Journal: Molecular Oncology

Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

doi: 10.1016/j.molonc.2013.08.011

Figure Lengend Snippet: Archazolid leads to growth inhibition in trastuzumab‐resistant tumor cells. (A) Western blot analysis of HER2 expression in SKBR3, JIMT‐1 and MCF‐7 cells. SKBR3 and JIMT‐1 cells were treated with increasing doses of trastuzumab ...

Article Snippet: P‐HER2 (Tyr1248) was labeled with Alexa Fluor 488 conjugated mAb Ab18 (Neomarkers, now Thermo‐Fisher Scientific) The lysosomal marker Lamp‐1 was obtained from the Developmental Studies Hybridoma Bank Iowa, the endosomal marker EEA1 from Santa Cruz Biotechnology (Santa Cruz, CA), the proliferation marker anti‐Ki67 from Dako (Glostrup, DK).

Techniques: Inhibition, Western Blot, Expressing

Journal: Molecular Oncology

Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

doi: 10.1016/j.molonc.2013.08.011

Figure Lengend Snippet: Archazolid impairs HER2‐related signaling. (A) SKBR3 cells were treated with increasing doses of trastuzumab or archazolid for 24 h and phosphorylation of AKT and P70S6K was analyzed by western blot. (B) JIMT‐1 cells were treated ...

Article Snippet: P‐HER2 (Tyr1248) was labeled with Alexa Fluor 488 conjugated mAb Ab18 (Neomarkers, now Thermo‐Fisher Scientific) The lysosomal marker Lamp‐1 was obtained from the Developmental Studies Hybridoma Bank Iowa, the endosomal marker EEA1 from Santa Cruz Biotechnology (Santa Cruz, CA), the proliferation marker anti‐Ki67 from Dako (Glostrup, DK).

Techniques: Western Blot

Journal: Molecular Oncology

Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

doi: 10.1016/j.molonc.2013.08.011

Figure Lengend Snippet: Archazolid leads to accumulation of HER2 in the cytosol. (A) JIMT‐1 and SKBR3 cells were treated with 1 and 10 nM archazolid for 24 or 48 h and HER2 level was measured on the cell surface by flow cytometry (*p ≤ 0,01, ...

Article Snippet: P‐HER2 (Tyr1248) was labeled with Alexa Fluor 488 conjugated mAb Ab18 (Neomarkers, now Thermo‐Fisher Scientific) The lysosomal marker Lamp‐1 was obtained from the Developmental Studies Hybridoma Bank Iowa, the endosomal marker EEA1 from Santa Cruz Biotechnology (Santa Cruz, CA), the proliferation marker anti‐Ki67 from Dako (Glostrup, DK).

Techniques: Flow Cytometry

Journal: Molecular Oncology

Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

doi: 10.1016/j.molonc.2013.08.011

Figure Lengend Snippet: Archazolid leads to accumulation of HER2 in endocytotic vesicles. Colocalization of HER2 (red) with lysosomes (Lamp‐1, green), autophagosomes (LC3, green) or endosomes (EEA1, green) after treatment with 10 nM archazolid for 24 h ...

Article Snippet: P‐HER2 (Tyr1248) was labeled with Alexa Fluor 488 conjugated mAb Ab18 (Neomarkers, now Thermo‐Fisher Scientific) The lysosomal marker Lamp‐1 was obtained from the Developmental Studies Hybridoma Bank Iowa, the endosomal marker EEA1 from Santa Cruz Biotechnology (Santa Cruz, CA), the proliferation marker anti‐Ki67 from Dako (Glostrup, DK).

Techniques:

Journal: Molecular Oncology

Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

doi: 10.1016/j.molonc.2013.08.011

Figure Lengend Snippet: Decreased tumor growth in SCID mice is coherent with decreased nuclear Ki67 expression and increased HER2 internalization of archazolid treated mice. (A) Growth of xenograft tumors is significantly inhibited by bi‐daily archazolid treatment (3 ng/g), ...

Article Snippet: P‐HER2 (Tyr1248) was labeled with Alexa Fluor 488 conjugated mAb Ab18 (Neomarkers, now Thermo‐Fisher Scientific) The lysosomal marker Lamp‐1 was obtained from the Developmental Studies Hybridoma Bank Iowa, the endosomal marker EEA1 from Santa Cruz Biotechnology (Santa Cruz, CA), the proliferation marker anti‐Ki67 from Dako (Glostrup, DK).

Techniques: Expressing

Journal: Oncotarget

Article Title: Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

doi: 10.18632/oncotarget.19958

Figure Lengend Snippet: (A) Sera from BALB/c mice (n=5) immunized with bivalent vaccine including a mixture of 100μg (BV100) or 400μg (BV400) of HER1-ECD and HER2-ECD, were obtained on days -2, 35, 56, 73 and 93. Quantification of specific IgG antibodies against HER1 or HER2 was performed by ELISA. Data was Log transformed (1/titer + 1) for graphic representation. Non statistical differences among the groups were found using the non-parametric Mann-Whitney U test. (B) H292 tumor cells were incubated for 30 minutes with immune sera obtained from mice immunized with BV100 or BV400, at extraction day 56 (diluted 1:100). Cells were stimulated for 10 min with EGF (100ng/mL). HER1 and HER2 expression and phosphorylation state was analysed by Western blot. Pooled pre-immune sera (PI) was included as negative control, while Tyrosine kinase inhibitor (TKI) AG1478 (1μM) was used as positive control. For densitometry the program ImageJ was employed. Representative results of one of two performed experiments are shown.

Article Snippet: Anti p-HER2 (Tyr1248) and total HER2 were obtained from Santa Cruz Biotechnology (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay, MANN-WHITNEY, Incubation, Expressing, Western Blot, Negative Control, Positive Control

Journal: Oncotarget

Article Title: Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

doi: 10.18632/oncotarget.19958

Figure Lengend Snippet: (A) Cells lines were incubated with the pooled immune sera (black-filled histogram) collected from day 35 and diluted 1: 200 in PBS at 2% of SCF, followed by goat anti-mouse IgG FITC (gray solid line) stain. Pre-immune serum (black dotted line) was used as negative control for recognition. (B) A431 lysate was incubated overnight with immune sera to immunoprecipitate HER1 and HER2. Detection of these receptors was performed by immunoblotting. Pre-immune serum was used as negative control, while MAbs nimotuzumab (nimo) and Herceptin were used as positive controls for specific HER1 and HER2 (respectively) precipitation. The pictures were processed to alter the order of the immunoprecipitation reactions (IP), to get a match between both immunoblotting (IB) conditions.

Article Snippet: Anti p-HER2 (Tyr1248) and total HER2 were obtained from Santa Cruz Biotechnology (USA).

Techniques: Incubation, Staining, Negative Control, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

doi: 10.18632/oncotarget.19958

Figure Lengend Snippet: (A) H292 cells were incubated during 8 hours with pooled immune sera (PAbs) from extraction day 56 (diluted 1:100), and further stimulated with EGF (10 min, 100ng/mL). STAT3, Akt and ERK1/2 expression and phosphorylation state was analysed by Western blot, and compared with cells incubated with pre-immune sera (PI) used as negative control. AG1478 (TKI) (10μM) was included as a positive control of receptor inhibition. (B) HER1 and HER2 expression was assessed by Western blot at different end points (from 30 minutes to 24 hours) in H292 cells treated with PAbs from day 35, diluted 1: 100. Cells treated with PI were considered as negative control. (C) Cells viability was determined by MTT assay after 24, 48 and 120 hours of incubation with PAbs (pattern box) or PI (white box) diluted 1: 20 and heated at 56°C for 30 minutes to inactivate the complement. Mitomycin C (MitoC, 25μg/mL) was considered as positive control of cell viability reduction (black box). The 100% of viability was referred to PI incubation. Mean ± S.D. of six experiments are represented. Statistical analysis was performed by Kruskall-Wallis test, followed by Games Howell pos-test. a vs b (p<0.05), a vs c (p< 0.01), a vs d (p<0.001).

Article Snippet: Anti p-HER2 (Tyr1248) and total HER2 were obtained from Santa Cruz Biotechnology (USA).

Techniques: Incubation, Expressing, Western Blot, Negative Control, Positive Control, Inhibition, MTT Assay

Journal: Oncotarget

Article Title: Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

doi: 10.18632/oncotarget.19958

Figure Lengend Snippet: (A) After 24 hours of incubation with immune sera (PAbs) from day 56 (dilution 1: 100), treatment was withdrawn and cells were maintained under culture conditions for additional 24 or 48 hours. Then, cells were lysed and HER1/HER2 expression, as well as ERK1/2 phosphorylation was evaluated by Western blot. Pooled pre-immune sera (PI) were considered as negative control. (B) After 24 or 48 hours of incubation with PAbs (pattern box) diluted 1: 20, cells were maintained in culture conditions until complete 96 hours. PI treated cells (white box) were considered as reference of maximum viability. Cells incubated with PAbs during 96 hours were included as control of maximum viability reduction. Mean ± S.D. of six experiments are represented. Statistic was performed by a Kruskall-Wallis test, followed by Games Howell pos-test. a vs b (p<0.05)/ a vs c (p< 0.001).

Article Snippet: Anti p-HER2 (Tyr1248) and total HER2 were obtained from Santa Cruz Biotechnology (USA).

Techniques: Incubation, Expressing, Western Blot, Negative Control

Journal: Cell Adhesion & Migration

Article Title: Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

doi: 10.1080/19336918.2023.2168231

Figure Lengend Snippet: LL-37 promoted EMT via HER2/EGFR-MAPK/ERK signaling in HCC cells. PLC/PRF-5 and Huh7 cells were transfected with pcDNA/LL-37 (a), or si-LL-37 (b), and the MMP14, MMP9 and p-ERK1/2 level was detected by western blot. PLC/PRF-5 and Huh7 cells were transfected with pcDNA/LL-37 for 48 h, then KO-947 (c) or Neratinib (d) was added. Western blots detected the levels of p-ERK1/2, ERK1/2, Vimentin, E-cadherin, N-cadherin, Slug and Snail. Transwell assay (e) and wound healing assay (f) were conducted to evaluate changes in invasion and migration after KO-947 or neratinib treatment, respectively. Multiple of invasion cells and the wound closure (%) compared with control (pcDNA3.0) were calculated from 4 independent experiments. Data are represented as the mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies against β-actin and p-HER2 (Tyr1248) were purchased from Abclonal (Wuhan, China), as well as anti-rabbit and anti-mouse secondary antibodies.

Techniques: Transfection, Western Blot, Transwell Assay, Wound Healing Assay, Migration

Journal: Cell Adhesion & Migration

Article Title: Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

doi: 10.1080/19336918.2023.2168231

Figure Lengend Snippet: The effect of LL-37 expression on EMT in xenograft tumors. PLC/PRF-5 and PLC/PRF-5 LL−37 (stable overexpressing LL-37 cells) were subcutaneously injected into nude mice to establish an xenograft mouse model, and the mice were assigned to three groups: PLC/PRF-5 xenografted mouse group (control group), PLC/PRF-5 LL−37 xenograft mouse group (OV-LL-37 group) and PLC/PRF-5 xenografted mouse group with si-LL-37 treatment group (si-LL-37 group). Mice were euthanatized on day 28. (a) Tumor tissues were crushed, lysed with RIPA lysis buffer and supernatants were collected to detect p-HER2, p-EGFR, p-ERK1/2 and EMT markers by western blot. The quantified data was normalized to the protein expression of control group cells (n = 4). (b) Total RNA from tumor tissues was extracted and the expression of EMT markers was detected by qRT-PCR. (c) Tumor tissues were embedded in paraffin and sectioned for IHC staining using antibodies against EMT markers. Representative images from each group are shown. Scale bars, 50 μm. Data are represented as means ± SEM. ns, no significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies against β-actin and p-HER2 (Tyr1248) were purchased from Abclonal (Wuhan, China), as well as anti-rabbit and anti-mouse secondary antibodies.

Techniques: Expressing, Injection, Lysis, Western Blot, Quantitative RT-PCR, Immunohistochemistry

Journal: Cell Adhesion & Migration

Article Title: Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

doi: 10.1080/19336918.2023.2168231

Figure Lengend Snippet: LL-37 silencing enhanced 1,25(OH) 2 D 3 -induced EMT in vivo. (a) PLC/PRF-5 xenograft tumors were crushed and lysed with RIPA lysis buffer, and then western blot detection was performed using the indicated antibodies. (b) Representative images of IHC of E-cadherin, N-cadherin and Slug in xenograft tumors. Scale bar: 50 μm. Data are represented as means ± SEM. (c) A schematic diagram summarizing the regulation mechanism of LL-37 on the EMT, migration and metastasis of HCC cells. 1,25(OH) 2 D 3 induces the secretory expression of LL-37 through VDR receptor signal. Then LL-37 phosphorylates EGFR/HER2 to stimulate MAPK/ERK signaling, regulate the expression of EMT-related transcription factors or markers. Ultimately affects the EMT, migration and metastasis of HCC cells. ns, no significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies against β-actin and p-HER2 (Tyr1248) were purchased from Abclonal (Wuhan, China), as well as anti-rabbit and anti-mouse secondary antibodies.

Techniques: In Vivo, Lysis, Western Blot, Migration, Expressing