Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 1 Elevated expression of erbB3 via stable transfection induces Akt activation and attenuates paclitaxel cyototoxicity in erbB2-overexpressing breast cancer cells. (a) MDA-MB-231 and SKBR3 cells were transfected with either pDsRed-Monimer-N1 or pDsRed-erbB3. After a 2-month selection with Geneticin, the stable clones of vector control (neo.1) or human erbB3 compli- mentary DNA (cDNA; B3.pool, B3.1, B3.2) were established, and the total cell lysates were subjected to western blot analyses with specific antibodies directed against erbB3, erbB2, epidermal growth factor receptor (EGFR), P-Akt, Akt, P- mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b, c) The series of stable transfectants derived from MDA-MB-231 or SKBR3 cells were plated onto 96-well plates with complete culture medium (Dulbecco’s modified Eagle medium (DMEM)/F12, 10% fetal bovine serum (FBS)). After 24 h, the medium was replaced with control medium (fresh DMEM/F12, 0.5% FBS) or same medium containing indicated concentrations of paclitaxel for another 72-h incubation. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)). Data shows the representative of at least three independent experiments. Bars represent s.d. values.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Stable Transfection, Activation Assay, Transfection, Selection, Clone Assay, Plasmid Preparation, Control, Western Blot, Derivative Assay, Incubation

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 2 Transient induction of erbB3 expression activates Akt and inhibits paclitaxel-induced apoptosis in erbB2-overexpressing breast cancer cells. MDA-MB-231 (MDA-231) and SKBR3 cells were infected with lentivirus containing either control vector or pLEX-erbB3 for 24 h. After selection with puromycin (1 mg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM for SKBR3 cells, 20 nM for MDA-MB-231 cells), for additional 24 h, were then collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP; F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp3, full-length caspase-3; C-Casp3, cleaved caspase-3) or b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Selection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 3 Specific knock down of erbB3 expression reduces P-Akt levels and significantly enhances paclitaxel-induced apoptosis. SKBR3 and MDA-MB-453 (MDA-453) cells were infected with lentivirus containing either ConshRNA or erbB3shRNA for 24 h. After selection with puromycin (1 mg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, erbB2, P-erbB2, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8 (F-Casp8, full-length caspase-8; C-Casp8, cleaved caspase-8), caspase-3 or b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Knockdown, Expressing, Infection, Selection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 4 The expression levels of erbB3 modulate Survivin expression in erbB2-overexpressing breast cancer cells through a PI-3K/ Akt-dependent mechanism. (a) MDA-MB-231, SKBR3 cells, their stable transfectants, and the MDA-MB-231 and SKBR3 cells infected with lentivirus containing either control vector or pLEX-erbB3, as performed in Figure 2, were collected and subjected to western blot analyses of Survivin, C-X-C chemokine receptor type 4 (CXCR4), Mcl-1, Bcl-xL or b-actin. (b) SKBR3, MDA-MB-453 and 435.eB1 cells infected with lentivirus containing either ConshRNA or erbB3shRNA, as performed in Figure 3, were collected and subjected to western blot analyses of Survivin, CXCR4, Mcl-1, Bcl-xL or b-actin. (c and d) SKBR3.B3.1 and SKBR3.B3.2 cells, untreated or treated with PD98059 (PD, 20 mM) or LY294002 (LY, 10 mM) for 6 h, were then collected and subjected to (c) western blot analyses of Survivin, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin and (d) MTS ((3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. The densitometry analyses of Survivin signals were shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Western Blot

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 5 Specific knockdown of Survivin expression abrogates erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells. (a) SKBR3.B3.1 and SKBR3.B3.2 cells infected with lentivirus containing either ConshRNA or SurshRNA (S3, S4 and S5) were subjected to the following experiments. (a) Western blot analyses of Survivin, erbB2, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to (c) western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8, caspase-3 or b-actin and (d) apoptosis enzyme-linked immunosorbent assay (ELISA). *Po0.005, **P ¼ 0.0133, ***P ¼ 0.0064 vs ConshRNA þ paclitaxel.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Knockdown, Expressing, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 6 Expression of erbB3 forms dimerization with erbB2, but not epidermal growth factor receptor (EGFR), and enhances erbB2 tyrosine kinase activation in breast cancer cells. (a) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to immunoprecipitation with anti- erbB3 mAb (mouse IgG was used as negative control with the same amount of total lysates of 435.eB1 and MDA-453 cells), and then followed by western blot analyses of erbB2, erbB3 or EGFR. (b) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to western blot analyses of erbB2, P-erbB2, erbB3, P-erbB3, EGFR, P-EGFR or b-actin. (c) Diagram of proposed model underlying the mechanism of erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Activation Assay, Infection, Control, Plasmid Preparation, Immunoprecipitation, Negative Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling

doi: 10.7150/ijbs.100276

Figure Lengend Snippet: Targeting the ESM1-epidermal growth factor receptor (EGFR) interaction by therapeutic peptides suppresses EGFR/human EGFR3 (HER3)-driven epithelial-mesenchymal transition (EMT) and cell mobility in gastric cancer (GC) cells. ( A ) Wild-type ESM1 (WT-ESM1) was introduced into AGS, Kato-III, and NCI-N87 cells, and then cells were subjected to a Western blot assay to evaluate the phosphorylation status of HER3. ( B, C ) Co-immunoprecipitation assays were conducted to assess the interaction between EGFR, ESM1, and HER3 in AGS/WT-ESM cells ( B ). Comparing the association of EGFR and HER3 in the AGS cells transfected with either WT-ESM1 or a control vector (Ctrl) ( C ). Subsequently, a Western blot analysis was performed to examine the formation of this complex. ( D ) Schematic diagram of two synthetic peptides including peptide 1 (1-27 aas) and peptide 2 (26-46 aas) of the ESM1 protein. ( E, F ) AGS cells were infected with a lentivirus carrying a control vector or WT-ESM1 following treatment of cells with synthetic peptides (1 µM) as indicated. The phosphorylation status of HER3, EGFR and Akt, and expressions of angiopoietin-2, Snail, and Slug were all detected by Western blotting ( E ). The migratory ability of cells was determined by a transwell migration assay ( F ). Differences are presented as the mean ± SD. *** p < 0.001, compared to the WT-ESM1-overexpressing only group.

Article Snippet: The following primary antibodies were used: endocan (LIA-1001; Lunginnov, Lille, France); phosphorylated (p)-EGFR (#3777), p-Akt (#9271), p-ERK (#4370), p-STAT3 (#9145), EGFR (#2239), Akt (#9272), STAT3 (#9139), ERK (#4695), E-cadherin (#3195), vimentin (#5741), Snail (#3879), Slug (#9585), p-HER3 (#2842), and HER3 (#12708) antibodies all obtained from Cell Signaling Technology (Danvers, MA, USA); angiopoietin-2 (bs-0677R-TR) and p-HER3 (bs-3491R) antibodies purchased from Bioss (Woburn, MA, USA); α-tubulin (#66031-1-Ig) antibodies respectively obtained from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Phospho-proteomics, Immunoprecipitation, Transfection, Control, Plasmid Preparation, Infection, Transwell Migration Assay

Journal: International Journal of Biological Sciences

Article Title: ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling

doi: 10.7150/ijbs.100276

Figure Lengend Snippet: A working model shows the molecular mechanism underlying the ability of ESM1 to promote progression of gastric cancer (GC) cells. The oncogenic role of ESM1 was attributed to triggering the epithelial-to-mesenchymal transition (EMT) by activating epidermal growth factor receptor (EGFR)/human EGFR3 (HER3) and their downstream signal, Akt. Angiopoietin-2 was highly correlated with ESM1 and interplayed with Akt to promote EMT progression. Blocking the interaction of ESM1 and the EGFR by synthetic ESM1 peptides attenuated the EGFR/HER3 activation-driven EMT, cell motility, and proliferation. This schematic representation was created using BioRender software.

Article Snippet: The following primary antibodies were used: endocan (LIA-1001; Lunginnov, Lille, France); phosphorylated (p)-EGFR (#3777), p-Akt (#9271), p-ERK (#4370), p-STAT3 (#9145), EGFR (#2239), Akt (#9272), STAT3 (#9139), ERK (#4695), E-cadherin (#3195), vimentin (#5741), Snail (#3879), Slug (#9585), p-HER3 (#2842), and HER3 (#12708) antibodies all obtained from Cell Signaling Technology (Danvers, MA, USA); angiopoietin-2 (bs-0677R-TR) and p-HER3 (bs-3491R) antibodies purchased from Bioss (Woburn, MA, USA); α-tubulin (#66031-1-Ig) antibodies respectively obtained from Proteintech (Rosemont, IL, USA).

Techniques: Blocking Assay, Activation Assay, Software

Journal: Oncotarget

Article Title: HER inhibitor promotes BRAF/MEK inhibitor-induced redifferentiation in papillary thyroid cancer harboring BRAFV600E

doi: 10.18632/oncotarget.15773

Figure Lengend Snippet: ( A ) Western blot of lysates of BCPAP cells treated with 0.1 μM dabrafenib with or without 1 μM lapatinib for indicated times. Lapatinib blocked the dabrafenib-induced HER3 and AKT phosphorylation and the pERK1/2 rebound. ( B ) BCPAP cells were treated with 2.5 μM selumetib with or without 1 μM lapatinib and collected at 0, 1, 8, and 48 h post-treatment. Lysates were Western blotted for HER3 and ERK.

Article Snippet: Membranes were hybridized with the following primary antibodies: p-Erk1/2, Erk1/2, p-HER3/ErbB3, HER3/ErbB3, HER2/ErbB2, p-AKT, AKT (Cell Signaling Technology), NIS, Tg, TPO, TSHR, GLUT1, and GLUT3 (Protein tech).

Techniques: Western Blot

Journal: Oncogene

Article Title: ERBB3-induced furin promotes the progression and metastasis of ovarian cancer via the IGF1R/STAT3 signaling axis

doi: 10.1038/s41388-020-1194-7

Figure Lengend Snippet: A and B , Cell lysates of NRG1 (20ng/ml) stimulation at the different timepoints were immunoblotted against ERBB receptors in OVCAR5 ( A ) and SKOV3 ( B ) cell lines. C and D , Cell lysates of NRG1 stimulation at the different time points cells were collected and immunoblotted against furin in OVCAR 5 ( C ) and SKOV3 ( D ). E and F . Cell lysates of ERBB3 inhibitor (AZD8931) treatment with or without NRG1 (20ng/ml) stimulation in the different times points were collected and immunoblotted against fruin and ERBB receptors in OVCAR 5 ( E ) and SKOV3 ( F ).

Article Snippet: We obtained, ERBB2 (#2165), p-ERBB2 (Tyr1248)(#2247), ERBB3 (#12708), p-ERBB3 (Tyr1289), IGF1R-β (#3027), GAPDH (#5174), mesothelin (#99966S), TGF-β (#3711), STAT3 (#9139), p-STAT3 (Tyr705)(#9145), p-STAT3 (Ser727)(#34911), JAK1 (#3344S), p-JAK1(Tyr1034/1035) (#74129S), JAK2 (3230S), p-JAK2 (Tyr1008) (#8082S) TYK2 (#14193S) and p-TYK2 (Tyr1054/1055) (#68790S) from Cell Signaling Technology (Danvers, MA), Furin (#sc-133142) was obtained from Santa Cruz Biotechnology.

Techniques: