Journal: American Journal of Cancer Research

Article Title: NRG1 secreted by cancer-associated fibroblasts contributes to enzalutamide resistance in prostate cancer cells

doi: 10.62347/ottr3398

Figure Lengend Snippet: Figure 4. Cancer associated fibroblasts secreted NRG1 in the in the co-culture system exposed with CAFs and promoted phosphorylation of HER3 in enzalutamide resistant PC3 cells (PC3-EnzR). Levels of NRG1 in the culture medium from PC3-EnzR or PC3-EnzR cells in the co-culture system exposed with CAFs were measured by ELISA (A). PC3-EnzR or PC3-EnzR cells in the co-culture system exposed with CAFs were harvested and western blotting was used to measure the protein expressions of pHER3, HER3, MMP1 and MMP9 (B-F). GAPDH was used as a loading control and the expressions were normalized to NC (PC3-EnzR culture). Results are shown as means ± SEM. ##P < 0.01, ###P < 0.001 compared to NC.

Article Snippet: Primary antibodies included NRG1 (Abcam #ab53104, 1:1000 dilution), HER3 (CST #12708, 1:1000 dilution), pHER3 (CST #12708, 1:2000 dilution), MMP1 (LSBio #LSC352518, 1:500 dilution), MMP9 (LSBio #LSC31757, 1:1000 dilution), and GAPDH (CST #2118, 1:1000 dilution).

Techniques: Co-Culture Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: American Journal of Cancer Research

Article Title: NRG1 secreted by cancer-associated fibroblasts contributes to enzalutamide resistance in prostate cancer cells

doi: 10.62347/ottr3398

Figure Lengend Snippet: Figure 5. Cancer associated fibroblasts secreted NRG1 in the in the co-culture system exposed with CAFs and promoted phosphorylation of HER3 in enzalutamide resistant DU145 cells (DU145-EnzR). Levels of NRG1 in the culture medium from DU145-EnzR or DU145-EnzR cells in the co-culture system exposed with CAFs were measured by ELISA (A). DU145-EnzR or DU145-EnzR cells in the co-culture system exposed with CAFs were harvested and western blotting was used to measure the protein expressions of pHER3, HER3, MMP1 and MMP9 (B-F). GAPDH was used as a loading control and the expressions were normalized to NC (DU145-EnzR culture). Results are shown as means ± SEM. ##P < 0.01, ###P < 0.001 compared to NC.

Article Snippet: Primary antibodies included NRG1 (Abcam #ab53104, 1:1000 dilution), HER3 (CST #12708, 1:1000 dilution), pHER3 (CST #12708, 1:2000 dilution), MMP1 (LSBio #LSC352518, 1:500 dilution), MMP9 (LSBio #LSC31757, 1:1000 dilution), and GAPDH (CST #2118, 1:1000 dilution).

Techniques: Co-Culture Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Journal of Thoracic Disease

Article Title: A subset of esophageal squamous cell carcinoma patient-derived xenografts respond to cetuximab, which is predicted by high EGFR expression and amplification

doi: 10.21037/jtd.2018.09.18

Figure Lengend Snippet: Correlation between protein level and tumor response

Article Snippet: For all of the samples, the relative EGFR protein expression level was determined by immunohistochemistry (IHC), anti-human antibodies including EGFR (CST, Beverly, MA, USA), P-EGFR (Abcam, Cambridge, MA, USA), HER3 (CST, Beverly, MA, USA), P-HER3 (CST, Beverly, MA, USA), MET (CST, Beverly, MA, USA), P-MET (CST, Beverly, MA, USA), Akt (CST, Beverly, MA, USA), P-Akt (CST, Beverly, MA, USA), ERK (CST, Beverly, MA, USA), P-ERK (CST, Beverly, MA, USA) were applied to stain the positive sections.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells

doi: 10.1186/s12964-019-0413-8

Figure Lengend Snippet: USP8-regulated ITCH interaction with c-FLIP mediates 9F7-F11-induced c-FLIP ubiquitination. BxPC3 cells were incubated with NRG1 or/and 9F7-F11 for various times. After cell lysis in CHAPS buffer, 2 mg of total protein extracts were co-immunoprecipitated with the anti-HER3 antibody 2F12 (Millipore) against HER3 C-terminal tail. Then, the first soluble supernatant was co-immunoprecipitated with the rabbit anti-c-FLIP polyclonal antibody H-202 (Santa Cruz Biotechnology) that targets both c-FLIP L and c-FLIP S . The presence of ITCH and USP8 in the two immunoprecipitates was assessed by western blotting. HER3 and c-FLIP ubiquitination status were assessed using the anti-K48 ubiquitin antibody. Whole cell lysates (WCL) were analyzed using the appropriate antibodies. Quantification of signal intensity (SI) with ImageJ software is indicated below the images, in comparison to SI = 1.0 ± .0 for untreated control. Significant increase or decrease of the densitometry, compared to control, is indicated in bold. β-actin was evaluated as loading control

Article Snippet: Rabbit monoclonal antibodies against HER3 and phosphorylated (p) HER3 (Tyr1289), PARP (clone 46D11), cleaved caspase-8 (Asp391; clone 18C8), caspase-9 and cleaved caspase-9, caspase-3 (clone 8G10) and cleaved caspase-3, XIAP, DR5 (clone D4E7), BIM, c-FLIP (clone D16A8), USP9X, β-actin and β-tubulin were from Cell Signaling Technology (Danvers, MA).

Techniques: Ubiquitin Proteomics, Incubation, Lysis, Immunoprecipitation, Western Blot, Software, Comparison, Control

Journal: Cell Communication and Signaling : CCS

Article Title: ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells

doi: 10.1186/s12964-019-0413-8

Figure Lengend Snippet: 9F7-F11 induces c-FLIP downregulation by proteasomal degradation . a Cancer cells were incubated with NRG1 or 9F7-F11. After cell lysis at different time points, c-FLIP L , USP8 and USP9X expression, as well as ITCH expression and phosphorylation (p) were analyzed by western blotting. b BxPC3 cells were incubated with 9F7-F11 for 3 h. HER3, c-FLIP L and c-FLIP S expression were analyzed by western blotting. c BxPC3 and MDA-MB-468 cells were incubated with NRG1 or 9F7-F11 for 48 h or 96 h, before detection of HER3 and c-FLIP L expression by western blotting. d After pre-incubation or not with 10 μM MG132 for 4 h, BxPC3 cells were incubated with 9F7-F11 with or without NRG1. After cell lysis, expression of HER3 and c-FLIP L was assessed in whole cell lysates by western blotting. The rabbit anti-HER3 polyclonal antibody C-17 (Santa Cruz Biotechnology) was used for detection. Protein level was measured with the ImageJ software and indicated as signal intensity (SI), relative to untreated control (SI = 1.0 ± .0). Significant increase or decrease of the densitometry, compared to control, is indicated in bold. β-tubulin was evaluated as loading control

Article Snippet: Rabbit monoclonal antibodies against HER3 and phosphorylated (p) HER3 (Tyr1289), PARP (clone 46D11), cleaved caspase-8 (Asp391; clone 18C8), caspase-9 and cleaved caspase-9, caspase-3 (clone 8G10) and cleaved caspase-3, XIAP, DR5 (clone D4E7), BIM, c-FLIP (clone D16A8), USP9X, β-actin and β-tubulin were from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Lysis, Expressing, Phospho-proteomics, Western Blot, Software, Control

Journal: Cell Communication and Signaling : CCS

Article Title: ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells

doi: 10.1186/s12964-019-0413-8

Figure Lengend Snippet: ITCH silencing or chemical inhibition blocks 9F7-F11-induced apoptosis and c-FLIP degradation. siSC- and siITCH-transfected BxPC3 cells were incubated with 9F7-F11 alone ( a ), or with NRG1 ( b ) for 48 h before detection by western blotting of ITCH and c-FLIP L expression, and PARP/caspase cleavage. MDA-MB-468 cells were incubated with increasing doses of chlorimipramine (CI) for 24 h ( c ), or with 15 μM CI ( d ) before incubation with 9F7-F11 for 24 h. Total protein extracts were analyzed by western blotting to evaluate PARP and caspase cleavage ( c ), ITCH ubiquitination and expression and c-FLIP and HER3 expression ( d ). Quantification of signal intensity (SI) with ImageJ software is indicated below the images. No protein expression was measured as 0.0 ± .0. Significant decrease or increase of the densitometry, compared to control, is indicated in bold. BxPC3 cells were left untreated or pre-incubated with ITCH chemical inhibitor CI for 48 h before treatment with 9F7-F11 with or without NRG1. Apoptosis was measured at 96 h by flow cytometry after cell labelling with Annexin V/7-AAD ( e ). siSC and siITCH-transfected BxPC3 cells were treated with 9F7-F11 alone or with NRG1. Western blot was performed to check ITCH reduction in siITCH-transfected BxPC3 cells. Apoptosis was measured at 96 h by flow cytometry after cell labelling with Annexin V/7-AAD ( f ). ** P < 0.01, *** P < 0.001, ns not significant

Article Snippet: Rabbit monoclonal antibodies against HER3 and phosphorylated (p) HER3 (Tyr1289), PARP (clone 46D11), cleaved caspase-8 (Asp391; clone 18C8), caspase-9 and cleaved caspase-9, caspase-3 (clone 8G10) and cleaved caspase-3, XIAP, DR5 (clone D4E7), BIM, c-FLIP (clone D16A8), USP9X, β-actin and β-tubulin were from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Transfection, Incubation, Western Blot, Expressing, Ubiquitin Proteomics, Software, Control, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells

doi: 10.1186/s12964-019-0413-8

Figure Lengend Snippet: 9F7-F11-induced tumor cell apoptosis occurs through caspase-8/9/3 activation and PARP cleavage. BxPC3, MDA-MB-468 or DU145 cancer cells were incubated with the anti-HER3 antibody 9F7-F11. Caspase-8, − 9 and − 3 and PARP cleavage were analyzed by western blotting and cell lysates prepared at different time points during antibody incubation. Quantification of signal intensity (SI) with ImageJ software is indicated below the images (relative to untreated control measured as 1.0 ± .0). Significant increase or decrease of the densitometry, compared to control, is indicated in bold. M: medium alone; p41/43 and p18 C8c: caspase-8 cleavage; p37 C9c: caspase-9 cleavage; p17/19 C3c: caspase-3 cleavage; PARPc: PARP cleavage. β-tubulin was evaluated as loading control

Article Snippet: Rabbit monoclonal antibodies against HER3 and phosphorylated (p) HER3 (Tyr1289), PARP (clone 46D11), cleaved caspase-8 (Asp391; clone 18C8), caspase-9 and cleaved caspase-9, caspase-3 (clone 8G10) and cleaved caspase-3, XIAP, DR5 (clone D4E7), BIM, c-FLIP (clone D16A8), USP9X, β-actin and β-tubulin were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Incubation, Western Blot, Software, Control

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