Journal: iScience

Article Title: Regulatory BC200 RNA promotes breast carcinogenesis by repressing BRCA1 gene expression

doi: 10.1016/j.isci.2026.115267

Figure Lengend Snippet: BC200 RNA represses expression of BRCA1 in MCF-10A cells (A) Transfection with BC200 RNA decreased protein levels of BRCA1 in MCF-10A cells (lanes 2–4). Transfection with BC200 RNA central domain mutant U + (which is translational control-incompetent) had no significant effect on BRCA1 expression (lane 5). Transfection with U6 RNA (30 nM) also did not have any significant effect (lane 6). Molecular masses are indicated. (B) Quantitative analysis confirmed that BC200 RNA effectively repressed the synthesis of BRCA1 in a dose-dependent manner, while BC200 RNA mutant U + and U6 RNA did not. Untransfected cells (UT, black bar) are presented for reference ( n = 8). Statistical analysis: one-way ANOVA (∗∗∗∗ p < 0.0001) followed by Dunnett’s multiple comparison analysis (comparison with BC200 RNA mutant U + -transfected cells): p = 0.95 for cells transfected with 7.5 nM BC200 RNA, ∗ p = 0.0111 for cells transfected with 15 nM BC200 RNA, ∗∗∗∗ p < 0.0001 for cells transfected with 30 nM BC200 RNA, and p = 0.9999 for cells transfected with U6 RNA. Error bars: SD. (C) Transfection with BC200 RNA did not significantly alter protein levels of Rb in MCF-10A cells (lane 2). Transfection with BC200 RNA mutant U + also did not impact Rb expression (lane 3) ( n = 5). Molecular masses are indicated. (D) Levels of BC200 RNA following transfection were measured by RT-qPCR. 72 h after transfection with 7.5, 15, or 30 nM wild-type BC200 RNA, 30 nM BC200 RNA mutant U + , or co-transfection with 30 nM BC200 RNA and 20 nM BC200 RNA-specific DsiRNA, BC200 RNA levels were measured and compared with those of untransfected MCF-10A cells. RNA levels in all samples were normalized to those of untransfected cells ( n = 6). Statistical analysis: one-way ANOVA (∗∗∗∗ p < 0.0001) followed by Dunnett’s multiple comparison analysis (comparison with untransfected cells): p = 0.92 for cells transfected with 7.5 nM BC200 RNA, ∗ p = 0.035 for cells transfected with 15 nM BC200 RNA, ∗∗∗∗ p < 0.0001 for cells transfected with 30 nM BC200 RNA, and ∗∗∗∗ p < 0.0001 for cells transfected with mutant U + . ∗∗∗∗ p < 0.0001 for dsiRNA compared with 30 nM BC200 RNA. Error bars: SD.

Article Snippet: BRCA1 , Cell Signaling Technology , 9010; RRID: AB_2228244.

Techniques: Expressing, Transfection, Mutagenesis, Control, Comparison, Quantitative RT-PCR, Cotransfection

Journal: iScience

Article Title: Regulatory BC200 RNA promotes breast carcinogenesis by repressing BRCA1 gene expression

doi: 10.1016/j.isci.2026.115267

Figure Lengend Snippet: BC200 RNA inhibits BRCA1-mediated DNA repair MCF-10A cells were transfected with BC200 RNA. After 48 h, they were exposed to UV light for 5 min. Comet assays were performed after 4 h. (A) Untransfected cells (UT) were able to recover from the DNA damage caused by UV light exposure, as little or no comet tail signal was detectable. (B) After having been transfected with 30 nM BC200 RNA, cells were unable to recover from DNA damage caused by UV light. DNA damage is evidenced by increased comet tail signals. (C) Co-transfection of BC200 RNA with DsiRNA specific for the 3′ region of BC200 RNA resulted in a restoration of cellular DNA repair competence as comet tail signals were now reduced. (D) Etoposide is a compound that causes DNA damage. Used at 20 μM, it produced significantly increased comet tail signals. Scale bar, 100 μm. (E) The extent of induced DNA damage is expressed as the extent tail moment (tail DNA % x tail length). The results are presented as violin plots with overlaid boxplots. A violin plot shows the distribution of the data as a density curve, where the frequency of the data points corresponds to the width of the curve. The violin plots show the distribution of the data, while the overlaid boxplots show summaries of the data as medians with error bars. We observe a clear and significant difference in the distribution of the data obtained with different treatments. Statistical analysis: one-way ANOVA (∗∗∗∗ p < 0.0001) followed by Dunnett’s multiple comparison analysis (comparison with cells transfected with BC200 RNA): ∗∗∗∗ p < 0.0001 for untransfected cells, ∗∗∗∗ p < 0.0001 for cells transfected with BC200 RNA and DsiRNA; ∗∗∗ p < 0.001 for cells treated with etoposide. Quantification was based on at least 10 images (i.e., fields) with a total of at least 100 cells analyzed for each treatment. Error bars: SD.

Article Snippet: BRCA1 , Cell Signaling Technology , 9010; RRID: AB_2228244.

Techniques: Transfection, Cotransfection, Produced, Comparison

Journal: iScience

Article Title: Regulatory BC200 RNA promotes breast carcinogenesis by repressing BRCA1 gene expression

doi: 10.1016/j.isci.2026.115267

Figure Lengend Snippet: BRCA1 cDNA rescues the BC200 RNA-induced phenotype (A) MCF-10A cells were transfected and seeded at low density in an agarose gel. After transfection, the ability of the cells to grow in soft agarose medium was assessed by measuring absorbance at 450 nm using a microtiter plate reader. Consistent with previous observations, MCF-10A cells transfected with 30 nM BC200 RNA exhibited a pronounced growth phenotype. Co-transfection with 30 nM of a DsiRNA specific for BC200 RNA significantly reduced this oncogenic phenotype. Finally, rescue experiments were performed in which BRCA1 expression was restored in BC200 RNA-expressing cells. BC200 RNA exerts its translational repression activity by inhibiting the activities of eIF4A and eIF4B, two translation initiation factors essential for the translation of mRNAs containing higher-order structural elements in their 5′ UTRs. To overcome BC200 RNA-mediated inhibition, cells in these rescue experiments were co-transfected with a plasmid expressing the coding sequence of BRCA1 mRNA. This cDNA lacks the structured 5′ UTR and is therefore not targeted by BC200 RNA-mediated translational repression. The results showed a significant decrease in malignant transformation. Quantitative analysis: one-way ANOVA (∗∗∗ p < 0.001) followed by Dunnett’s multiple comparison analysis (comparison with cells transfected with 30 nM BC200 RNA): ∗∗∗ p < 0.001 for untransfected cells, ∗∗ p = 0.002 for cells transfected with 30 nM BC200 RNA and 30 nM dsiRNA, ∗ p = 0.016 for cells transfected with 30 nM BC200 RNA and eGFP-BRCA1 ( n = 5). Error bars: SD. (B) Levels of BC200 RNA following transfection were measured by RT-qPCR 48 h after transfection with 30 nM wild-type BC200 RNA, co-transfection with 30 nM BC200 RNA and 20 nM BC200 RNA-specific DsiRNA, or co-transfection with 30 nM BC200 RNA and 1.5 μg eGFP-BRCA1 plasmid per culture. BC200 RNA levels were measured and compared with those of untransfected MCF-10A cells. BC200 RNA levels in all samples were normalized to those of untransfected cells ( n = 4). Statistical analysis: one-way ANOVA (∗∗∗∗ p < 0.0001) followed by Dunnett’s multiple comparison analysis (comparison with untransfected cells): ∗∗∗ p < 0.001 for cells transfected with 30 nM BC200 RNA, ∗∗∗ p < 0.001 for cells transfected with 30 nM BC200 RNA and eGFP-BRCA1. ∗∗∗ p < 0.0004 for cells transfected with 30 nM BC200 RNA and DsiRNA vs. cells transfected with 30 nM BC200 RNA. Error bars: SD. (C) Representative photomicrographs show differential expression of eGFP fluorescence in the samples. MCF-10A cells were transfected with 30 nM BC200 RNA, 30 nM BC200 RNA and 20 nM BC200 RNA-specific DsiRNA, or co-transfected with 30 nM BC200 RNA and 1.5 μg eGFP-BRCA1 plasmid per culture. 48 h after transfection, the differential expression of eGFP was examined. Cells were then plated in soft agarose for the transformation assay. The top row of images presents brightfield photomicrographs, while the bottom row of images presents fluorescence photomicrographs documenting differential eGFP expression levels. Only the sample transfected with the eGFP-BRCA1 plasmid shows a fluorescence signal. Scale bar, 100 μm.

Article Snippet: BRCA1 , Cell Signaling Technology , 9010; RRID: AB_2228244.

Techniques: Transfection, Agarose Gel Electrophoresis, Cotransfection, Expressing, Activity Assay, Inhibition, Plasmid Preparation, Sequencing, Transformation Assay, Comparison, Quantitative RT-PCR, Quantitative Proteomics, Fluorescence

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A . MCF7 cells were treated for 1 h with DMSO or 50 μM PUGNAc ± 10 μM veliparib, irradiated with 6 Gy, fixed after 2 h and stained for 53BP1 and BRCA1. Representative images with 53BP1 foci (green), BRCA1 foci (red), and DAPI (blue) are shown. B , C . Plots of 53BP1 ( B ) and BRCA1 ( C ) foci per nucleus at 2 h after 6 Gy in DAPI Low and DAPI High cells. D . Cells were treated as in A but fixed after 24 h. E, F . Plots of 53BP1 ( E ) and BRCA1 ( F ) foci per nucleus at 24 h after 6 Gy in DAPI Low and DAPI High cells. For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: The primary antibodies used were 53BP1, BRCA1 (Santa Cruz Biotechnology; sc-6954; 1:1,000), and RAD51 (Novus Biologicals; NB100–148; 1:1,000).

Techniques: Irradiation, Staining

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A . MCF7 cells were incubated with 10 μM BrdU for 24 h, then treated for 1 h before 6 Gy with DMSO ± 10 μM veliparib or 50 μM PUGNAc + 10 μM veliparib, fixed after 24 h and single strand DNA detected with anti-BrdU antibody under non-denaturing conditions. Representative images show BrdU foci in red with DAPI in blue. B-D . Cells were treated for 1 h before 6 Gy with DMSO ± 10 μM veliparib or 50 μM PUGNAc + 10 μM veliparib, fixed after 24 h and probed for RPA ( B ), RAD51 ( C ) or BRCA1 ( D ). E-H . Plots of BrdU ( E ), RPA ( F ), RAD51 ( G ), and BRCA1 ( H ) foci per nucleus in at 24 h after 6 Gy. For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: The primary antibodies used were 53BP1, BRCA1 (Santa Cruz Biotechnology; sc-6954; 1:1,000), and RAD51 (Novus Biologicals; NB100–148; 1:1,000).

Techniques: Incubation

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A, B . shScr, shOGT, and shOGA were induced for 48 h with 1 μg/ml doxycycline and cells were treated with 0 or 10 μM veliparib for 1 h prior to 6 Gy, fixed after 24 h and stained for BRCA1. Shown are representative images ( A ) and plots of BRCA1 foci per nucleus ( B ). C, D . Cells were treated as in A and stained for RPA. Shown are representative images ( C ) and plots of RPA foci per nucleus ( D ). E . shScr, shOGT, and shOGA cells were treated as in A and then lightly permeabilized, fixed and then stained with anti-DNA to detect cytosolic DNA (cytoDNA). Shown are representative pseudo-colored images, with cytoDNA in red, DAPI counterstain in blue, and overlays. For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: The primary antibodies used were 53BP1, BRCA1 (Santa Cruz Biotechnology; sc-6954; 1:1,000), and RAD51 (Novus Biologicals; NB100–148; 1:1,000).

Techniques: Staining