Journal: bioRxiv

Article Title: Orai1 is required for Ca 2+ -dependent plasma membrane repair and mechanoadaptation

doi: 10.64898/2026.05.13.724989

Figure Lengend Snippet: A. Western blot confirmation of loss of Orai1 expression upon CRISPR/Cas9-mediated KO in MDA-MB-231 and Hs578T cells; shown are pools of three clones maintained individually. B-C. Impairment of SOCE upon Orai1 KO in TNBC cells. SOCE was measured using thapsigargin-induced ER Ca 2+ depletion. Typical profiles are shown on left and quantification of fold-change in peak fluorescence intensity from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. D-E. Orai1-KO impairs the repair of mechanically induced plasma membrane injury. WT vs. Orai1-KO cells were subjected to mechanical injury to plasma membrane and repair assay was performed as in . Representative confocal images are shown in D and quantified data are shown in E. Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. F-G. Orai1-KO impairs the repair of SLO-induced membrane injury. Plasma membrane damage using SLO and the repair assay on MDA-MB-231 ( F ) and Hs578T ( G ) cell lines were as in . Representative confocal images are shown on left. Quantification of PI-stained cells from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. H-J. Orai1 inhibitors impair plasma membrane repair. Panel H shows Thapsigargin (TG)-induced SOCE measurements (initial peak, Ca 2+ store release in the absence of extracellular Ca 2+ ; second peak, SOCE in the presence of extracellular Ca 2+ ) performed on the indicated cell lines cultured with or without CM4620 (10 μM, 4 hours pretreatment). Note significant SOCE inhibition by CM4620 in WT cells but not in Orai1-KO cells, supporting Orai1-selelctive effect of CM4620. Data represents mean +/- SEM of three experiments, Welch’s t test, ***p<0.001. The I panel shows the impairment of mechanically induced plasma membrane repair by CM4620 in MDA-MB-231. Cells were cultured without or with pretreatment with CM4620 (10 μM) and analyzed for repair as in . Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. The J panel shows the impairment of SLO-induced membrane damage in MDA-MB-231. Left panel shows representative FACS analysis of membrane repair in cells without or with CM4620 treatment. Right panel shows quantification of cells that failed to repair (PI staining) from 3 independent experiments. Data represents mean +/-SEM of three experiments. Welch’s t test, ***, p<0.001.

Article Snippet: Hs578T cell line (ATCC) was cultured in α-MEM medium supplemented as above plus 1 μg/mL hydrocortisone and 12.5 ng/mL epidermal growth factor (Millipore Sigma, St. Louis, MO).

Techniques: Western Blot, Expressing, CRISPR, Clone Assay, Fluorescence, Clinical Proteomics, Membrane, Staining, Cell Culture, Inhibition

Journal: bioRxiv

Article Title: Orai1 is required for Ca 2+ -dependent plasma membrane repair and mechanoadaptation

doi: 10.64898/2026.05.13.724989

Figure Lengend Snippet: A. TEAD inhibition reduces CAV1 expression. MDA-MB-231 or Hs578T cells cultured without or with the pan-TEAD inhibitor GNE-7883 (10 μM) for 48 hours were analyzed by WB for CAV1 expression; Hsc70, loading control. Fold-change in signals quantified by densitometry and analyzed using Image J are shown on right. Welch’s t test, *, p<0.05; **, p<0.01. B-C. TEAD inhibition reduces the Thapsigargin (TG)-induced SOCE. Cells were pretreated with GNE-7883 (10 μM) for 24 hours. Left, representative plots; right, quantified data from three experiments. Welch’s t test; **p<0.001. D. TEAD inhibition impairs the mechanical force-induced Ca 2+ import. AFM cantilevers were used to apply force and measure Ca 2+ import from the extracellular space using fluorescence intensity of transfected Ca 2+ reporter (R-RECO1.2) as the readout. Cells were pretreated with GNE-7883 (10 μM) for 24 hours where indicated. Data points represent cells analyzed through three independent experiments (n=33). Welch’s t test, ***, p<0.001. E-F. TEAD inhibition impairs the repair of induced plasma membrane injuries. Shown is the impact of GNE-7883 on the repair of mechanically induced ( E ) and SLO-induced ( F ) plasma membrane damage, assessed as in and , respectively. GNE-7883 pre-treatment (10 μM), where indicated, was for 72 hours. Representative confocal images ( E ; top panel) or FACS plots ( F ; right panel) are shown. Quantified data shown are from 3 independent experiments and presented as mean +/- SEM of 3 experiments. Two-way ANOVA or Welch’s t test applied to data in E and F , respectively, *p<0.05. Scale bar, 20 µm.

Article Snippet: Hs578T cell line (ATCC) was cultured in α-MEM medium supplemented as above plus 1 μg/mL hydrocortisone and 12.5 ng/mL epidermal growth factor (Millipore Sigma, St. Louis, MO).

Techniques: Inhibition, Expressing, Cell Culture, Control, Fluorescence, Transfection, Clinical Proteomics, Membrane

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Hypoxia promotes enrichment and DNA damage-independent oxidative activation of ataxia telangiectasia mutated. A: Flow cytometry analysis of CD44+/CD24- cell populations in Hs578T and MDA-MB-231 cells after exposure to hypoxia or normoxia; B: Quantitative reverse transcriptase polymerase chain reaction analysis of cancer stem cells-associated genes ( c-Myc , octamer-binding protein 4, Kruppel-like factor 4, sex-determining region Y-box 2, NANOG ) in mammosphere cultures under hypoxia; C-E: Western blot analysis of phosphorylated ataxia telangiectasia mutated, γH2AX, and 53BP1 in Hs578T and MDA-MB-231 cancer stem cells under normoxia, hypoxia, or H 2 O 2 treatment. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01; ns: not significant. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; OCT4: Octamer-binding protein 4; p-ATM: Phosphorylated ataxia telangiectasia mutated.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Activation Assay, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Knockdown, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 promote cancer stem cells enrichment and stemness maintenance in triple-negative breast cancer. A: Effects of serine hydroxymethyltransferase 2 knockdown on mammosphere formation, size, and number in Hs578T and MDA-MB-231 cancer stem cells; B: Effects of methylenetetrahydrofolate dehydrogenase 2 knockdown on mammosphere formation, size, and number; C and D: Western blot analysis of stemness-associated proteins Kruppel-like factor 4 and sex-determining region Y-box 2 after serine hydroxymethyltransferase 2 or methylenetetrahydrofolate dehydrogenase 2 knockdown. Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Article Snippet: The human breast cancer cell lines Hs578T and MDA-MB-231 were acquired from the American Type Culture Collection.

Techniques: Knockdown, Western Blot

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