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ATCC female human cell lines mcf10a
Female Human Cell Lines Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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female human cell lines mcf10a - by Bioz Stars, 2026-06

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a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating used for analysis throughout this publication. b.) Scatterplot of DNA content versus median cyclin B1 intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. c.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. d.) Quantification of mean cyclin B1 intensity in MCF10A cells with non-targeting or cyclin B1 knockdowns to demonstrate cyclin B1 antibody specificity. A Mann-Whitney test was conducted to assess significance. Error bars are representative of 10-90 percentile range. P value: <0.0001. e.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Quantification of integrated SLBP intensity in MCF10A cells with non-targeting or SLBP knockdowns to demonstrate SLBP antibody specificity. Error bars are representative of 10-90 percentile range. A Mann-Whitney test was conducted to assess significance. P value: <0.0001. g.) Breakdown of quantification of median SLBP nuclear intensity across the cell cycle from into individual graphs. Data are presented as mean ± SEM. h.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 5μM RO-3306, or a combination of 5μM AZD6738 and 5μM RO-3306 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. i.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 1μM NU6140, or a combination of 5μM AZD6738 and 1μM NU6140 for 1hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating used for analysis throughout this publication. b.) Scatterplot of DNA content versus median cyclin B1 intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. c.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. d.) Quantification of mean cyclin B1 intensity in MCF10A cells with non-targeting or cyclin B1 knockdowns to demonstrate cyclin B1 antibody specificity. A Mann-Whitney test was conducted to assess significance. Error bars are representative of 10-90 percentile range. P value: <0.0001. e.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Quantification of integrated SLBP intensity in MCF10A cells with non-targeting or SLBP knockdowns to demonstrate SLBP antibody specificity. Error bars are representative of 10-90 percentile range. A Mann-Whitney test was conducted to assess significance. P value: <0.0001. g.) Breakdown of quantification of median SLBP nuclear intensity across the cell cycle from into individual graphs. Data are presented as mean ± SEM. h.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 5μM RO-3306, or a combination of 5μM AZD6738 and 5μM RO-3306 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. i.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 1μM NU6140, or a combination of 5μM AZD6738 and 1μM NU6140 for 1hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Article Snippet: Human MCF10A cells (ATCC, CRL-10317) were grown in DMEM/F12 medium (ThermoFisher Scientific, 11330057) supplemented with 100U/mL penicillin/streptomycin (Thermo Fisher Scientific, 15140122), 5% horse serum (ThermoFisher Scientific, 16050122), 20ng/ml epidermal growth factor (EGF) (Sigma-Aldrich, E5036), 0.5 mg/mL hydrocortisone, 10μg/mL insulin (Sigma-Aldrich, I1882-100MG), and 100ng/mL cholera toxin (Sigma-Aldrich, C8052).

Techniques: MANN-WHITNEY

a.) Representative images of cyclin B1 and EdU in MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Arrows indicate nuclei positive for both EdU and cyclin B1. Scale bar is equal to 40μm. b.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Data are presented as mean ± SEM. c.) Scatterplots of DNA content (integrated DAPI intensity) versus mean cyclin B1 cytoplasmic intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by EdU intensity (gating of negative/positive EdU values in ) as denoted by the associated scale. Representative of n=4 biological replicates. d.) Scatterplots of DNA content (integrated DAPI intensity) versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by mean cyclin B1 intensity as denoted by the associated scale. Representative of n=4 biological replicates. e.) Quantification of mean cyclin B1 intensity values from in EdU positive cells. Error bars are representative of 10-90 percentile range. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: <0.0001 (DMSO versus AZD6738) and <0.0001 (DMSO versus LY2603618). f.) Quantification of mean EdU intensity values from in cells with high cyclin B1 levels (gating of low/high cyclin B1 values in ). Error bars are representative of 10-90 percentile range. P values: <0.0001 (DMSO versus AZD6738) and <0.0001 (DMSO versus LY2603618). g.) Representative images of SLBP and EdU in S phase MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=3 biological replicates. Scale bar is equal to 10μm. h.) Quantification of median SLBP nuclear intensity across the cell cycle in MCF10A cells treated with DMSO, 5μM AZD6738, 5μM VE822, 2μM LY2603618, or 250nM ChIR124 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Representative images of cyclin B1 and EdU in MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Arrows indicate nuclei positive for both EdU and cyclin B1. Scale bar is equal to 40μm. b.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Data are presented as mean ± SEM. c.) Scatterplots of DNA content (integrated DAPI intensity) versus mean cyclin B1 cytoplasmic intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by EdU intensity (gating of negative/positive EdU values in ) as denoted by the associated scale. Representative of n=4 biological replicates. d.) Scatterplots of DNA content (integrated DAPI intensity) versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by mean cyclin B1 intensity as denoted by the associated scale. Representative of n=4 biological replicates. e.) Quantification of mean cyclin B1 intensity values from in EdU positive cells. Error bars are representative of 10-90 percentile range. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: <0.0001 (DMSO versus AZD6738) and <0.0001 (DMSO versus LY2603618). f.) Quantification of mean EdU intensity values from in cells with high cyclin B1 levels (gating of low/high cyclin B1 values in ). Error bars are representative of 10-90 percentile range. P values: <0.0001 (DMSO versus AZD6738) and <0.0001 (DMSO versus LY2603618). g.) Representative images of SLBP and EdU in S phase MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=3 biological replicates. Scale bar is equal to 10μm. h.) Quantification of median SLBP nuclear intensity across the cell cycle in MCF10A cells treated with DMSO, 5μM AZD6738, 5μM VE822, 2μM LY2603618, or 250nM ChIR124 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM.

Techniques:

a.) Representative images of cyclin B1, EdU, and yH2AX in MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Arrows indicate nuclei positive for EdU, cyclin B1 and yH2AX. Scale bar is equal to 50μm. b.) Scatterplots of DNA content (integrated DAPI intensity) versus mean yH2AX intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by high or low cyclin B1 intensity as denoted by the associated scale. Representative of n=4 biological replicates. c.) Quantification of median cyclin B1 cytoplasmic intensity in MCF10A cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of mitotic failure (defined in ) in control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Percentages were calculated based on counts of 4 images per treatment for each biological replicate. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.9964 (siControl versus siCCNB1 + DMSO), <0.0001 (siControl versus siCCNB1 + AZD6738), and 0.0003 (siControl versus siCCNB1 + LY2603618). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Representative images of cyclin B1, EdU, and yH2AX in MCF10A cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Arrows indicate nuclei positive for EdU, cyclin B1 and yH2AX. Scale bar is equal to 50μm. b.) Scatterplots of DNA content (integrated DAPI intensity) versus mean yH2AX intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Each dot is representative of values from a singular cell. The plot is colored by high or low cyclin B1 intensity as denoted by the associated scale. Representative of n=4 biological replicates. c.) Quantification of median cyclin B1 cytoplasmic intensity in MCF10A cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of mitotic failure (defined in ) in control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Percentages were calculated based on counts of 4 images per treatment for each biological replicate. A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.9964 (siControl versus siCCNB1 + DMSO), <0.0001 (siControl versus siCCNB1 + AZD6738), and 0.0003 (siControl versus siCCNB1 + LY2603618). Data are presented as mean ± SEM.

Techniques: Control, Knockdown

a.) Quantification of median cyclin B1 cytoplasmic intensity in RPE-1 cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. b.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. c.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Representative images of cells undergoing normal mitosis versus mitotic failure. Scale bar is equal to 15μm.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Quantification of median cyclin B1 cytoplasmic intensity in RPE-1 cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. b.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. c.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Representative images of cells undergoing normal mitosis versus mitotic failure. Scale bar is equal to 15μm.

Techniques: Control, Knockdown

a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating of EdU negative cells with >3n DNA content (referred to as G2) used for analysis of chromatin-bound replication component retention. b.) Scatterplots of mean chromatin-bound PCNA intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The dotted line and color of the points indicate the gating for PCNA positive and negative cells. c.) Scatterplots of mean EdU intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The points are colored by status of being PCNA negative/positive as denoted by the associated key. Representative of n=3 biological replicates. d.) Scatterplots of mean chromatin-bound MCM2 intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The dotted line and color of the points indicate the gating for MCM2 positive and negative cells. E.) Scatterplots of mean EdU intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The points are colored by status of being MCM2 negative/positive as denoted by the associated key. Representative of n=3 biological replicates.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating of EdU negative cells with >3n DNA content (referred to as G2) used for analysis of chromatin-bound replication component retention. b.) Scatterplots of mean chromatin-bound PCNA intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The dotted line and color of the points indicate the gating for PCNA positive and negative cells. c.) Scatterplots of mean EdU intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The points are colored by status of being PCNA negative/positive as denoted by the associated key. Representative of n=3 biological replicates. d.) Scatterplots of mean chromatin-bound MCM2 intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The dotted line and color of the points indicate the gating for MCM2 positive and negative cells. E.) Scatterplots of mean EdU intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The points are colored by status of being MCM2 negative/positive as denoted by the associated key. Representative of n=3 biological replicates.

Techniques:

a.) Scatterplots of mean chromatin-bound PCNA intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. b.) Quantification of mean PCNA intensities from in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population (Supplemental Fig. 3a). Error bars are representative of 10-90 percentile range. c.) Scatterplots of mean MCM2 intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. d.) Quantification of mean MCM2 intensities from in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population. Error bars are representative of 10-90 percentile range. e.) Quantification of integrated DAPI intensities to measure DNA content in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population (n=3 biological replicates). Error bars are representative of 10-90 percentile range. f.) Quantification of the percentage of PCNA (left) or MCM2 (right) positive pre-extracted MCF10A cells (gating of PCNA and MCM2 shown in and ) in the defined G2 population of control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed for each plot to assess biological significance. P values for PCNA plot: >0.9999 (DMSO siControl versus siCCNB1), <0.0001 (AZD6738 siControl versus siCCNB1), and <0.0001 (LY2603618 siControl versus siCCNB1). P values for MCM2 plot: 0.9694 (DMSO siControl versus siCCNB1), 0.0021 (AZD6738 siControl versus siCCNB1), and 0.0081 (LY2603618 siControl versus siCCNB1). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Scatterplots of mean chromatin-bound PCNA intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. b.) Quantification of mean PCNA intensities from in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population (Supplemental Fig. 3a). Error bars are representative of 10-90 percentile range. c.) Scatterplots of mean MCM2 intensity versus DNA content in pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. d.) Quantification of mean MCM2 intensities from in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population. Error bars are representative of 10-90 percentile range. e.) Quantification of integrated DAPI intensities to measure DNA content in pre-extracted MCF10A cells with low or high yH2AX in the defined G2 population (n=3 biological replicates). Error bars are representative of 10-90 percentile range. f.) Quantification of the percentage of PCNA (left) or MCM2 (right) positive pre-extracted MCF10A cells (gating of PCNA and MCM2 shown in and ) in the defined G2 population of control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed for each plot to assess biological significance. P values for PCNA plot: >0.9999 (DMSO siControl versus siCCNB1), <0.0001 (AZD6738 siControl versus siCCNB1), and <0.0001 (LY2603618 siControl versus siCCNB1). P values for MCM2 plot: 0.9694 (DMSO siControl versus siCCNB1), 0.0021 (AZD6738 siControl versus siCCNB1), and 0.0081 (LY2603618 siControl versus siCCNB1). Data are presented as mean ± SEM.

Techniques: Control, Knockdown

a.) Representative images of PCNA, yH2AX, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Cell cycle status is indicated in the figure. Representative of n=3 biological replicates. Scale bar is equal to 10μm. b.) Representative images of MCM2, yH2AX, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Cell cycle status is indicated in the figure. Representative of n=3 biological replicates. Scale bar is equal to 10μm. C.) Representative images of PCNA, MCM2, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The DMSO treated nuclei shows a cell in late S phase, while the nuclei from the other treatments show EdU negative cells that have halted replication. Whit boxes indicate areas of the nuclei that have been magnified. Representative of n=3 biological replicates. Scale bar is equal to 10μm.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Representative images of PCNA, yH2AX, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Cell cycle status is indicated in the figure. Representative of n=3 biological replicates. Scale bar is equal to 10μm. b.) Representative images of MCM2, yH2AX, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Cell cycle status is indicated in the figure. Representative of n=3 biological replicates. Scale bar is equal to 10μm. C.) Representative images of PCNA, MCM2, and EdU in pre-extracted MCF10A nuclei treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. The DMSO treated nuclei shows a cell in late S phase, while the nuclei from the other treatments show EdU negative cells that have halted replication. Whit boxes indicate areas of the nuclei that have been magnified. Representative of n=3 biological replicates. Scale bar is equal to 10μm.

Techniques:

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Techniques:

a.) Quantification the percentage of PCNA positive pre-extracted cells in the defined G2 population of control, USP37, or TRAIP knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.5397 (DMSO siControl versus siUSP37), 0.6511 (DMSO siControl versus siTRAIP), <0.0001 (AZD6738 siControl versus siUSP37), 0.0068 (AZD6738 siControl versus siTRAIP), 0.0003 (LY2603618 siControl versus siUSP37), and 0.0101 (LY2603618 siControl versus siUSP37). Data are presented as mean ± SEM. b.) Scatterplots of mean PCNA intensity versus DNA content in control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. c.) Quantification of the percentage of MCM2 positive pre-extracted cells in the defined G2 population of control, USP37, or TRAIP knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.0837 (DMSO siControl versus siUSP37), 0.8341 (DMSO siControl versus siTRAIP), <0.0001 (AZD6738 siControl versus siUSP37), 0.0433 (AZD6738 siControl versus siTRAIP), 0.0410 (LY2603618 siControl versus siUSP37), and 0.5357 (LY2603618 siControl versus siUSP37). Data are presented as mean ± SEM. d.) Scatterplots of mean MCM2 intensity versus DNA content in control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. e.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Quantification the percentage of PCNA positive pre-extracted cells in the defined G2 population of control, USP37, or TRAIP knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.5397 (DMSO siControl versus siUSP37), 0.6511 (DMSO siControl versus siTRAIP), <0.0001 (AZD6738 siControl versus siUSP37), 0.0068 (AZD6738 siControl versus siTRAIP), 0.0003 (LY2603618 siControl versus siUSP37), and 0.0101 (LY2603618 siControl versus siUSP37). Data are presented as mean ± SEM. b.) Scatterplots of mean PCNA intensity versus DNA content in control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. c.) Quantification of the percentage of MCM2 positive pre-extracted cells in the defined G2 population of control, USP37, or TRAIP knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.0837 (DMSO siControl versus siUSP37), 0.8341 (DMSO siControl versus siTRAIP), <0.0001 (AZD6738 siControl versus siUSP37), 0.0433 (AZD6738 siControl versus siTRAIP), 0.0410 (LY2603618 siControl versus siUSP37), and 0.5357 (LY2603618 siControl versus siUSP37). Data are presented as mean ± SEM. d.) Scatterplots of mean MCM2 intensity versus DNA content in control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Plots are divided by EdU negative/positive cells and by treatment. The plot is colored by mean yH2AX intensity as denoted by the associated scale. Representative of n=3 biological replicates. e.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control, USP37, or TRAIP knockdown and pre-extracted MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Techniques: Control, Knockdown

a.) Schematic of ATRi resistant cell line development (ATRi-R). b.) Cell proliferation plot of ATRi-R cells compared to controls under 5μM AZD6738 treatment. c.) Representative images of yH2AX in MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Representative of n=3 biological replicates. Scale bar is equal to 50μm. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 1mM hydroxyurea or a combination of 5μM AZD6738 and 1mM hydroxyurea 1.5hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Representative images of DAPI stained nuclei in MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Representative of n=3 biological replicates. Arrows indicate cells with mitotic slippage. Scale bar is equal to 20μm. g.) Quantification of mitotic failure (defined in ) in control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.9983 (DMSO MCF10A versus ATRi-R), 0.0027 (AZD6738 MCF10A versus ATRi-R), 0.0007 (LY2603618 MCF10A versus ATRi-R). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Schematic of ATRi resistant cell line development (ATRi-R). b.) Cell proliferation plot of ATRi-R cells compared to controls under 5μM AZD6738 treatment. c.) Representative images of yH2AX in MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Representative of n=3 biological replicates. Scale bar is equal to 50μm. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 1mM hydroxyurea or a combination of 5μM AZD6738 and 1mM hydroxyurea 1.5hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Representative images of DAPI stained nuclei in MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Representative of n=3 biological replicates. Arrows indicate cells with mitotic slippage. Scale bar is equal to 20μm. g.) Quantification of mitotic failure (defined in ) in control and cyclin B1 knockdown MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). A two-way analysis of variance (ANOVA) was performed to assess biological significance. P values: 0.9983 (DMSO MCF10A versus ATRi-R), 0.0027 (AZD6738 MCF10A versus ATRi-R), 0.0007 (LY2603618 MCF10A versus ATRi-R). Data are presented as mean ± SEM.

Techniques: Staining, Control, Knockdown

a.) Quantification of mean yH2AX nuclear intensity across the cell cycle from ATRi-R cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data from the parental MCF10A cell line is also presented in Supplementary Fig. 1c. Arrows indicate plots of ATRi-R values with scaled Y axis. Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from ATRi-R cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data from the parental MCF10A cell line is also presented in Supplementary Fig. 1d. Arrows indicate plots of ATRi-R values with scaled Y axis. Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Quantification of mean yH2AX nuclear intensity across the cell cycle from ATRi-R cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data from the parental MCF10A cell line is also presented in Supplementary Fig. 1c. Arrows indicate plots of ATRi-R values with scaled Y axis. Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from ATRi-R cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data from the parental MCF10A cell line is also presented in Supplementary Fig. 1d. Arrows indicate plots of ATRi-R values with scaled Y axis. Data are presented as mean ± SEM.

Techniques:

a.) Representative images of cyclin B1, EdU, and yH2AX in MCF10A and ATRi-R cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Scale bar is equal to 50μm. b.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Note: MCF10A data was also shown in Fig.1b. Data are presented as mean ± SEM. c.) Representative images of pFOXM1 (T600) and EdU in MCF10A and ATRi-R cells treated with DMSO or 5μM AZD6738 for 1hr. Representative of n=3 biological replicates. Scale bar is equal to 10μm. d.) Quantification of median pFOXM1 (T600) nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Representative images of cyclin B1, EdU, and yH2AX in MCF10A and ATRi-R cells treated with DMSO or 5μM AZD6738 for 16hrs. Representative of n=4 biological replicates. Scale bar is equal to 50μm. b.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=4 biological replicates). Note: MCF10A data was also shown in Fig.1b. Data are presented as mean ± SEM. c.) Representative images of pFOXM1 (T600) and EdU in MCF10A and ATRi-R cells treated with DMSO or 5μM AZD6738 for 1hr. Representative of n=3 biological replicates. Scale bar is equal to 10μm. d.) Quantification of median pFOXM1 (T600) nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. e.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A and ATRi-R cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM.

Techniques:

Mucin 1 (MUC1) expression in human breast cancer. A , MUC1 expression levels in breast cancer (UALCAN website). B , MUC1 expression levels in different subtypes of breast cancer (UALCAN website). C , MUC1 protein expression in breast cancer (Human Protein Atlas database). D , MUC1 expression in human normal mammary epithelial cells MCF10A, TNBC cells (MDA-MB-231), and TNBC tissues (scale bar=100 μm). Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test. BRCA: breast cancer gene; TCGA: The Cancer Genome Atlas; TNBC: triple-negative breast cancer.

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Mucin 1 (MUC1) expression in human breast cancer. A , MUC1 expression levels in breast cancer (UALCAN website). B , MUC1 expression levels in different subtypes of breast cancer (UALCAN website). C , MUC1 protein expression in breast cancer (Human Protein Atlas database). D , MUC1 expression in human normal mammary epithelial cells MCF10A, TNBC cells (MDA-MB-231), and TNBC tissues (scale bar=100 μm). Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test. BRCA: breast cancer gene; TCGA: The Cancer Genome Atlas; TNBC: triple-negative breast cancer.

Article Snippet: The human triple-negative breast cancer cell line MDA-MB-231 and the normal human breast epithelial cells MCF10A were obtained from the Procell Biotechnology Company (China).

Techniques: Expressing, Two Tailed Test

PCMT1 is involved in PTX resistance of BC cells, a process potentially involving COX-2-mediated AA metabolism. (A) RT-qPCR analysis of PCMT1 mRNA levels in tumor tissues and paired adjacent normal tissues from 30 BC patients treated with PTX. (B) Immunoblotting analysis of PCMT1 and COX-2 protein levels in tumor tissues and paired adjacent normal tissues from PTX-treated BC patients (n = 10; 5 PTX-sensitive and 5 PTX-resistant). (C) Correlation between PCMT1 and COX-2 expression was assessed using the Pearson correlation coefficient. (D) RT-qPCR analysis of PCMT1 mRNA levels in MCF10A, BC cells (MDA-MB-231, MCF-7), and PTX-resistant cells (MDA-MB-231/PTX, MCF-7/PTX) (n = 3). (E) Immunoblotting analysis of PCMT1 and COX-2 protein levels in BC cells and PTX-resistant cells (n = 3). (F) IC50 values of BC cells and PTX-resistant cells assessed using the CCK-8 assay (n = 3). Values are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: LITAF suppresses breast cancer and paclitaxel resistance by ubiquitinating and degrading PCMT1 to inhibit COX-2-dependent arachidonic acid metabolism

doi: 10.3389/fphar.2026.1706420

Figure Lengend Snippet: PCMT1 is involved in PTX resistance of BC cells, a process potentially involving COX-2-mediated AA metabolism. (A) RT-qPCR analysis of PCMT1 mRNA levels in tumor tissues and paired adjacent normal tissues from 30 BC patients treated with PTX. (B) Immunoblotting analysis of PCMT1 and COX-2 protein levels in tumor tissues and paired adjacent normal tissues from PTX-treated BC patients (n = 10; 5 PTX-sensitive and 5 PTX-resistant). (C) Correlation between PCMT1 and COX-2 expression was assessed using the Pearson correlation coefficient. (D) RT-qPCR analysis of PCMT1 mRNA levels in MCF10A, BC cells (MDA-MB-231, MCF-7), and PTX-resistant cells (MDA-MB-231/PTX, MCF-7/PTX) (n = 3). (E) Immunoblotting analysis of PCMT1 and COX-2 protein levels in BC cells and PTX-resistant cells (n = 3). (F) IC50 values of BC cells and PTX-resistant cells assessed using the CCK-8 assay (n = 3). Values are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human normal mammary epithelial cells MCF10A (ATCC, USA) were cultured in DMEM/F12 (Procell) supplemented with HS (5%), EGF (20 ng/mL), hydrocortisone (0.5 μg/mL), insulin (0.5 μg/mL), NEAA (1%), and P/S (1%).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay

siSPRR3 depletion results in a reduction in the number of nucleoli per nucleus in MCF10A cells. A , siSPRR3 depletion with siGENOME siRNAs reduces the number of nucleoli per nucleus. Images and histograms from a genome-wide screen using Dharmacon/Horizon siGENOME siRNAs . The images show nuclei (Hoechst, blue ) and nucleoli (anti-fibrillarin, red ) after 72 h of treatment with the negative control (siGFP), positive control (siUTP4), or siSPRR3 siRNAs. Histograms show the distribution of cells that have the indicated number of nucleoli per nucleus. Light grey shows the distribution for the negative control, black shows the test condition, and dark grey shows overlap of the two frequencies. B , siSPRR3 depletion with siON-TARGET (siONT) SMARTPool siRNA reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (non-targeting siRNA, siNT), positive control (siUTP4), or siSPRR3 siRNAs. The shading in the histograms is as in A ). The data were collected across four replicates which are pooled in the histogram. C , siSPRR3 depletion with individual siONT SMARTPool siRNAs (deconvolution) reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (siNT), positive control (siNOL11), or representative siSPRR3 individual siRNAs (SPRR3-si1 and SPRR3-si2). The shading in the histograms is as in ( A ). The histograms include all three replicates pooled. The table summarizes each of four individual siONT SMARTPool siRNAs, showing that a reduction in nucleolar number correlates with a loss in cell viability. D , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 mRNA levels in MCF10A cells. RT-qPCR data of SPRR3 mRNA demonstrating knockdown after 72 h. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. The mean ± SEM are shown alongside individual data points, colored by replicate. E , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 protein levels in MCF10A cells. Western blot of SPRR3 protein demonstrating decreased levels after 72 h. Protein levels were normalized to total protein (trichloroethanol total protein stain), then to siNT. The mean ± SEM are shown alongside individual data points, colored by replicate. This sample was run on the same Western blot as in . After imaging total protein on the membrane, the blot was cut between 10 to 15 kD markers to stain separately for SPRR3 ( E , above) or RPS28 . Data in ( D and E ) were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗∗, p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: siSPRR3 depletion results in a reduction in the number of nucleoli per nucleus in MCF10A cells. A , siSPRR3 depletion with siGENOME siRNAs reduces the number of nucleoli per nucleus. Images and histograms from a genome-wide screen using Dharmacon/Horizon siGENOME siRNAs . The images show nuclei (Hoechst, blue ) and nucleoli (anti-fibrillarin, red ) after 72 h of treatment with the negative control (siGFP), positive control (siUTP4), or siSPRR3 siRNAs. Histograms show the distribution of cells that have the indicated number of nucleoli per nucleus. Light grey shows the distribution for the negative control, black shows the test condition, and dark grey shows overlap of the two frequencies. B , siSPRR3 depletion with siON-TARGET (siONT) SMARTPool siRNA reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (non-targeting siRNA, siNT), positive control (siUTP4), or siSPRR3 siRNAs. The shading in the histograms is as in A ). The data were collected across four replicates which are pooled in the histogram. C , siSPRR3 depletion with individual siONT SMARTPool siRNAs (deconvolution) reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (siNT), positive control (siNOL11), or representative siSPRR3 individual siRNAs (SPRR3-si1 and SPRR3-si2). The shading in the histograms is as in ( A ). The histograms include all three replicates pooled. The table summarizes each of four individual siONT SMARTPool siRNAs, showing that a reduction in nucleolar number correlates with a loss in cell viability. D , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 mRNA levels in MCF10A cells. RT-qPCR data of SPRR3 mRNA demonstrating knockdown after 72 h. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. The mean ± SEM are shown alongside individual data points, colored by replicate. E , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 protein levels in MCF10A cells. Western blot of SPRR3 protein demonstrating decreased levels after 72 h. Protein levels were normalized to total protein (trichloroethanol total protein stain), then to siNT. The mean ± SEM are shown alongside individual data points, colored by replicate. This sample was run on the same Western blot as in . After imaging total protein on the membrane, the blot was cut between 10 to 15 kD markers to stain separately for SPRR3 ( E , above) or RPS28 . Data in ( D and E ) were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗∗, p < 0.001.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Genome Wide, Negative Control, Positive Control, Quantitative RT-PCR, Knockdown, Comparison, Western Blot, Staining, Imaging, Membrane

SPRR3 depletion reduces pre-rRNA transcription. A , schematic of the major steps in ribosome biogenesis in human cells. B , SPRR3 depletion inhibits nucleolar rRNA biogenesis. Representative images and quantification of control or SPRR3-depleted MCF10A cells (siONT SMARTpool) following anti-fibrillarin (FBL) staining and 5-EU incorporation. Scale bars are 10 μm. siNT is a non-targeting negative control siRNA. siPOLR1A is a positive control targeting the RNAPI subunit, POLR1A. The overall mean percent inhibition ± SEM is shown for each treatment, with each dot representing one well. Each well in the siSPRR3 condition represents a separate day of testing and control datapoints are distributed across the three testing days. 0% inhibition is determined by the mean value of siNT, and 100% inhibition is set to the mean value of the siPOLR1A positive control. C , RT-qPCR analysis shows decreased 47S/45S pre-rRNA levels upon SPRR3 depletion in MCF10A cells. The mean ± SEM are shown alongside individual data points, colored by replicate. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , dual-luciferase reporter assay shows RNAPI promoter activity is reduced after SPRR3 depletion. The mean ± SEM are shown alongside individual data points, colored by replicate. The controls in these data have also been published in , without the inclusion of siSPRR3. All the data in this figure were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion reduces pre-rRNA transcription. A , schematic of the major steps in ribosome biogenesis in human cells. B , SPRR3 depletion inhibits nucleolar rRNA biogenesis. Representative images and quantification of control or SPRR3-depleted MCF10A cells (siONT SMARTpool) following anti-fibrillarin (FBL) staining and 5-EU incorporation. Scale bars are 10 μm. siNT is a non-targeting negative control siRNA. siPOLR1A is a positive control targeting the RNAPI subunit, POLR1A. The overall mean percent inhibition ± SEM is shown for each treatment, with each dot representing one well. Each well in the siSPRR3 condition represents a separate day of testing and control datapoints are distributed across the three testing days. 0% inhibition is determined by the mean value of siNT, and 100% inhibition is set to the mean value of the siPOLR1A positive control. C , RT-qPCR analysis shows decreased 47S/45S pre-rRNA levels upon SPRR3 depletion in MCF10A cells. The mean ± SEM are shown alongside individual data points, colored by replicate. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , dual-luciferase reporter assay shows RNAPI promoter activity is reduced after SPRR3 depletion. The mean ± SEM are shown alongside individual data points, colored by replicate. The controls in these data have also been published in , without the inclusion of siSPRR3. All the data in this figure were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Techniques: Control, Staining, Negative Control, Positive Control, Inhibition, Quantitative RT-PCR, Comparison, Luciferase, Reporter Assay, Activity Assay

SPRR3 depletion causes reduced global translation (protein synthesis). A , representative image and quantification of puromycin incorporation shows reduced translation after SPRR3 depletion in MCF10A cells. α-puromycin shows puromycin incorporation as a proxy for global protein synthesis. Total protein is the trichloroethanol total protein stain loading control. Images were quantified with Bio-Rad Image Lab. The mean ± SEM are shown alongside individual data points, colored by replicate. siRPL4 is the positive control. Data were normalized to a non-targeting siRNA (siNT), then graphed and analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗∗∗, p < 0.001. The control data in this puromycin Western blot have also been published in without the SPRR3 data. B , summary table describing the effects of SPRR3 depletion on ribosome biogenesis and the nucleolus. SPRR3 depletion reduces nucleolar number, nucleolar rRNA biogenesis (5-EU incorporation assay), pre-rRNA transcript levels (RT-qPCR of 47S/45S rRNA), rDNA promoter activity (luciferase reporter assay), and global protein synthesis (puromycin incorporation assay). SPRR3 depletion was found to have no effect on the ratio of 18S to 28S rRNA nor to produce changes in pre-rRNA northern blots, indicating that SPRR3 does not affect rRNA processing. Taken together, this suggests that SPRR3 plays a role in pre-rRNA transcription and nucleolar rRNA biogenesis that is essential to the normal translational activity of ribosomes.

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes reduced global translation (protein synthesis). A , representative image and quantification of puromycin incorporation shows reduced translation after SPRR3 depletion in MCF10A cells. α-puromycin shows puromycin incorporation as a proxy for global protein synthesis. Total protein is the trichloroethanol total protein stain loading control. Images were quantified with Bio-Rad Image Lab. The mean ± SEM are shown alongside individual data points, colored by replicate. siRPL4 is the positive control. Data were normalized to a non-targeting siRNA (siNT), then graphed and analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗∗∗, p < 0.001. The control data in this puromycin Western blot have also been published in without the SPRR3 data. B , summary table describing the effects of SPRR3 depletion on ribosome biogenesis and the nucleolus. SPRR3 depletion reduces nucleolar number, nucleolar rRNA biogenesis (5-EU incorporation assay), pre-rRNA transcript levels (RT-qPCR of 47S/45S rRNA), rDNA promoter activity (luciferase reporter assay), and global protein synthesis (puromycin incorporation assay). SPRR3 depletion was found to have no effect on the ratio of 18S to 28S rRNA nor to produce changes in pre-rRNA northern blots, indicating that SPRR3 does not affect rRNA processing. Taken together, this suggests that SPRR3 plays a role in pre-rRNA transcription and nucleolar rRNA biogenesis that is essential to the normal translational activity of ribosomes.

Techniques: Staining, Control, Positive Control, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Northern Blot

SPRR3 depletion causes the nucleolar stress response in MCF10A and A549 cells. A , schematic of the nucleolar stress response in human cells. Disruption of ribosome biogenesis causes accumulation of free 5S RNP which inhibits the ubiquitin ligase MDM2, leading to accumulation of TP53 and increased transcription of CDKN1A . B , TP53 stabilization after SPRR3 depletion in MCF10A cells. Representative images and quantification of TP53 Western blots after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. TP53 levels were normalized to total protein (trichloroethanol stain), then siNT. C , CDKN1A mRNA levels are elevated after SPRR3 depletion in MCF10A cells. After 72 h of siSPRR3 treatment, CDKN1A mRNA transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. These data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , siSPRR3 reduces SPRR3 mRNA levels in A549 cells. After 72 h of siSPRR3 treatment, SPRR3 transcript levels were detected by RT-qPCR. The data were normalized as in ( C ). E , siSPRR3 reduces SPRR3 protein levels in A549 cells. After 72 h of treatment with siSPRR3, SPRR3 protein levels were detected by Western blot. SPRR3 levels were normalized as in ( B ). F , SPRR3 depletion lowers 47S/45S pre-rRNA levels in A549 cells. Primers to the 5′ ETS of the 47S/45S rRNA were used to detect pre-rRNA levels after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. The data were normalized as in ( C and D ). G , TP53 stabilization after SPRR3 knockdown in A549 cells. Representative images and quantification of TP53 Western blots after 48h or 72h of treatment with siSPRR3 are shown. Protein levels were normalized as in ( B and E ). H , CDKN1A levels are elevated after SPRR3 depletion in A549 cells. After 72 h of treatment with siSPRR3, CDKN1A transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. Data were normalized as in ( C ). The mean ± SEM are shown alongside individual data points. For graphs with two conditions, data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. For graphs with more than two conditions, data were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes the nucleolar stress response in MCF10A and A549 cells. A , schematic of the nucleolar stress response in human cells. Disruption of ribosome biogenesis causes accumulation of free 5S RNP which inhibits the ubiquitin ligase MDM2, leading to accumulation of TP53 and increased transcription of CDKN1A . B , TP53 stabilization after SPRR3 depletion in MCF10A cells. Representative images and quantification of TP53 Western blots after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. TP53 levels were normalized to total protein (trichloroethanol stain), then siNT. C , CDKN1A mRNA levels are elevated after SPRR3 depletion in MCF10A cells. After 72 h of siSPRR3 treatment, CDKN1A mRNA transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. These data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , siSPRR3 reduces SPRR3 mRNA levels in A549 cells. After 72 h of siSPRR3 treatment, SPRR3 transcript levels were detected by RT-qPCR. The data were normalized as in ( C ). E , siSPRR3 reduces SPRR3 protein levels in A549 cells. After 72 h of treatment with siSPRR3, SPRR3 protein levels were detected by Western blot. SPRR3 levels were normalized as in ( B ). F , SPRR3 depletion lowers 47S/45S pre-rRNA levels in A549 cells. Primers to the 5′ ETS of the 47S/45S rRNA were used to detect pre-rRNA levels after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. The data were normalized as in ( C and D ). G , TP53 stabilization after SPRR3 knockdown in A549 cells. Representative images and quantification of TP53 Western blots after 48h or 72h of treatment with siSPRR3 are shown. Protein levels were normalized as in ( B and E ). H , CDKN1A levels are elevated after SPRR3 depletion in A549 cells. After 72 h of treatment with siSPRR3, CDKN1A transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. Data were normalized as in ( C ). The mean ± SEM are shown alongside individual data points. For graphs with two conditions, data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. For graphs with more than two conditions, data were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Techniques: Disruption, Ubiquitin Proteomics, Western Blot, Positive Control, Staining, Quantitative RT-PCR, Comparison, Knockdown

SPRR3 drives AKT phosphorylation and maintains POLR1A levels. A , AKT phosphorylation at serine 473 (pAKT) is decreased after a 72h SPRR3 depletion in MCF10A cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). Blots were probed for pAKT, then stripped and re-probed for total AKT. Signal was measured in Bio-Rad Image Lab. pAKT levels were normalized to total protein and total AKT. Total AKT was normalized to total protein, ensuring no significant difference in overall AKT levels upon SPRR3 depletion. B , phosphorylated AKT (pAKT) levels are decreased after 72h SPRR3 depletion in A549 cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). C , POLR1A levels are decreased upon 72h SPRR3 depletion. Representative image of Western blotting and quantification of POLR1A levels in MCF10A cells. siPOLR1A was used as a positive control. D , summary of effects of siSPRR3 depletion that we have confirmed in both MCF10A cells and A549 cells. For all graphs in this figure, the mean ± SEM are shown alongside individual data points. Data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 drives AKT phosphorylation and maintains POLR1A levels. A , AKT phosphorylation at serine 473 (pAKT) is decreased after a 72h SPRR3 depletion in MCF10A cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). Blots were probed for pAKT, then stripped and re-probed for total AKT. Signal was measured in Bio-Rad Image Lab. pAKT levels were normalized to total protein and total AKT. Total AKT was normalized to total protein, ensuring no significant difference in overall AKT levels upon SPRR3 depletion. B , phosphorylated AKT (pAKT) levels are decreased after 72h SPRR3 depletion in A549 cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). C , POLR1A levels are decreased upon 72h SPRR3 depletion. Representative image of Western blotting and quantification of POLR1A levels in MCF10A cells. siPOLR1A was used as a positive control. D , summary of effects of siSPRR3 depletion that we have confirmed in both MCF10A cells and A549 cells. For all graphs in this figure, the mean ± SEM are shown alongside individual data points. Data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Techniques: Phospho-proteomics, Staining, Western Blot, Positive Control

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