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mcf 7  (ATCC)
99
ATCC mcf 7
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 <t>and</t> <t>MCF-7</t> 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.
Mcf 7, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcf  (ATCC)
99
ATCC mcf
(a) Fluorescence confocal images of HeLa, 4T1, <t>MCF-7,</t> 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.)
Mcf, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcf - by Bioz Stars, 2026-03
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mcf 7 atcc  (ATCC)
99
ATCC mcf 7 atcc
(a) Fluorescence confocal images of HeLa, 4T1, <t>MCF-7,</t> 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.)
Mcf 7 Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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breast cancer cell line mcf  (ATCC)
99
ATCC breast cancer cell line mcf
Knockdown of GGCT inhibited the proliferation and caused significant changes in gene expression profiles detected by transcriptomic highthroughput sequencing <t>in</t> <t>MCF-7</t> 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.
Breast Cancer Cell Line Mcf, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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breast cancer cell line mcf - by Bioz Stars, 2026-03
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mcf 7 cells  (ATCC)
99
ATCC mcf 7 cells
CDK4/6i triggers an altered splicing program affecting cell cycle genes and RNA quality control genes. (A) Barplot of AS events detected as significant by rMATS. Positive = more included in Palbo. Negative = less included in Palbo. (B) Overlap between differentially expressed genes as described in Fig. and alternatively spliced genes. P = 5.04e-03, hypergeometric test. (C) GO analysis of genes affected at both the expression and AS level. (D) Left: visualization of RNA sequencing coverage over INCENP exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.04e-03, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of INCENP exon skipping. Lower: Alternative exon coding region in INCENP protein. (E) Left: Visualization of genome coverage in SMARCA1 exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.58e-02, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of SMARCA1 exon skipping. Lower: Alternative exon coding region in SMARCA1 protein. (F) Left: Visualization of genome coverage in MCM7 retained intron event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of MCM7 intron retention. (G) Left: visualization of genome coverage in DHX34 exon skipping event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of DHX34 exon inclusion. P = 7.5e-05, Student’s two-sided t -test. Lower: Validation of lowered DHX34 protein expression by Western blot. P = 1.36e-02, Student’s two-sided t -test. (H) RT-PCR of DHX34 after treatment with CDK4/6i and NMD inhibitors. All conditions compared to DMSO, P -values calculated via Student’s t -test. SMG1i: P = 1.41e-03, CHX: P = 5.69e-03, Palbo: P = 1.42e-02, Ribo: P = 2.77e-02, Abema: P = 4.89e-02. (I) Overlap between Palbo-induced splicing events in T-47D and NMDi-induced splicing events in <t>MCF-7.</t> P = 9.35e-55, hypergeometric test.
Mcf 7 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


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.

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

(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.)

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

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.

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

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.

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

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.

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

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.

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

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.

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

CDK4/6i triggers an altered splicing program affecting cell cycle genes and RNA quality control genes. (A) Barplot of AS events detected as significant by rMATS. Positive = more included in Palbo. Negative = less included in Palbo. (B) Overlap between differentially expressed genes as described in Fig. and alternatively spliced genes. P = 5.04e-03, hypergeometric test. (C) GO analysis of genes affected at both the expression and AS level. (D) Left: visualization of RNA sequencing coverage over INCENP exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.04e-03, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of INCENP exon skipping. Lower: Alternative exon coding region in INCENP protein. (E) Left: Visualization of genome coverage in SMARCA1 exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.58e-02, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of SMARCA1 exon skipping. Lower: Alternative exon coding region in SMARCA1 protein. (F) Left: Visualization of genome coverage in MCM7 retained intron event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of MCM7 intron retention. (G) Left: visualization of genome coverage in DHX34 exon skipping event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of DHX34 exon inclusion. P = 7.5e-05, Student’s two-sided t -test. Lower: Validation of lowered DHX34 protein expression by Western blot. P = 1.36e-02, Student’s two-sided t -test. (H) RT-PCR of DHX34 after treatment with CDK4/6i and NMD inhibitors. All conditions compared to DMSO, P -values calculated via Student’s t -test. SMG1i: P = 1.41e-03, CHX: P = 5.69e-03, Palbo: P = 1.42e-02, Ribo: P = 2.77e-02, Abema: P = 4.89e-02. (I) Overlap between Palbo-induced splicing events in T-47D and NMDi-induced splicing events in MCF-7. P = 9.35e-55, hypergeometric test.

Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: CDK4/6i triggers an altered splicing program affecting cell cycle genes and RNA quality control genes. (A) Barplot of AS events detected as significant by rMATS. Positive = more included in Palbo. Negative = less included in Palbo. (B) Overlap between differentially expressed genes as described in Fig. and alternatively spliced genes. P = 5.04e-03, hypergeometric test. (C) GO analysis of genes affected at both the expression and AS level. (D) Left: visualization of RNA sequencing coverage over INCENP exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.04e-03, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of INCENP exon skipping. Lower: Alternative exon coding region in INCENP protein. (E) Left: Visualization of genome coverage in SMARCA1 exon skipping event. Right: Validation of AS by RT-PCR and corresponding quantification ( P = 1.58e-02, Student’s two-sided t -test). Gel shows one representative replicate of RT-PCR verification of SMARCA1 exon skipping. Lower: Alternative exon coding region in SMARCA1 protein. (F) Left: Visualization of genome coverage in MCM7 retained intron event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of MCM7 intron retention. (G) Left: visualization of genome coverage in DHX34 exon skipping event. Right: Validation of AS by RT-PCR. Gel shows one representative replicate of RT-PCR verification of DHX34 exon inclusion. P = 7.5e-05, Student’s two-sided t -test. Lower: Validation of lowered DHX34 protein expression by Western blot. P = 1.36e-02, Student’s two-sided t -test. (H) RT-PCR of DHX34 after treatment with CDK4/6i and NMD inhibitors. All conditions compared to DMSO, P -values calculated via Student’s t -test. SMG1i: P = 1.41e-03, CHX: P = 5.69e-03, Palbo: P = 1.42e-02, Ribo: P = 2.77e-02, Abema: P = 4.89e-02. (I) Overlap between Palbo-induced splicing events in T-47D and NMDi-induced splicing events in MCF-7. P = 9.35e-55, hypergeometric test.

Article Snippet: T-47D and MCF-7 cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM) complete medium (Gibco, Cat #11-965-092) supplemented with 10% fetal bovine serum (EMD Millipore, Cat #F2442).

Techniques: Control, Expressing, RNA Sequencing, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Western Blot

Palbociclib induces expression of estrogen-response factors. (A) Heat map showing log 2 foldchange of all detected cyclin transcripts. Legend: Red: upregulated genes, blue: downregulated genes. (B) Plot comparing log 2 foldchange of genes upregulated by Palbo and genes in the core enrichment subset of the “Estrogen Response Early” gene set from Fig. (top). Red: significantly detected genes passing log 2 foldchange ≥ 0.75, black: not significant (ns). (C) Western blot showing expression of indicated proteins in T-47D (left) and MCF-7 (right) cells after treatment with 1 μM Palbo or 1 μM fulvestrant alone and in combination for 24 h. (D) Western blot showing expression of indicated proteins in T-47D cells treated with 1 μM Palbo and collected at the indicated times.

Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: Palbociclib induces expression of estrogen-response factors. (A) Heat map showing log 2 foldchange of all detected cyclin transcripts. Legend: Red: upregulated genes, blue: downregulated genes. (B) Plot comparing log 2 foldchange of genes upregulated by Palbo and genes in the core enrichment subset of the “Estrogen Response Early” gene set from Fig. (top). Red: significantly detected genes passing log 2 foldchange ≥ 0.75, black: not significant (ns). (C) Western blot showing expression of indicated proteins in T-47D (left) and MCF-7 (right) cells after treatment with 1 μM Palbo or 1 μM fulvestrant alone and in combination for 24 h. (D) Western blot showing expression of indicated proteins in T-47D cells treated with 1 μM Palbo and collected at the indicated times.

Article Snippet: T-47D and MCF-7 cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM) complete medium (Gibco, Cat #11-965-092) supplemented with 10% fetal bovine serum (EMD Millipore, Cat #F2442).

Techniques: Expressing, Western Blot

Combined inhibition of CDK4/6 and CDK7. (A) MCF-7 cell count after 5-day treatment of samuraciclib (CDK7i) or Palbo alone and in combination. (B) Western blot showing increased suppression of p-CDK2 with combined Palbo and CDK7i treatment. (C) Schematic summarizing pathways described throughout this study, integrating gene and protein regulation and essentiality. The symbols next to each gene/gene family represent observations from the Palbo dataset. Symbol positions: the first position corresponds to transcriptomic DE, the second position corresponds to proteomic DE, and the third position corresponds to gene essentiality. Symbol colors: Red arrow corresponds to upregulation, blue arrow corresponds to downregulation, gray dash corresponds to no significance (n.s.) or no data available (n.d.). A green arrow at the third represents a gene within the top 1000 T-47D essential genes (z-scored) from DepMap 24Q4 data; a green dash at the third position represents a gene outside of the top 1000 essential T-47D genes.

Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: Combined inhibition of CDK4/6 and CDK7. (A) MCF-7 cell count after 5-day treatment of samuraciclib (CDK7i) or Palbo alone and in combination. (B) Western blot showing increased suppression of p-CDK2 with combined Palbo and CDK7i treatment. (C) Schematic summarizing pathways described throughout this study, integrating gene and protein regulation and essentiality. The symbols next to each gene/gene family represent observations from the Palbo dataset. Symbol positions: the first position corresponds to transcriptomic DE, the second position corresponds to proteomic DE, and the third position corresponds to gene essentiality. Symbol colors: Red arrow corresponds to upregulation, blue arrow corresponds to downregulation, gray dash corresponds to no significance (n.s.) or no data available (n.d.). A green arrow at the third represents a gene within the top 1000 T-47D essential genes (z-scored) from DepMap 24Q4 data; a green dash at the third position represents a gene outside of the top 1000 essential T-47D genes.

Article Snippet: T-47D and MCF-7 cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM) complete medium (Gibco, Cat #11-965-092) supplemented with 10% fetal bovine serum (EMD Millipore, Cat #F2442).

Techniques: Inhibition, Cell Characterization, Western Blot