hs578t wild type cell line  (ATCC)

Journal: Biomedicines

Article Title: Integrative RNA-Seq and TCGA-BRCA Analyses Highlight the Role of LINC01133 in Triple-Negative Breast Cancer

doi: 10.3390/biomedicines14020268

Figure Lengend Snippet: Representative scheme of the experimental design used to investigate the role of lncRNA LINC01133 in the human TNBC cell line Hs578T. The parental Hs578T cell line, namely Hs578T_wt, was genetically modified via lentiviral transfection to generate a control cell line, Hs578T_ctr, and a LINC01133 knockout cell line. After validation of the knockout, an RNA-Seq was performed in triplicate for each condition. Transcriptomic analyses included comparisons between Hs578T_ko and Hs578T_ctr; Hs578T_ko and Hs578T_wt; and Hs578T_wt and Hs578T_ctr to identify the DEGs of interest, which were dominant in Hs578T_ko (called KO-dominants). Subsequently, ontological pathway enrichment and translational integration with transcriptomic data from TNBC tumors of the TCGA-BRCA cohort were performed to evaluate the expression profile of KO-dominant DEGs in clinical samples.

Article Snippet: The Hs578T wild-type cell line (Hs578T_wt, ATCC, Manassas, VA, USA) was used as the parental cell line in biomolecular assays and served as the basal cell line for the generation of genetically modified (GMO) cell lines.

Techniques: Genetically Modified, Transfection, Control, Knock-Out, Biomarker Discovery, RNA Sequencing, Expressing

Journal: Biomedicines

Article Title: Integrative RNA-Seq and TCGA-BRCA Analyses Highlight the Role of LINC01133 in Triple-Negative Breast Cancer

doi: 10.3390/biomedicines14020268

Figure Lengend Snippet: Generation and validation of the Hs578T_ko cell line. ( A ) Schematic panel of the CRISPR/Cas9-based gene editing strategy used for the knockout of LINC01133 in Hs578T_wt using lentiviral particle production in 293T cells and subsequent transduction into the target cell line. In ( B ) is shown the analysis of LINC01133 expression by qRT-PCR in the Hs578T cell lines, showing that the generated Hs578T_ko 1 + 3 cell line presented a complete knockout compared to the other strategies and the Hs578T_ctr cell line. In ( C ), we evaluated the stability of the Hs578T_ko knockout across multiple cell passages, confirming the absence or low expression of the LINC01133 transcript. In ( D ), genome editing was confirmed by PCR using internal and external primers to the LINC01133 gene locus, showing non-amplification in Hs578T + ko 1 + 3. qRT-PCRs were performed in technical and biological triplicate, using three reference genes ( HPRT1 , GAPDH , and HMBS ). Statistical analysis was performed to obtain FoldChange using the 2 −ΔΔCT method, with comparative analysis via One-Way ANOVA and multiple comparisons via Dunnett’s test, with error based on SEM. p -value: * ≤ 0.05; **** ≤ 0.0001; ns: non-significant.

Article Snippet: The Hs578T wild-type cell line (Hs578T_wt, ATCC, Manassas, VA, USA) was used as the parental cell line in biomolecular assays and served as the basal cell line for the generation of genetically modified (GMO) cell lines.

Techniques: Biomarker Discovery, CRISPR, Knock-Out, Transduction, Expressing, Quantitative RT-PCR, Generated, Amplification

Journal: Biomedicines

Article Title: Integrative RNA-Seq and TCGA-BRCA Analyses Highlight the Role of LINC01133 in Triple-Negative Breast Cancer

doi: 10.3390/biomedicines14020268

Figure Lengend Snippet: Identification of DEGs and definition of KO-dominant genes in the knockout cell for LINC01133. On the left, we present the heatmap of DEGs obtained from the transcriptional comparisons, processed with DESeq2 , normalized to z-scores, and clustered based on DEGs. Above, the Venn diagram illustrates the overlap among the DEGs identified in the transcriptional comparisons. Below are volcano plots for the three transcriptomic comparisons, highlighting the distribution of DEGs as a function of log2FC and adjusted p -values. On the right, a specific volcano plot of the KO-dominant DEGs obtained shows genes whose magnitude of alteration in the Hs578T_ko condition exceeded that observed in Hs578T_wt and Hs578T_ctr by at least 0.5 log2FC units. Differential expression analyses were adjusted for multiple testing using the Benjamini–Hochberg method . Genes with FDR < 0.05 were considered differentially expressed. Visualizations were generated in R, v. 4.6, using the ComplexHeatmap package .

Article Snippet: The Hs578T wild-type cell line (Hs578T_wt, ATCC, Manassas, VA, USA) was used as the parental cell line in biomolecular assays and served as the basal cell line for the generation of genetically modified (GMO) cell lines.

Techniques: Knock-Out, Quantitative Proteomics, Generated

Journal: Biomedicines

Article Title: Integrative RNA-Seq and TCGA-BRCA Analyses Highlight the Role of LINC01133 in Triple-Negative Breast Cancer

doi: 10.3390/biomedicines14020268

Figure Lengend Snippet: Integration of dominant DEGs with TNBC data from the TCGA-BRCA cohort. In ( A ), we show the distribution of LINC01133 expression levels in TNBC samples from the TCGA-BRCA cohort (n = 199), ordered by log2(TPM + 1) transcript expression levels. In ( B ), we present heatmaps showing the expression of the 30 most upregulated and 30 most downregulated dominant genes in Hs578T_ko, evaluated in the TNBC samples (n = 199 samples) and organized according to the LINC01133 expression levels’ gradient. TCGA/BRCA data were obtained via the TCGABiolinks package . The TNBC delimitation was performed in R using the AIMS package . The data were normalized using z-scores and clustered relative to the DEGs. The visualizations were generated in the R environment (v. 4.6) using the ComplexHeatmap package .

Article Snippet: The Hs578T wild-type cell line (Hs578T_wt, ATCC, Manassas, VA, USA) was used as the parental cell line in biomolecular assays and served as the basal cell line for the generation of genetically modified (GMO) cell lines.

Techniques: Expressing, Generated

Journal: Biomedicines

Article Title: Integrative RNA-Seq and TCGA-BRCA Analyses Highlight the Role of LINC01133 in Triple-Negative Breast Cancer

doi: 10.3390/biomedicines14020268

Figure Lengend Snippet: Trend of dominant DEG expression levels along the LINC01133 gradient in TCGA-BRCA TNBC. Analysis of the relationship between LINC01133 expression levels and the normalized mean expression of dominant DEGs associated with knockout in TNBC tumors from the TCGA-BRCA cohort. Samples were stratified into five expression percentiles of LINC01133. The graphs show trends for the upregulated and downregulated gene sets in Hs578T_ko, identifying clusters that exhibit inhibitory or activating trends along the gradient. Trend analyses were calculated using the mean expression levels (z-score) of the upregulated and downregulated KO-dominant gene sets obtained from TCGA-BRCA transcriptomic data. Analyses were carried out in the R environment, and visualizations were generated using the ggplot2 package .

Article Snippet: The Hs578T wild-type cell line (Hs578T_wt, ATCC, Manassas, VA, USA) was used as the parental cell line in biomolecular assays and served as the basal cell line for the generation of genetically modified (GMO) cell lines.

Techniques: Expressing, Knock-Out, Generated