Journal: Cancer Research

Article Title: AREG and EREG Are Predictive Biomarkers of Response to EGFR Inhibition in Gastroesophageal Cancer

doi: 10.1158/0008-5472.CAN-25-0073

Figure Lengend Snippet: A subset of gastroesophageal tumors not displaying EGFR gene amplification is sensitive to anti-EGFR drugs. A, Heatmap showing the viability (in percentage vs. untreated) of 27 primary cell lines treated for 6 days with the indicated doses of the mAb cetuximab. The heatmap represents the average of three independent experiments. B, Boxplot showing the sensitivity of the 27 primary cells (divided into resistant and sensitive groups on the basis of the response to cetuximab) to the indicated drugs. Each dot represents the mean AUC computed by the mean of three independent experiments. Statistical significance was calculated using the Student t test. The labeled dot indicates a HER2-amplified cell line (GTR0233). C, Boxplots showing the percentage of nonviable cells (positive for trypan blue staining), untreated (−) cells, or cells treated (+) with cetuximab (CTX; 5 µg/mL for 6 days). The average of four independent experiments performed on three cetuximab-sensitive (GTR0539, GTR0640, and GTR0247) and three cetuximab-resistant cell lines (GTR0125, GTR0498, and GTR0687) is shown. Statistical significance was calculated using the ratio paired t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: The following primary antibodies were used: HER3 (Cell Signaling Technology #12708 RRID: AB_2721919), HER2 (Cell Signaling Technology #2165 RRID: AB_10692490), vinculin (Millipore 05-386 RRID: AB_11212640), and human EGFR (described in ref. ).

Techniques: Amplification, Labeling, Staining

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 6. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a mouse anti–human SKP2 antibody and a goat anti-mouse IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. # p < 0.01 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 7. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a rabbit anti-human SKP2 antibody and a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. * p < 0.05 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 8. Knocking down CYP1A1, SKP2, or SRSF5 abolishes enhanced oral keratinocyte proliferation induced by L-Arginine treatment under high-glucose conditions. TIGK cells were transfected with 20 nM siRNAs of CYP1A1, SKP2, or SRSF5 and then treated with 500 µM Arg in a culture medium with high glucose (48 mM). (A) mRNA expression of CYP1A1, SKP2, or SRSF5 48 h after transfection. (B) The proliferation of TIGK cells after knocking down CYP1A1, SKP2, or SRSF5 assessed by cell count at 48 h and MTS assay at 72 and 120 h after transfection. (C) Representative images of cell density at 48 h after treatment. Scale bar: 100 µm. * p < 0.05, # p < 0.01.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Transfection, Expressing, Cell Counting, MTS Assay

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 knockout inhibits autophagy in ERBB2-positive SKBR3 breast cancer cells. ( A ) Knockout of ERBB2 was performed by a double-nickase Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system and demonstrated by Western blotting. Autophagy was measured by Western blotting LC3-II and p62. Chloroquine (CQ, 20 µM) was used to block lysosomal degradation for autophagic flux measurement (the same hereafter). No AA, amino acid starvation. Anti-actin beta (ACTB) was used as a loading control (the same hereafter). Uncropped Western blot images and quantification of the protein band intensities were shown in . ( B ) Autophagy was measured by demonstrating the mRFP-LC3 puncta in cells and counting the numbers of mRFP-LC3 puncta per cell. Data represent or demonstrate the results from at least three independent experiments (the same hereafter). More example images of mRFP-LC3 puncta are shown in the . *, p < 0.5; **, p < 0.01; ***, p < 0.001 (the same hereafter). NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, CRISPR, Western Blot, Blocking Assay, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Overexpression of ERBB2 promotes autophagy in ERBB2-negative and -ERBB2-positive breast cancer cells. Western blotting demonstration of ERBB2 overexpression in the ERBB2-negative MDA-MB-231 cells and the ERBB2-positive MCF7 cells ( A ). ERBB2 overexpression increased the basal levels of autophagy ( B ) and amino acid starvation (No AA)-induced levels of autophagy ( C ). Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Over Expression, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of lapatinib on autophagy in NIH3T3 cells expressing EGFR or ERBB2. ( A ) Western blotting showed the expression of EGFR and ERBB2. ( B ) Effects of EGFR and ERBB2 on basal autophagy. ( C ) Effects of lapatinib (1 µM) on autophagy in cells expressing vector alone, EGFR or ERBB2. Autophagy was measured by Western blotting LC3-II and SQSTM1/p62 in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 regulates the expression of autophagy genes. ( A ) Bioinformatics analysis of the mRNA levels of different autophagy genes (ATG genes) in ERBB2-positive and -negative human breast tumor tissues. The data were extracted from the Cancer Genome Atlas (TCGA) database. RE, relative mRNA expression of an autophagy gene in ERBB2-positive breast tumors compared to that in ERBB2-negative breast tumors. The mRNA expression was quantified by RNA-seq by Expectation–Maximization (RSEM) followed by log2 transformation to estimate the gene-level transcription. ( B ) Western blotting demonstration of the effects of ERBB2 overexpression and knockout on protein expression of autophagy proteins ULK1, FIP200 (RB1CC1), BECN1, ATG7, ATG5, and ATG12. Overexpression of ERBB2 in MDA-MB-231 and MCF7 cells and ERBB2 knockout in SKBR3 cells are demonstrated in A and A, respectively. Expression of ATG5 or ATG12 was demonstrated by the level of ATG12–ATG5 complex since it is well known that ATG5 and ATG12 form a conjugate in the form of ATG12–ATG5 during autophagy. Uncropped Western blot images and quantification of the protein band intensities are shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, RNA Sequencing, Transformation Assay, Western Blot, Over Expression, Knock-Out, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: The Kaplan–Meier plots of the relapse-free survival (RFS)-based survival probability of patients with ERBB2-positive and -negative breast tumors. The plots were downloaded from the Kaplan–Meier Plotter website ( http://kmplot.com/analysis/index.php?p=service&cancer=breast (accessed on 19 November 2019)). ( A ) Data of patients with ATG12 expression ( ATG12 mRNA) in breast tumors. ( B ) Data of patients with MAP1LC3B (LC3B) expression ( MAP1LC3B mRNA) in breast tumors. ( C ) Data of patients with BECN1 expression ( BECN1 mRNA) in breast tumors.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Autophagy is ATG12-dependent in ERBB2-positive and -negative breast cancer cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( A ) Western blotting demonstration of siRNA knockdown of ATG12 in the ERBB2-positive SKBR3 breast cancer cells and ATG12 overexpression in the ERBB2-negative MDA-MB-231 breast cancer cells. ( B ) ATG12 knockdown decreased the basal level and amino acid starvation (No AA)-induced level of autophagy in SKBR3 cells. ( C ) ATG12 overexpression increased the basal level and amino acid starvation (No AA)-induced level of autophagy in MDA-MB-231 cells. Uncropped Western blot images and quantification of the protein band intensities are shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Western Blot, Knockdown, Over Expression

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 promotion of autophagy inhibits cell death induced by chemotherapy drug Taxol in breast cancer cells. ( A ) ERBB2 knockout decreased autophagy induced by Taxol (paclitaxel, 10 µM) in SKBR3 cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl (30 mM). ERBB2 knockout is demonstrated in A. ( B ) Cell death induced by Taxol was increased by ERBB2 knockout or ATG12 knockdown in SKBR3 cells. Knockdown of ATG12 and the related effect on autophagy are demonstrated in A,B. ( C ) ATG12 overexpression increased Taxol-induced autophagy in MDA-MB-231 cells. Overexpression of ERBB2 is demonstrated in A. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl. ( D ) Cell death induced by Taxol was decreased by overexpression of ERBB2 or ATG12 in MDA-MB-231 cells. ATG12 overexpression and the related effect on autophagy are demonstrated in A,C. NH 4 Cl, instead of CQ, was chosen to measure Taxol-induced autophagy because CQ can induce higher cell death than NH 4 Cl ( and unpublished data). Uncropped Western blot images and quantification of the protein band intensities were shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells. *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, Western Blot, Knockdown, Over Expression, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of ERBB2 antibody on ATG12 expression, autophagy, and cell death. An anti-human ERBB2 antibody (Ab) (research-grade trastuzumab biosimilar) was used to treat SKBR3 cells. ( A ) ERBB2 Ab treatment reduced the protein levels of ERBB2 and ATG12. ( B ) Real-time PCR experiments showed that ERBB2 Ab treatment decreased ATG12 mRNA level. ( C ) Immunoprecipitation (IP) demonstration of the interaction between ERBB2 and ATG12. ( D ) ERBB2 Ab treatment inhibited autophagy at the basal and amino acid starvation (No AA)-induced levels. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( E ) ERBB2 Ab treatment increased cell death without and with Taxol treatment. Uncropped Western blot images and quantification of the protein band intensities are shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: A graphic model for ERBB2 promotion of autophagy and its effect on cancer treatment and outcome to patients. ERBB2 expression upregulates the protein levels autophagy-related (ATG) proteins ULK1, FIP200, ATG5, ATG7, and ATG12 increasing ATG12-dependent autophagy. This leads to treatment resistance of breast cancer and a worse outcome for patients with ERBB2-positive breast cancer. Treatment with ERBB2 antibody (Herceptin or others) causes degradation of ERBB2 and downregulation of ATG12 resulting in autophagy inhibition.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Inhibition