Journal: EMBO molecular medicine

Article Title: A novel epigenetic CREB-miR-373 axis mediates ZIP4-induced pancreatic cancer growth.

doi: 10.1002/emmm.201302507

Figure Lengend Snippet: Figure 2. ZIP4 induces the expression of miR‐373 through activation of the transcription factor CREB. A. CREB phosphorylation was downregulated in AsPC-shZIP4 and upregulated in MIA-ZIP4 cells. B. Schematic structure of the 2.5 kb genomic region upstream of miR-373. There are seven potential CREB binding sites in this region. C. Promoter activity. The promoter reporter construct was co-transfected with pRL-TK into AsPC-1 cells, the promoter activity was determined by a chemiluminescence reader (significant p-value <0.0001, t-test, n ¼ 4). D. Mutational analysis of miR-373 promoter. The wild type and mutant promoter constructs were co-transfected with pRL-TK into AsPC-1 cells. The promoter activity was determined by a luciferase as described above (from left to right, significant p-value ¼ 0.0002, 0.0035; t-test, n ¼ 4). E. ChIP binding assay with anti-CREB and anti-p-CREB antibody in AsPC-1 cells confirmed the binding of CREB to the miR-373 promoter region. F. Silencing of CREB leads to decreased expression of miR-373 in pancreatic cancer cells. AsPC-1 cells were transfected with siRNA against CREB (Mission siRNA from Sigma). The miR-373 level was determined by real time PCR (p ¼ 0.0014; t-test, n ¼ 3). Data were expressed as the mean SD of triplicate values.

Article Snippet: To investigate the functional involvement of the miR‐373 target genes TP53INP1, LATS2 and CD44, shRNA retrovirus vectors (Origene, Rockville, MD) were used to select stable knockdown cells in MIA PaCa‐2 cells (Li et al, 2009).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Binding Assay, Activity Assay, Construct, Transfection, Mutagenesis, Luciferase, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: SE-associated LINC01013 was transcriptionally activated by CEBPB in hPASMCs. A Prediction of candidate transcription factors and binding sites. (Transcription factor related databases: JASPAR: https://jaspar.elixir.no/ ; PROMO: https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ; GENECARD: https://www.genecards.org/ ; AnimalTFDB: http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/ ; Super enhancer related database: LncSEA: https://bio.liclab.net/LncSEA/ ). B The schematic diagram illustrates the LINC01013 promoter, divided into segments P1-P4, and the binding sites of transcription factors. C Four constituents (E1-E4) of SE region of LINC01013 derived from the WashU Epigenome Browser databases ( http://epigenomegateway.wustl.edu/browser/ ). D , E hPASMCs were subjected to ChIP analysis using antibodies against H3K27ac, H3K4me1 and CEBPB. The association with the SE region (D, E1-E4) and promoter region (E, P1-P4) of LINC01013 was quantified by RT‒qPCR ( n = 3). F hPASMCs were treated with CEBPB siRNA and subjected to ChIP analysis using antibodies against H3K27ac. The association with the E2 of SE (left) and P1-P3 promoter regions (right) of LINC01013 was quantified by RT-qPCR ( n = 3). G Schematic diagram of transcribing LINC01013 in hPASMCs. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; IP, immunoprecipitation; IgG, Immunoglobulin G; TSS, transcription initiation site

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Binding Assay, Derivative Assay, Quantitative RT-PCR, Negative Control, Immunoprecipitation

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

Journal: Oncotarget

Article Title: Regulation of SOX10 stability via ubiquitination-mediated degradation by Fbxw7α modulates melanoma cell migration

doi:

Figure Lengend Snippet: A. Endogenous interaction between SOX10 and Fbxw7α was detected in melanoma cells. MM200 cells were lysed and subjected to IP using IgG, anti-Fbxw7α, or anti-SOX10, as indicated, and then, the cells were analyzed by Western blotting ( n = 3). B. The SOX10 protein level was inversely correlated with Fbxw7α. Various melanoma cell lines as indicated were lysed, and the protein levels of SOX10, Fbxw7α and GAPDH were evaluated by Western blotting. C. Overexpression of Fbxw7α and GSK3β downregulates the SOX10 and MIA protein levels ( n = 3). MM200 cells were lysed, and the indicated protein levels were detected by Western blotting ( n = 3). D. Silencing of Fbxw7α upregulates the SOX10 and MIA protein levels. SK-Mel-Bcl2 cells were lysed, and the indicated protein levels were detected by Western blotting ( n = 3).

Article Snippet: Human anti-MIA (melanoma inhibitory activity) was purchased from Abnova (PAB27627) antibodies.

Techniques: Western Blot, Over Expression