mcf 10a cell line  (ATCC)

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF interacts with eIF4E at the m 7 G cap, independent of intact eIF4G1. ( a ) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′ UTR luciferase reporters in untreated and TGF-β-treated MCF-10A cells. Data were normalized to Firefly luciferase expression and are presented as fold change of this normalized signal relative to CXCR4 reporter in untreated MCF-10A cells. ( b ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of indicated Renilla luciferase reporters (pRL-TK) in untreated and TGF-β-treated MCF-10A cells. Data were normalized to endogenous ACTB expression. ( c ) As in (a), with the indicated 3′ UTR luciferase reporters driven from an EMCV internal ribosomal entry site (IRES) in untreated and TGF-β-treated MCF-10A cells. ( d ) As in (b), for reporter assays in panel (c). ( e ) Right six lanes—immunoblots of indicated immunoprecipitates from whole-cell lysates derived from MCF-10A cells treated with TGF-β for 72 h. One half of each total immunoprecipitate was digested with RNase A prior to immunoblotting with the indicated antibodies. Right six lanes—as in the left six lanes, but lysates were digested with coxsackievirus 2A protease to cleave eIF4G1 before immunoprecipitation. CT = C-terminal; FL = full length; NT = N-terminal. ( f ) m 7 GTP cap analog binding assays utilizing cytosolic extracts derived from MCF-10A cells treated with TGF-β for 72 h. As above, one half of each extract was digested with coxsackievirus 2A protease to cleave eIF4G1 before the assay. ( g ) Proximity ligation assays using the indicated pairs of antibodies on MCF-10A cells treated with TGF-β for 72 h. In all panels, results are representative of at least three independent experiments and error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Reporter Assay, Luciferase, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Derivative Assay, Immunoprecipitation, Binding Assay, Ligation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 stimulates translation of GRE-containing EMT effector mRNAs in the context of reduced eIF4G1 function. ( a ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using anti-CELF1, anti-eIF4E, and anti-eIF4G1 antibodies or mouse and rabbit IgG. ACTB and GAPDH are non-GRE-containing negative control mRNAs. ( b ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using tandem anti-eIF4E/anti-CELF1 immunoprecipitation, tandem anti-eIF4E/anti-eIF4G1 immunoprecipitation, or tandem immunoprecipitation with mouse and rabbit IgGs. ACTB and GAPDH are non-GRE-containing negative controls. ( c, d ) Efficiency of in vitro translation of indicated capped and polyadenylated Renilla luciferase reporter mRNAs in mock or 2A protease-digested cell-free extract. ( e, f ) Efficiency of in vitro translation of reporter mRNAs as described in panels (c) and (d), but with mock-depleted ( Beads ) cell-free extract, eIF4G1-immunodepleted ( ID ) cell-free extract, or eIF4G1-immunodepleted cell-free extract reconstituted by addition of 20 nM enriched eIF4G1 and/or an equivalent concentration of recombinant CELF1. In panels (c–f), all extracts were derived from TGF-β-treated MCF-10A cells transiently transfected with shRNAs targeting either GLB1 (c, e) or CELF1 (d, f). CXCR4 = control, WT = wild-type 3′ UTR, ΔGRE =3′ UTR with deletion of GRE. In all panels, results are representative of at least three independent experiments. Error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 ( a, b, e, f : ANOVA with Dunnet’s post-hoc test; c, d : Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Immunoprecipitation, In Vitro, Luciferase, Concentration Assay, Recombinant, Derivative Assay, Transfection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: Phosphorylation of eIF4E is required for CELF1-driven EMT in MCF-10A cells. ( a ) Immunoblots of lysates derived from MCF-10A cells stably expressing either HA-tagged WT or S209A mutant murine EIF4e and shRNA targeting human EIF4E or control shRNA, and either mock transfected or transiently transfected with a CELF1 overexpression construct for 72 h. GAPDH = loading control. ( b ) Immunoblot of indicated immunoprecipitates from lysates derived from TGF-β-treated MCF-10A cells, stably expressing either HA-tagged WT or S209A mutant murine Eif4e and shRNA targeting human EIF4E or control shRNA. IgG: negative immunoprecipitation control. ( c ) Polysomal profiles from MCF-10A cells in which endogenous EIF4E expression had been knocked down via shRNA and then rescued via stable transduction of either WT or S209A mutant Eif4e . ( d ) qRT-PCR validation of polyribosomal enrichment and depletion of indicated mRNAs via total and polysomal mRNA from MCF-10A cells stably expressing WT or S209A mutant Eif4e , treated with TGF-beta for 72 h. ( e ) MCF-10A cells in which endogenous EIF4E expression had been knocked down via shRNA and then rescued via stable transduction of either WT or an S209A mutant Eif4e were transiently transfected with CELF1 expression construct. After 72 h, extracts were assessed via immunoblot for relative protein expression of CELF1-regulated EMT effectors. In all panels, results are representative of at least three independent experiments. Error bars in panel (d) depict mean ± standard deviation (SD). NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Stable Transfection, Expressing, Mutagenesis, shRNA, Control, Transfection, Over Expression, Construct, Immunoprecipitation, Transduction, Quantitative RT-PCR, Biomarker Discovery, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 directly binds eIF4E via interactions via the canonical dorsal cleft region and the lateral hydrophobic patch. ( a ) Schematic of CELF1 domain structure and candidate eIF4E binding motifs. RRM, RNA-recognition motif. ( b ) Immunoblots of immunoprecipitations from lysates of MCF-10A cells transfected with WT or indicated mutant GFP-CELF1 plasmids for 72 h. ( c ) Immunoblots of binding assays using affinity-purified phosphomimic eIF4E ( GST-EIF4E S209D ), affinity-purified WT CELF1 ( 6xHis-CELF1 ), or affinity-purified mutant CELF1 ( 6x-His-CELF1 Δ365–71 ). ( d ) Immunoblots of immunoprecipitations derived from lysates of MCF-10A cells stably expressing an shRNA targeting the 3′ UTR of EIF4E and co-expressing either WT or EIF4E W73A mutant, treated with TGF-β or transiently transfected with a GFP-CELF1 plasmid for 72 h. IgG: negative control. ( e ) Immunoblots of binding assays in which affinity-purified 6xHis-CELF1 was mixed with affinity-purified phosphomimic ( GST-EIF4E S209D ) or mutant ( GST-EIF4E S209D/W73A ) eIF4E. ( f ) HSQC-NMR spectra of 15 N-labeled eIF4E S209D , alone or mixed with a seven-fold excess of CELF1 YAAAALP-containing peptide (sequence shown). Representative shifts are magnified. ( g ) Dorsal surface view of the crystal structure of eIF4E complexed with m 7 GTP (PDB 1IPC - ), depicting chemical shifts observed in HSQC-NMR. The canonical eIF4E binding cleft is colored in red, and chemical shifts induced by the CELF1 peptide are indicated in blue. Shifts overlapping the canonical binding cleft are indicated in purple. ( h ) As in (g), rotating the eIF4E structure ninety degrees along a roughly fifteen-degree bearing for depiction of the lateral surface mediating non-canonical binding. Coloring and annotations are as in (g). ( i ) Purified, untagged CELF1 was mixed with purified eIF4E, eIF4E S209D , eIF4E S209D/W73A (disrupts canonical dorsal binding), or eIF4E S209D/I63A/I79A (disrupts non-canonical lateral binding) and then immunoprecipitated with IgG (negative control) or anti-CELF1 antibody and immunoblotted with the indicated antibodies. Results in (a–e, i) are representative of at least three individual experiments.

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Binding Assay, Western Blot, Transfection, Mutagenesis, Affinity Purification, Derivative Assay, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Negative Control, Labeling, Sequencing, Purification, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: Interaction of CELF1 and eIF4E is required for CELF1-driven EMT and experimental metastasis. ( a ) Immunoblot analysis of indicated EMT markers in lysates derived from MCF-10A cells transfected with WT or indicated mutant GFP-CELF1 plasmids for 72 h. ( b ) Immunoblot analysis of indicated EMT markers in lysates derived from MCF-10A cells expressing either WT or W73A mutant human EIF4E and shRNA targeting the 3′ UTR of human EIF4E and induced to undergo EMT via stable expression of GFP-CELF1 or TGF-β treatment for 72 h. ( c ) Immunoblot analysis of indicated EMT markers and GFP-CELF1 in lysates derived from parental MCF-10AT1 cells ( left column ) and MDA-MB-468 ( right column ), or each cell line stably transduced with either WT or Δ365–71 mutant GFP-CELF1 . GAPDH = loading control in panels (a), (b), and (c); black line in panels (a) and (b) denotes lysates derived from the same experiment, but gels processed in parallel. All results (a–c) are representative of at least three independent experiments. Quantification of relative in vitro cellular migration ( d, f ) and invasion ( e, g ) in transwell assays in parental MCF-10AT1 and MDA-MB-468 cells, respectively, or stably transduced with either WT or Δ365–371 mutant GFP-CELF1 . Data represents mean ± SD of at least three independent experiments, each performed in triplicate. * P -value < 0.05 (ANOVA with Dunnet’s post-hoc test). ( h, i ) Parental MCF-10AT1 cells, or cells stably overexpressing either WT or Δ365–71 mutant GFP-CELF1 , were injected into the tail vein of athymic nude mice. The incidence and progression of metastasis were measured by luciferin injection and bioluminescence imaging of Firefly luciferase (h), and ex vivo excised lungs on day 15 (i). ( j ) Representative hematoxylin and eosin (H&E) ( top ) and immunohistochemical (IHC) ( bottom ) staining, respectively, of the lungs from mice shown in panel (h). Scale bar, 200 µm ( top ); 50 µm ( bottom ). Black arrows ( bottom ) indicate micrometastases. Dotted lines indicate area shown in corresponding H&E staining of serial sections shown in panel (j). For (h–j), representative images are from n = 4 for parental, n = 4 for WT GFP-CELF1 , and n = 6 for mutant GFP-CELF1 Δ365–71 experimental groups.

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Western Blot, Derivative Assay, Transfection, Mutagenesis, Expressing, shRNA, Stable Transfection, Transduction, Control, In Vitro, Migration, Injection, Imaging, Luciferase, Ex Vivo, Immunohistochemical staining, Staining

Journal: Redox Biology

Article Title: Unravelling the anti-cancer mechanisms elicited by non-covalent thioredoxin reductase inhibitors for triple negative breast cancer therapy

doi: 10.1016/j.redox.2025.103980

Figure Lengend Snippet: TXNRD(i)s increase redox stress which do not correlate with cell viability. A. Gene set enrichment analysis of Oxidative Stress Response gene set by WikiPathways. NES and nominal p-value were determined by the GSEA software. B. Redox stress quantification using H 2 DCFDA by flow cytometry of MDA-MB-231 (top) (n = 2), HCC1806 (middle) (n = 3), and MCF-10A (bottom) (n = 3) cells. Cells were treated with 10 μM 8VP101 for 0, 2, 6,18, and 24 h, harvested and incubated with 1 μM H 2 DCFDA for 15 min. Representative histograms (left) of measured DCF mean fluorescence intensity (MFI) with the grey peak representing unstained control. Statistical significance was determined by an unpaired t -test relative to 0-h time point. C. Cell viability determined by crystal violet. MDA-MB-231 (top), HCC1806 (middle), and MCF-10A (bottom) cells treated with 10 μM 8VP101 for 24 h. Cells were fixed and stained with crystal violet, then solubilized and absorption values are normalized to vehicle control set as 100 %. Statistical significance was determined by an unpaired t -test. Data is presented as the mean±sem. ∗p ≤ 0.05; ∗∗p ≤ 0.005; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns: not significant.

Article Snippet: MCF-10A cells were obtained from ATCC and maintained in DMEM-F12 medium (Gibco, 11330032) supplemented with 10 % fetal bovine serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin.

Techniques: Software, Flow Cytometry, Incubation, Fluorescence, Control, Staining

Journal: Redox Biology

Article Title: Unravelling the anti-cancer mechanisms elicited by non-covalent thioredoxin reductase inhibitors for triple negative breast cancer therapy

doi: 10.1016/j.redox.2025.103980

Figure Lengend Snippet: Treatment with TXNRD(i)s inhibits cells proliferation and disrupts cells cycle by inducing G1 arrest and reducing S phase . A. Gene set enrichment analysis of DNA Replication by Gene Ontology Biological Processes. NES and nominal p-value were determined by GSEA software. B. EdU incorporation assay by flow cytometry of MDA-MB-231 cells. Cells were treated with 4 μM or 10 μM 8VP101 for 24 h and loaded with 1 μM EdU for 3 h before harvest and analysis. Representative histograms of EdU incorporation are shown on the left. Statistical significance was determined by an unpaired t -test. C. Gene expression by RT-QPCR in MDA-MB-231 cells treated with 10 μM 8VP101 for 24 h. Relative gene expression is calculated using the ΔΔCt method normalized to β-actin. Statistical significance was determined by an unpaired t -test. D. Cell cycle analysis using PI by flow cytometry of MDA-MB-231 (top), HCC1806 (middle), and MCF-10A (bottom) cells. Cells were treated with 4 μM or 10 μM 8VP101 for 24 h. Statistical significance was determined by Kruskal-Wallis test followed by Dunns test. E. Protein levels of p21 assessed by western blot. MDA-MB-231 cells were treated with 10 μM 8VP101 for 6 h. β-actin was used as the loading control. Data is presented as the mean±sem. ∗p ≤ 0.05; ∗∗p ≤ 0.005; ∗∗∗p ≤ 0.001; ns: not significant.

Article Snippet: MCF-10A cells were obtained from ATCC and maintained in DMEM-F12 medium (Gibco, 11330032) supplemented with 10 % fetal bovine serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin.

Techniques: Software, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Cell Cycle Assay, Western Blot, Control

Journal: Redox Biology

Article Title: Unravelling the anti-cancer mechanisms elicited by non-covalent thioredoxin reductase inhibitors for triple negative breast cancer therapy

doi: 10.1016/j.redox.2025.103980

Figure Lengend Snippet: TXNRD(i)s inhibit TXNRD1 and TXNRD2 enzymes and both enzymes are critical for growth of TNBC cells. A. HCC1806 cells were pretreated with 8VP101 or vehicle control for 1 h, then treated with 10 μM of TRFS-green for 4 h, followed by incubation with MitoTracker-red. Live cells were imaged on a Nikon Ti2E inverted microscope at 20x. Representative images shown. Cytosolic and mitochondrial TXNRD activity on average intensity per cell per field basis was quantified for 4 μM 8VP101 treatment. Average intensity was calculated from n = 3 biological replicates and n = 9 technical replicates. ∗∗∗∗p ≤ 0.0001. B. MDA-MB-231, HCC1806 and MCF-10A cells were transfected with siNeg, siTXNRD1, siTXNRD2, or both siTXNRD1 and siTXNRD2, 10 nM each, and monitored for cell growth every 12 h over 120 h using BioTek BioSpa. Data is shown as fold change normalized to the initial timepoint. Growth curves are averages −/+ sem from n = 3 biological replicates. End-point statistical significance was determined. ∗∗p ≤ 0.005; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. C. MDA-MB-231 cells were treated with DMSO, 8VP101 or 9VP19 at 10 μM each and monitored for cells growth as in (B). End-point statistical significance was determined. ∗∗∗∗p ≤ 0.0001. D. Cell cycle analysis using PI by flow cytometry of MDA-MB-231 cells. Cells were transfected with 10 nM siTXNRD1, 10 nM siTXNRD2, or a combination of 10 nM siTXNRD1 and 10 nM siTXNRD2 for 24 h. Statistical significance was determined by Kruskal-Wallis test followed by Dunns test comparing siTXNRD1, siTXNRD2, and both siTXNRD1 and siTXNRD2 to siNeg control. Data is presented as the mean±sem. ∗p ≤ 0.05; ns: not significant.

Article Snippet: MCF-10A cells were obtained from ATCC and maintained in DMEM-F12 medium (Gibco, 11330032) supplemented with 10 % fetal bovine serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin.

Techniques: Control, Incubation, Inverted Microscopy, Activity Assay, Transfection, Cell Cycle Assay, Flow Cytometry

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

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.

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

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.

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

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.

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

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.

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

Journal: International Journal of Molecular Sciences

Article Title: Breast-Cancer-Derived Secretomes from MCF-7 Cells Modulate Bacterial Pathogenic Traits

doi: 10.3390/ijms27042073

Figure Lengend Snippet: Scanning Electron Microscopy (SEM) images illustrating the dose-dependent effects of breast cells’ conditioned media (CM) on P . aeruginosa and E . faecalis morphology. Panels ( a – h ) show representative SEM micrographs of P . aeruginosa ( a – d ) and E . faecalis ( e – h ) after 24 h incubation with cell-free conditioned-media at the indicated concentrations and sources: ( a , e ) 10% MCF-10A conditioned medium; ( b , f ) 10% MCF-7 conditioned medium; ( c , g ) 15% MCF-10A conditioned medium; ( d , h ) 15% MCF-7 conditioned medium. Yellow arrows indicate prominent structural alterations such as membrane irregularities, aggregation, and surface disruptions, most apparent in bacteria exposed to MCF-7 conditioned media. All images were acquired at 25,000× magnification and are representative of three independent experiments. Scale bars are indicated.

Article Snippet: MCF-7 (luminal A breast cancer cell line) and MCF-10A (normal breast epithelial cells) cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Electron Microscopy, Incubation, Membrane, Bacteria

Journal: International Journal of Molecular Sciences

Article Title: Breast-Cancer-Derived Secretomes from MCF-7 Cells Modulate Bacterial Pathogenic Traits

doi: 10.3390/ijms27042073

Figure Lengend Snippet: Quantitative analysis of biofilm formation by bacteria exposed to breast cell conditioned media. Bar graphs show the logarithmic OD 630 values representing biofilm biomass formed by ( A ) P. aeruginosa , ( B ) E. faecalis and ( C ) E. coli after 24 h incubation in microtiter plates with 5%, 10% and 15% ( v / v ) concentrations of MCF-10A (CM-10A) or MCF-7 (CM-7), after normalized to their respective serum-free media control (CFM-10A and CFM-7). Statistically significant differences between treatment groups are indicated by asterisks (** p < 0.01, *** p < 0.0001). All experiments were conducted in triplicate, and data are presented as mean ± SD.

Article Snippet: MCF-7 (luminal A breast cancer cell line) and MCF-10A (normal breast epithelial cells) cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Bacteria, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Breast-Cancer-Derived Secretomes from MCF-7 Cells Modulate Bacterial Pathogenic Traits

doi: 10.3390/ijms27042073

Figure Lengend Snippet: Temporal changes in virulence gene abundance in P. aeruginosa genomic DNA following exposure to breast-cell-derived conditioned media. The relative abundance of virulence-associated genes ( A ) pilB , ( B ) lasB , ( C ) exoS , ( D ) algD , ( E ) plcH , and ( F ) exoU in P. aeruginosa was quantified by real-time PCR after incubation for 24, 48, and 72 h with 10% ( v / v ) conditioned media derived from MCF-10A (CM-10A) or MCF-7 (CM-7) cells. Quantitative PCR was performed using genomic DNA as the template. Data are presented as mean ± SD ( n = 3), normalized to the 16S rRNA gene and to the corresponding cell-free medium controls (CFM-7 or CFM-10A), and expressed relative to CM-10A at each time point. Statistically significant differences are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.0001). ns denotes that the result is not statistically significant.

Article Snippet: MCF-7 (luminal A breast cancer cell line) and MCF-10A (normal breast epithelial cells) cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Breast-Cancer-Derived Secretomes from MCF-7 Cells Modulate Bacterial Pathogenic Traits

doi: 10.3390/ijms27042073

Figure Lengend Snippet: Temporal changes in virulence gene abundance in E. faecalis following exposure to breastcell−derived conditioned media. The relative abundance of virulence−associated genes ( A ) fsrC, ( B ) esp, ( C ) ace, ( D ) asa1, and ( E ) efa in E. faecalis was quantified by real−time PCR after incubation for 24, 48, and 72 h with 10% ( v / v ) conditioned media derived from MCF-10A (CM-10A) or MCF-7 (CM-7) cells. Quantitative PCR was performed using genomic DNA as the template. Data are presented as mean ± SD ( n = 3), normalized to the 16S rRNA gene and to the corresponding cell−free medium controls (CFM-10A or CFM-7), and expressed relative to CM-10A at each corresponding time point. Statistically significant differences between CM-7– and CM-10A–treated cultures are indicated by asterisks (*** p < 0.0001).

Article Snippet: MCF-7 (luminal A breast cancer cell line) and MCF-10A (normal breast epithelial cells) cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Breast-Cancer-Derived Secretomes from MCF-7 Cells Modulate Bacterial Pathogenic Traits

doi: 10.3390/ijms27042073

Figure Lengend Snippet: Temporal changes in virulence gene abundance in E. coli following exposure to breast cell−derived conditioned media. The relative abundance of adhesion−associated virulence genes ( A ) papG , ( B ) papC , ( C ) draA , and ( D ) fimH in E. coli was quantified by real−time PCR after incubation for 24, 48, and 72 h with 10% ( v / v ) conditioned media derived from MCF-10A (CM-10A) or MCF-7 (CM-7) cells. Quantitative PCR was performed using genomic DNA as the template. Data are presented as mean ± SD ( n = 3), normalized to the 16S rRNA gene and to the corresponding cell−free medium controls (CFM-10A or CFM-7), and expressed relative to CM-10A at each corresponding time point. Statistically significant differences between CM-7– and CM-10A–treated cultures are indicated by asterisks (** p < 0.01, *** p < 0.0001).

Article Snippet: MCF-7 (luminal A breast cancer cell line) and MCF-10A (normal breast epithelial cells) cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Incubation