Journal: bioRxiv

Article Title: Sensitivity to Vγ9Vδ2TCR T cells is imprinted after single mutations during early oncogenesis

doi: 10.1101/2024.11.19.624272

Figure Lengend Snippet: (A) MCF10a cells were transduced with different ErbB2 variants and co-cultured with either Vγ9Vδ2TCR or HER2-CAR transduced T cells. Tumor cells were pre-treated with the PI3K kinase inhibitor Pictilisib at 2uM overnight. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (B) Furthermore, tumor cells were isolated and stained for BTN2A1 via TCR tetramer staining and (C) BTN3A cell surface expression. (D) Protein expression of HER2, phosphorylated AKT (pAKT) and total AKT in MCF10a mutant lines cultured for 24h in full culture medium (F) or medium without additional growth factors (S) . (E) Multiple tumor cell lines were co-cultured with Vγ9Vδ2TCR T-cells after pre-treatment with either the PI3K kinase inhibitor, AKT inhibitor or MEK inhibitor. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (F) Multiple tumor cell lines were pre-treated with either PI3K kinase inhibitor, AKT inhibitor, mTOR inhibitor Rapamycin or mTOR inhibitor Torin1 and subsequently co-cultured with Vγ9Vδ2TCR T cells. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (G) Heatmap illustrating the expression of genes from the ‘PI3K_and_AKT_family’ gene list, in the WT-AKPS model, with or without PAM. (H) Heatmap of Pearson’s correlation of the expression of genes from the ‘PI3K_and_AKT_family’ and BTNx genes in the WT-AKPS model, with or without PAM.

Article Snippet: pBABE-Puro retroviral vector (EV) and pBABE-Puro vectors carrying ERBB2 wild-type and mutants were co-transfected independently with pUMVC (Addgene #8449) and VSV-G (Addgene #8454) retroviral packaging plasmids into HEK-293T cells using PEI-transfection.

Techniques: Transduction, Cell Culture, Co-Culture Assay, Isolation, Staining, Expressing, Mutagenesis

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Validation of successful ectopic overexpression of ERBB2 in the CRC (HCT116 and HT29) and normal colon (CCD33 and CCD841) cell lines. ( A ) Relative ERBB2 expression in the normal colon cell lines CCD33 and CCD841 and the CRC cell lines HT29 and HCT116 as determined by qRT-PCR. Data were normalized to the expression of the internal control 18S rRNA gene and fold expressions were plotted relative to expression in the CCD33 cells. Data represent the mean ± SD of three independent experiments. ( B ) Relative ERBB2 expression in non-transfected and either empty pcDNA3 vector or pcDNA3- ERBB2 transfected HCT116, HT29, CCD33, and CCD841 cells as determined by qRT-PCR. Data were normalized to the expression of the internal control 18S rRNA gene and fold expressions were plotted relative to expression in the non-transfected cells. Data represent the mean ± SD of three independent experiments. *** p < 0.001; ns: not significant. ( C ) Same as B, but relative HER2 protein expression was determined in the different experimental conditions. Blots were re-probed with anti-β-Actin antibody to confirm equal loading across the lanes. The representative blots from three independent experiments are shown.

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Biomarker Discovery, Over Expression, Expressing, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Heatmap of the differentially expressed genes in CRC (HCT116 and HT29) and normal colon (CCD33 and CCD841) cell lines transfected with empty vector or ERBB2, either clustered based on expression ( A ) or grouped based on transfection and phenotype ( B ).

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Genome-wide gene expression changes between ERBB2 + and ERBB2 − CRC patients. ( A ) Principal component analysis (PCA) was performed to determine batch effects among the 14 patients’ samples. Comparison of PC1 and PC2 variation sequestered the samples based on ERBB2 expression. ( B ) Volcano plot of differentially expressed genes between ERBB2 - and ERBB2 + patients’ samples from input RNA-seq. Genes that are expressed significantly higher and lower based on log2 fold change in HER2+ samples are highlighted by green and blue dots, respectively. Unchanged transcripts are demarcated as grey circles ( p > 0.05). ( C ) Heatmap of the top 100 differentially expressed genes.

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Genome Wide, Gene Expression, Comparison, Expressing, RNA Sequencing