Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: TBP and TBPL1 genes, but not TBPL2 , are expressed in healthy and breast cancer cell lines. Total RNA was isolated with Trizol and end-point RT-PCR was performed with the oligonucleotides shown in Table . ( A ) Electrophoresis on 1% bleach agarose gel showing the integrity of the RNA28S and RNA18S ribosomal genes in MCF-12F and MCF-10A healthy cell lines (lanes1 and 2, respectively), and MCF-7, T47D, SK-BR-3, and MDA-MB-231 breast cancer cell lines (lanes 3 to 7, respectively). ( B ) Amplification of a 232 bp DNA fragment corresponding to the TBP gene in all breast cell lines. ( C ) Amplification of a 289 bp DNA fragment corresponding to the TBPL1 gene in all cell lines. ( D ) TBPL2 gene was not expressed in any of the healthy cell lines or breast cancer cell lines. ( E ) As an internal control, a 298 bp DNA fragment corresponding to the B2M gene was amplified in all the breast cell lines studied.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Amplification, Control

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: TBP and TBPL1 genes are differentially expressed in breast cancer cell lines according to the stage and molecular classification. ( A ) Relative TBP mRNA expression levels were evaluated by RT-qPCR using the RNA18SN1 gene as a housekeeping gene and normalized to the values obtained for MCF10A. ( B ) RT-qPCR evaluated relative TBPL1 mRNA expression levels. The results shown are from three independent experiments in triplicate, and the analysis of the results was performed with the 2 (-∆∆Ct) algorithm. Statistics analysis was carried out with unpaired Student’s t-test with Welch’s correction., and the asterisks show the p-value for significance.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: Intracellular localization of TBPL1 protein by immunofluorescence and confocal microscopy in healthy and breast cancer cell lines. Cells were fixed and incubated with antibodies against TBPL1 (1:200 dilution) and stained with Rhodamine-Phalloidin (1:100 dilution) for actin cytoskeleton and with Hoechst 33,342 for the nuclei.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Immunofluorescence, Confocal Microscopy, Incubation, Staining

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: Determination of the TBPL1 gene expression levels in untransfected and transfected healthy and breast cancer cell lines with the U6-gRNA Cas-92A + GFP-RM CRISPR plasmid. The TBPL1 gene mRNA levels were measured by end-point RT-PCR in all cell lines. As a control, the B2M mRNA expression level was also determined in all samples. RNA samples of the three independent experimental replicates for nontransfected and transfected cell lines were used as templates for RT-PCR assays before sending them for RNAseq analyses. ( A , C , E , G ) MCF-12, T47D, SK-BR-3, and MDA-MB-2321 nontransfected (NT) cell lines. ( B , D , F , H ) MCF-12, T47D, SK-BR-3, and MDA-MB-231 transfected (T) cell lines. ( I ) RNA gel electrophoresis shows the integrity of RNA18S and RNA28S ribosomal genes for all healthy and breast cancer cell lines.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Gene Expression, Transfection, CRISPR, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Nucleic Acid Electrophoresis

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: Determination of the TBPL1 protein expression level by Western blot. Protein extracts from nontransfected and transfected cell lines with the U6-gRNA Cas-92A + GFP-RM CRISPR plasmid were analyzed by Western blot. Membranes were probed with anti-TBPL1 and anti-Tubulin antibodies and revealed for chemoluminescence as described in Methods.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Expressing, Western Blot, Transfection, CRISPR, Plasmid Preparation

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: Gene expression profiles of the wild type and TBPL1 knocked out gene determined in healthy and breast cancer cell lines. Knock-out cell lines were obtained by transfection of the U6-gRNA Cas-92A + GFP-RM CRISPR plasmid with Lipofectamine as described in Materials and Methods. Comparisons of RNAseq data were then carried out between non-transfected and transfected cell lines. Volcano plot of the genes identified in MCF-12F (A), T47D ( B ), SK-BR-3 ( C ), and MDA-MB-231 ( D ). ( E ) Principal Component Analysis (PCA) of the data obtained for the three replicates for each cell line. ( F ) Hierarchical clustering of the genes found overexpressed and subexpressed in the healthy and breast cancer cell lines.

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Gene Expression, Knock-Out, Transfection, CRISPR, Plasmid Preparation

Journal: Scientific Reports

Article Title: RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures

doi: 10.1038/s41598-025-11634-2

Figure Lengend Snippet: Enriched pathways (KEGG) and gene expression levels affected by the knocking out of the TBPL1 gene. Pathways and genes affected by the TBPL1 gene knocking out in relation to the wild-type cells: MCF-12F ( A , B ), T47D ( C , D ), SK-BR-3 ( E , F ), MDA-MB-231 ( G , H .).

Article Snippet: The membrane was subsequently blocked with a 5% skim milk solution in PBS containing 0.05% Tween 20 in PBS (PBS-T) overnight at 4 °C under gentle shaking, followed by overnight incubation at 4 °C with a 1:1,000 dilution of each anti-human TBPL1 rabbit monoclonal antibody (Boster Bio-Tech) and anti-tubulin mouse monoclonal primary antibody (Invitrogen) diluted in PBS-T with 5% skim milk under gentle shaking.

Techniques: Gene Expression