mcf10a  (ATCC)

Journal: Bioactive Materials

Article Title: Ribbon-shaped microgels as bioinks for 3D bioprinting of anisotropic tissue structures

doi: 10.1016/j.bioactmat.2025.12.040

Figure Lengend Snippet: μRB bioinks support multicellular patterning to model breast cancer-bone invasion at tissue interface. (A) Schematic of experimental design: bioprinting of MSCs and osteogenic differentiation to derive bone grid, followed by injection of breast cancer bioink, and monitoring invasion over time using confocal microscopy. (B) Confocal images of MSCs (red) after printing (Scale bar = 1 mm). (C) Confocal images of scaffold sections containing CellTracker-labeled MSCs (red) after 28 days of osteogenic differentiation and GFP + MDA-MB-231 cells extruded into the open pores of the grids (green) (Scale bar = 200 μm). (D) Confocal images of patterned MSC-derived bone (red) with MDA-MB-231 and MCF-7 breast cancer cells (green) after 14 days of co-culture (Scale bar = 1 mm). (E) Quantification of breast cancer cell invasion: percentage that remain in open pores vs. invading into the MSC-bone compartment (n = 5 per group). Values are reported as mean ± S.D. and p-values were determined by two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.005, ∗∗∗∗p ≤ 0.001.

Article Snippet: Breast cancer cell lines MDA-MB-231 (ATCC) and MCF-7 (ATCC) were lentivirus transduced to express GFP and cultured in DMEM media (4.5 g L −1 glucose) supplemented with 10 % (v/v) fetal bovine serum and 1 % (v/v) penicillin-streptomycin.

Techniques: Injection, Confocal Microscopy, Labeling, Derivative Assay, Co-Culture Assay

Journal: Materials Today Bio

Article Title: Expanding the toolbox of bioorthogonal activation of photosensitizers for precise photodynamic therapy through transition metal-mediated deallylation

doi: 10.1016/j.mtbio.2026.102797

Figure Lengend Snippet: (a) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The HeLa human cervical cancer cells (ATCC, CCL-2), 4T1 murine mammary carcinoma cells (ATCC, CRL-2539), MCF-7 human breast cancer cells (ATCC, HTB-22), and NIH 3T3 murine embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher, cat. no. 11965092) supplemented with fetal calf serum (10 %) and penicillin-streptomycin (100 unit/mL and 100 μg/mL, respectively).

Techniques: Fluorescence, Incubation, Positive Control, Staining, Irradiation, Viability Assay, Binding Assay

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: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: Knockdown of GGCT inhibited the proliferation and caused significant changes in gene expression profiles detected by transcriptomic highthroughput sequencing in MCF-7 cells. (A) mRNA expression of GGCT was analyzed by qRT-PCR 3 days after transfection. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. (B) Western blotting analysis of GGCT and α-tubulin 4 days after transfection of MCF-7 cells with GGCT-siRNA or non-target control siRNA. (C) The relative survival number of trypan blue-negative viable MCF-7 cells at 1, 4, 7 days post-transfection. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using one-way ANOVA followed by Tukey’s post hoc test. (D) Representative images at 4 days post-transfection. (E) Hierarchical clustering analysis of differentially expressed genes (DEGs) detected by RNA-seq analysis. The color scale indicates log10(FPKM) and intensity increases from green to red, indicating down-regulation and up-regulation, respectively. (F) Numbers of significantly up-regulated (red) and down-regulated (green) DEGs in siGGCT-transfected MCF-7 cells at days 1, 2, and 3 post-transfection, identified by RNA-seq analysis using the filtering criteria of |log₂ fold change| >1 and q-value <0.01. Scale bar: 50 μm. ANOVA, Analysis of variance; FC, fold change; FPKM, fragments per kilobase of exon model per million mapped reads; GGCT, γ-glutamylcyclotransferase; qRT-PCR, quantitative reversetranscription- polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Knockdown, Gene Expression, High Throughput Screening Assay, Sequencing, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Control, RNA Sequencing, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of DEGs in MCF-7 cells after GGCT knockdown. (A) GO enrichment analysis for biological processes (up-regulated and down-regulated genes), and KEGG pathway analysis of DEGs, using samples collected 3 days after knockdown. The top 10 biological process terms ranked according to p-Values are shown. (B) Hypothetical schema of cell cycle regulation following GGCT knockdown in MCF-7 cells, constructed based on DEGs enriched in the KEGG pathway ‘hsa04110: Cell cycle’ (http://www.kegg.jp). (C) Relative mRNA expression patterns of GGCT, TGF-β2, CDKN1A (p21 Cip1 ), and CDKN2B (p15 INK4b ) were measured with qRT-PCR. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. CDKN1A, Cyclin dependent kinase inhibitor 1A; CDKN2B, cyclin dependent kinase inhibitor 2B; DEGs, differentially expressed genes; GGCT, γ-glutamylcyclotransferase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; TGF-β2, transforming growth factor-β2.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Knockdown, Construct, Expressing, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: Up-regulation of p15 INK4b and p21 Cip1 contributes to G0/G1 cell cycle arrest and subsequent cell growth inhibition in GGCT-depleted MCF-7 cells. (A) The levels of p15I NK4b and p21 Cip1 proteins were normalized to levels of α-tubulin and the fold change relative to the control was calculated at 4 days after the indicated siRNA transfection. (B) The cell cycle distribution and (C) representative histograms in FACS analysis in MCF-7 cells 4 days after transfection with the indicated siRNAs. n=3 per group; *p<0.05; **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. (D) The number of viable cells in the trypan blue dye exclusion test and (E) representative images of MCF-7 cells at 7 days after transfection with the indicated siRNAs. Scale bar: 50 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; FACS, fluorescence-activated cell sorting; GGCT, γ-glutamylcyclotransferase; PCR, polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Inhibition, Control, Transfection, Fluorescence, FACS, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: TGF-β2/SMAD signaling axis regulates p15 INK4b and p21 Cip1 expression in GGCT-depleted MCF-7 cells. (A) mRNA expression levels of GGCT and TGF-β2 were analyzed by qRT-PCR 3 days after transfection with GGCT- and/or TGF-β2-siRNA. (B) Western blot analysis of GGCT and TGF-β2 expression 4 days after transfection with GGCT- and/or TGF-β2-siRNA. (C) Western blotting analysis of p15 INK4b , p21 Cip1 , phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3, GGCT, and α-tubulin in MCF-7 at 4 days after transfection with GGCT-siRNA and/or TGF-β2-siRNA, or non-target control siRNA. (D) Western blotting analysis of p15 INK4b , p21 Cip1 , pSMAD3, SMAD3, GGCT, and α-tubulin in MCF-7 cells at 4 days after transfection with GGCT-siRNA and/ or SMAD3-siRNA, or non-target control siRNA. (E) The number of viable cells in the trypan blue dye exclusion test and (F) representative images of MCF- 7 cells at 4 days after transfection with the indicated siRNAs. Scale bar: 200 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; pSMAD, phosphorylated SMAD; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: p15 INK4b , p21 Cip1 , and their upstream regulator TGF-β2 are involved in the induction of cellular senescence following GGCT-KD in MCF-7 cells. (A) Representative images of SA-β-Gal staining 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT, p15, and p21. Scale bar: 50 μm. (B) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. (C) Representative images of SA-β-Gal staining at 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT and TGF-β2. Scale bar: 50 μm. (D) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. n=3 per group; *p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; KD, knockdown; SA-β-Gal, senescence-associated β-galactosidase; siRNA, smallinterfering RNA; TGF-β2, transforming growth factor-β2.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Staining, Transfection, Knockdown