-VAD-fmk for 1 h as indicated and then treated with 200 μm etoposide for 0-16 h. In A-C, the HER-2 cleavage products are indicated by arrows. Apoptotic nuclei were scored at each time point in parallel experiments, and the percentage of apoptotic cells is indicated.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Western Blot

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: HER-2 is cleaved by caspases at four sites in the cytoplasmic tail. Four putative caspase cleavage sites (Asp1016, Asp1019, Asp1087, and Asp1125) were mutated to glutamic acid residues, and the mutant HER-2 proteins were examined for sensitivity to caspase proteolysis. A, MDA-MB-231 human breast cancer cells were transiently transfected with WT HER-2 or mutant HER-2 cDNAs as indicated. The next day, cells were preincubated with 100 nm epoxomicin for 1 h and then treated with 0.5 μg/ml TRAIL for 6 h. HER-2 was detected by immunoblotting using a carboxyl-terminal HER-2 mAb (cleavage products are indicated by arrows). B, schematic representation of HER-2 fragments (yellow) resulting from caspase proteolysis at each of the sites identified in A. The caspase cleavage-resistant D1016E/D1019E/D1087E/D1125E HER-2 mutant is designated 4× HER-2. The 25-kDa product (purple) is not detected by the carboxyl-terminal HER-2 Ab (red). C, 35S-labeled 158-5E/WT HER-2 or 158-5E/4× HER-2 proteins were incubated with control (C) buffer or the indicated amounts (ng) of caspase-3 or -8.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Mutagenesis, Transfection, Western Blot, Labeling, Incubation

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: Cleavage-resistant and truncated HER-2 confer greater protection against apoptosis than WT HER-2. A, immunoblot analysis of MDA-MB-231 pools stably expressing vector, WT, caspase-truncated (amino acids 1-1016, designated Trun), or caspase cleavage-resistant 4× HER-2. B, FACS analysis of cell surface expression of HER-2 proteins in MDA-MB-231 pools. C, MDA-MB-231 pools were treated with 0-5 μg/ml TRAIL for 24 h, and apoptotic nuclei were scored (mean ± S.E., n = 3). **, p < 0.01; ***, p < 0.001 for the indicated comparisons by two-way ANOVA with Bonferroni post-test. D, immunoblot analysis of MDA-MB-231 pools treated with TRAIL (0 or 5 μg/ml) for 24 h. E, MDA-MB-231 pools were serum-starved for 24 h, treated with 100 ng/ml EGF for 10 min, and examined for phospho-Akt (serine 473) and total Akt levels by immunoblotting. F, MDA-MB-231 pools were treated with 5 μg/ml TRAIL for 24 h. Phospho-Akt (serine 473), total Akt levels, phospho-p42/p44 MAPK (Thr202/Tyr204), and total p42/p44 MAPK were determined by immunoblotting. A positive control lysate for phospho-Akt (serine 473; Cell Signaling) was also immunoblotted.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Positive Control

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: The 47- and 25-kDa HER-2 products induce apoptosis by a caspase-dependent and Bcl-2-suppressible mechanism. A, schematic representation of the HER-2 products generated by caspase cleavage. B, MDA-MB-231 cells were co-transfected with cDNAs encoding FLAG-tagged HER-2 cleavage products (25, 22, or 47 kDa) or FLAG vector and pEGFP-N1 with or without a 1-h pretreatment with 100 μm Z-VAD-fmk. GFP-positive cells were scored for apoptotic nuclei 24 h later (mean ± S.E., n = 3). ***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. C, immunoblot confirms expression of an amino-terminal FLAG-tagged 22-kDa HER-2 cDNA in transfected MDA-MB-231 cells. D, MDA-MB-231 cells were co-transfected with cDNAs encoding the 25-kDa HER-2 (left) or the 47-kDa HER-2 cleavage product (right), pEGFP-N1, and vector, dominant negative (DN) FADD, CrmA, Bcl-2, or p35 cDNA. Apoptosis was scored as in B. **, p < 0.01 versus vector by one-way ANOVA with Tukey's multiple comparison post-test.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Generated, Transfection, Plasmid Preparation, Western Blot, Expressing, Dominant Negative Mutation

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: The 47- and 25-kDa HER-2 products localize to mitochondria and induce cytochrome c release. A, confocal images of MDA-MB-231 cells transfected with cDNAs encoding FLAG-tagged 25- or 47-kDa HER-2 cleavage products. Transfected MDA-MB-231 cells were immunostained with FLAG mAb (green). Mitochondria were labeled with Mitotracker Deep Red 633 (red), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Colocalization (yellow) is shown in the merged image. Bar, 10 μm. B, immunoblot analysis of cytochrome c levels in cytosolic and mitochondrial fractions of MDA-MB-231 cells transfected with vector or HER-2 products. C, confocal analysis of cytochrome c release. MDA-MB-231 cells stably expressing pBABE vector (left) or pBABE-Bcl-xL (right) were co-transfected with the FLAG-tagged 25- or 47-kDa HER-2 product (or tBid) and pEGFP-N1 in the presence of Z-VAD-fmk. GFP-positive cells (green), cytochrome c immunostaining (red), and 4′,6-diamidino-2-phenylindole staining (DAPI, blue) are shown. In the MDA-MB-231/pBABE cells (left), cells with cytochrome c release (indicated by an arrow in the GFP panels) exhibit diffuse or nearly absent cytochrome c staining. In the MDA-MB-231/pBABE-Bcl-xL (right), cells with intact mitochondrial cytochrome c (indicated by an arrow in the GFP panels) show punctate staining. D, quantitation of the cytochrome c release data shown in C (mean ± S.E., n = 5). ***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. E, immunoblots of cytosolic and mitochondrial fractions from HER-2-overexpressing SKBR3 cells treated with 2.5 μg/ml TRAIL and 1 μg/ml cycloheximide for the indicated number of hours.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Transfection, Labeling, Staining, Western Blot, Plasmid Preparation, Stable Transfection, Expressing, Immunostaining, Quantitation Assay

Journal:

Article Title: Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector * S⃞

doi: 10.1074/jbc.M802156200

Figure Lengend Snippet: Caspase-cleaved HER-2 contains a BH3-like domain and mimics the actions of Bad. A, sequence alignment of the 47- and 25-kDa HER-2 cleavage products and the BH3 domains of Bcl-2 family members. The absolutely conserved Leu and Asp residues in the BH3 domain that are required for proapoptotic activity are highlighted in yellow. B, the conserved Leu1120 and Asp1125 in human HER-2 were mutated to glutamic acid, either alone (L1120E or D1125E) or in combination (L1120E/D1125E, designated 2×E). MDA-MB-231 cells were co-transfected with cDNAs encoding the 25-kDa HER-2 product (WT or BH3 mutants) and pEGFP-N1. GFP-positive cells were scored for apoptotic nuclei 24 h later. C, apoptosis induced by the HER-2 products is Bax/Bak-dependent. WT or Bax/Bak DKO MEFs were infected with a bicistronic retrovirus encoding GFP and vector, GFP and HER-2 product (25 or 47 kDa), or GFP-tagged tBid for 48 h, and the percentage of GFP-positive cells with apoptotic nuclei was scored. D, 20-mer peptides (amino acids 1113-1132 of human HER-2) containing the WT HER-2 BH3 domain, a mutant 2×E HER-2 BH3 domain, or a Bid BH3 domain were incubated with isolated Jurkat cell mitochondria, and the released cytochrome c in the supernatant was analyzed by enzyme-linked immunosorbent assay. Readings were normalized to DMSO treatment. E, the 25-kDa HER-2 cleavage product binds to Bcl-xL. MDA-MB-231 pools stably expressing Bcl-xL or empty vector were immunoprecipitated (IP) with a rabbit Bcl-xL antibody and immunoblotted with a mouse FLAG (left) or mouse Bcl-x antibody (right). F, the 25-kDa HER-2 acts synergistically with tBid to induce apoptosis, mimicking the actions of Bad. MDA-MB-231 cells stably expressing Bcl-xL were transiently co-transfected with tBid and a bicistronic plasmid co-expressing GFP and vector, BAD, Noxa, or the 25-kDa HER-2 product. GFP-positive cells were scored for apoptotic nuclei 24 h later. G, the 25- and 47-kDa HER-2 products cooperate with Noxa to induce apoptosis. MCF-7 cells stably expressing caspase-3 were co-transfected with cDNAs encoding the 25- or 47-kDa HER-2 product, Bad (B), Noxa (N), or empty vector (alone and in combination) and pEGFP-N1. GFP-positive cells were scored for apoptotic nuclei 24 h later. H, MDA-MB-231 cells were transiently co-transfected with pEGFP-N1 and empty vector or cDNAs encoding full-length WT or mutant 2×E HER-2. Twenty-four h later, cells were treated with TRAIL for 24 h, and GFP-positive cells were scored for apoptotic nuclei. In B-D and F-H, data are the mean ± S.E. (n = 3). B and D,*, p < 0.05; **, p < 0.01 for the indicated comparisons by two-tailed unpaired t test. C, ***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. F, ***, p < 0.001 versus tBid + vector by one-way ANOVA with Bonferroni post-test. G,*, p < 0.05; **, p < 0.01 versus Noxa by one-way ANOVA with Bonferroni post-test. H, ***, p < 0.001 versus WT HER-2 by two-way ANOVA with Bonferroni post-test.

Article Snippet: WT full-length human HER-2 cDNA, kindly provided by Dr. Gibbes Johnson ( 30 ), was subcloned into the XhoI sites of the retroviral vector pLXSN (Clontech).

Techniques: Sequencing, Activity Assay, Transfection, Infection, Plasmid Preparation, Mutagenesis, Incubation, Isolation, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Biomolecular Detection and Quantification

Article Title: Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

doi: 10.1016/j.bdq.2014.08.001

Figure Lengend Snippet: Sixteen cancer assay sequences and probe chemistries evaluated in addition to EGFR L858R and T790M.

Article Snippet: Human Epidermal Growth Factor Receptor 2 ( HER 2) p.L755S, c.2264T>C , WT PROBE , 6FAM-TCCCTCAACACTTTG-MGBNFQ , TAQMAN MGB , LIFE TECHNOLOGIES.

Techniques: Sequencing, Mutagenesis, Binding Assay, Activity Assay