human breast cancer cell line t47d  (ATCC)

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: METTL14 was associated with the progression of ERα + breast cancer and reduced the sensitivity of breast cancer cells to CDK4/6i therapy. A Representative image of IHC staining for METTL14 in clinical breast cancer samples. Scale bar = 50 μm. B The present study investigated the potential correlation between the expression of METTL14 in clinical samples of patients with breast cancer and overall survival. C METTL14 expression in tumor tissues (n = 80) and normal tissues (n = 80, t test). D The study explored the potential association between METTL14 expression and breast cancer stage (n = 17, 45 and 18 for histopathological grades I, II and III, respectively) in the aforementioned clinical samples (one-way ANOVA). E Correlation between METTL14 expression in clinical breast cancer samples and lymph node metastasis (n = 48 for nonmetastatic samples and n = 32 for metastatic samples) ( t test). F Representative image of IHC staining for METTL14 in samples from patients with ERα + breast cancer with and without relapse after CDK4/6i therapy. G METTL14 expression in samples from patients with ERα + breast cancer with relapse (n = 4) and without relapse (n = 4) after CDK4/6i therapy ( t test). H T47D-Par cells stably overexpressing METTL14 were generated. Changes in METTL14 expression were confirmed using western blotting. NC: cells transfected with a blank control vector. I The proliferative ability of T47D-Par cells stably overexpressing METTL14 was investigated via assessment of EdU incorporation (green), and representative images are shown. Scale bar = 25 μm. Nuclei were stained with DAPI. Breast cancer cells were treated with a CDK4/6i. J The proliferative ability of T47D-Par cells stably overexpressing METTL14 was investigated via a colony formation assay, and the numbers of colonies were determined (t test). Breast cancer cells were treated with a CDK4/6i. K T47D-Par cells stably overexpressing METTL14 were treated with graded concentrations of a CDK4/6i. The IC50 was measured by a cell counting kit-8 (CCK-8) assay after 48 h. L T47D-Res cells with stable METTL14 knockdown were generated. Changes in METTL14 expression were confirmed using western blotting. M The proliferative ability of T47D-Res cells with stable METTL14 knockdown was investigated via an EdU incorporation assay, and representative images are shown. Scale bar = 25 μm. Breast cancer cells were treated with a CDK4/6i. N The proliferative ability of T47D-Res cells with stable METTL14 knockdown was investigated via a colony formation assay, and the numbers of colonies were determined (one-way ANOVA test). Breast cancer cells were treated with a CDK4/6i. O T47D-Res cells with stable METTL14 knockdown were treated with graded concentrations of a CDK4/6i. The IC50 was measured by a cell counting kit-8 (CCK-8) assay after 48 h. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing, Stable Transfection, Generated, Western Blot, Transfection, Control, Plasmid Preparation, Staining, Colony Assay, Cell Counting, CCK-8 Assay, Knockdown

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Studies in ex vivo and in vivo breast cancer models showed that METTL14 promoted CDK4/6i resistance. A – C Biopsy samples from clinical patients who received CDK4/6i therapy were obtained, and breast cancer organoids were generated. Resistant organoids were established by treatment with a CDK4/6i at sequentially increasing concentrations. We subsequently overexpressed METTL14 in parental BC organoids and knocked down METTL14 in CDK4/6i-resistant BC organoids. A Representative images of parental and CDK4/6i-resistant BC organoids. B The percentage growth of parental BC organoids with or without METTL14 overexpression after treatment with a CDK4/6i ( t test). C The percentage growth of CDK4/6i-resistant BC organoids with or without METTL14 knockdown after treatment with a CDK4/6i (one-way ANOVA). D – F T47D-Par cells with or without stable METTL14 overexpression were inoculated into the mammary fat pads of nude mice, and the mice were treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. D Representative images of in vivo bioluminescence in each treatment group after the last treatment. E Representative images of IHC staining for METTL14 in tumor sections from the indicated groups. Scale bar = 25 μm. F Data analysis of in vivo bioluminescence in each treatment group was performed at the indicated time points ( t test). G – I T47D-Res cells with or without stable METTL14 knockdown were inoculated into the mammary fat pads of nude mice, which were subsequently treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Subsequently, the mice were euthanized, and the tumors were excised and weighed. G Representative images of in vivo bioluminescence in each treatment group after the last treatment. H Representative images of IHC staining for METTL14 in tumor sections from the indicated groups. Scale bar = 25 μm. I Data analysis of in vivo bioluminescence in each treatment group was performed at the indicated time points (one-way ANOVA). J – M MMTV-PyMT-METTL14 ± and MMTV-PyMT-METTL14 +/+ mice were treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. J Schematic diagram illustrating the genetic approach used to generate MMTV-PyMT-METTL14 ± mice. K The tumor volume was measured at the indicated time points ( t test). L The appearance of the tumors in the indicated groups. M The weights of the tumors in the indicated groups ( t test). The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Ex Vivo, In Vivo, Generated, Over Expression, Knockdown, Injection, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: METTL14-mediated m6A modification of E2F1 mRNA maintains its IGF2BP2-dependent stability. A A schematic illustration of the RNA-seq and MeRIP-seq results in T47D-Par cells with or without METTL14 overexpression. B KEGG enrichment analysis of RNA-seq data from T47D-Par cells with or without METTL14 overexpression. The “cell cycle”-related gene signature is labeled in black font. C Density distribution of m6A peaks across mRNA transcripts, as determined by MeRIP-seq. D Venn diagram illustrating the overlapping genes between the differentially expressed genes identified through MeRIP-seq and those identified through RNA-seq. E MeRIP-qPCR analysis was used to investigate changes in m6A modification of the E2F1 gene in T47D-Par cells with or without METTL14 overexpression (IgG was used as a negative control, t test). F Changes in E2F1 protein expression in T47D-Par cells with or without METTL14 overexpression were confirmed by western blotting. G Luciferase reporters containing either the wild-type or mutant E2F1 3′UTR were constructed. H The luciferase reporter assay was used to determine the abundances of transcripts generated from the luciferase vectors containing the wild-type and the mutated E2F1 3′-UTR, and the relative ratio of firefly luciferase activity to Renilla luciferase activity was calculated ( t test). I Decay rate of E2F1 mRNA after treatment with actinomycin D (5 g/ml) in T47D-Par cells with or without METTL14 overexpression ( t test). J Real-time PCR analysis of E2F1 expression in T47D-Par cells after individual knockdown of IGF2BP1, IGF2BP2 or IGF2BP3 (one-way ANOVA). K T47D cells were lysed and incubated with magnetic beads coated with IgG or an antibody specific for IGF2BP2. The RNA‒protein complexes were immunoprecipitated, and the relative interaction between E2F1 and IGF2BP2 was quantified via qPCR (with IgG serving as a negative control; t test). L Correlation between IGF2BP2 and E2F1 expression in ERα + breast cancer tissues from the TCGA database (n = 1226). The data are presented as the means ± SDs. ns P > 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Modification, RNA Sequencing Assay, Over Expression, Labeling, Negative Control, Expressing, Western Blot, Luciferase, Mutagenesis, Construct, Reporter Assay, Generated, Activity Assay, Real-time Polymerase Chain Reaction, Knockdown, Incubation, Magnetic Beads, Immunoprecipitation

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: CDK4/6i inhibits SPOP-mediated ubiquitination and degradation of METTL14 in CDK4/6i-resistant breast cancer cells. A This figure shows a schematic depiction of the domains of SPOP that interact with METTL14, as well as the mutations frequently associated with breast cancer. B The left panel shows the molecular docking of SPOP and METTL14, with METTL14 represented in yellow and SPOP represented in various colors. The diagram in the right panel highlights the interaction between SPOP and METTL14, which occurs specifically through the MATH domain encompassing amino acids 80–140. The red area indicates the active region of interaction, while the green area indicates the noninteracting regions. C Analysis of whole-cell lysates (WCLs) and SPOP-interacting products derived from T47D-Res cells was conducted using co-IP. Wild-type SPOP and SPOP proteins with mutations frequently associated with breast cancer are shown (IgG served as a negative control). D A cellular ubiquitination assay was performed on WCLs and His precipitates derived from T47D-Res cells transfected with specific constructs. The resulting reaction products were then subjected to immunoblot (IB) analysis. E In vitro ubiquitination assays were carried out using commercial E1, E2, and Ub proteins as well as purified Flag-METTL14 and different HA-SPOP proteins. The reaction products from these assays were also subjected to IB analysis. F T47D-Res cells were subjected to a 24-h treatment with either CDK4/6i or vehicle. Subsequently, cell lysates were collected for immunoblot analysis, which revealed that CDK4/6i treatment resulted in downregulation of SPOP expression and a concurrent increase in the METTL14 protein level

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Derivative Assay, Co-Immunoprecipitation Assay, Negative Control, Ubiquitin Assay, Transfection, Construct, Western Blot, In Vitro, Purification, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Screening of METTL14 inhibitors. A Molecular docking structure of METTL14. B A CCK-8 assay was used to evaluate the therapeutic effects of 70 candidate compounds combined with a CDK4/6i in T47D-Res cells, and 20 candidate compounds (1 μM, 48 h) that could improve the therapeutic effect of CDK4/6i treatment in T47D-Res cells are shown. Compound #43 was WKYMVM-NH2. C Dot blot assay results showing the effect of the 20 candidate compounds in B on the m6A abundance in CDK4/6i-resistant T47D cells. A1-A7, compounds #04, #06, #08, #09, #13, #16, and #43; B1-B7, compounds #25, #28, #32, #33, #40, #48, and #53; C1-C7, compounds #55, #59, #61, #65, #68, and #70 and the blank control (treated with PBS). D Molecular docking analysis results showing the interaction between METTL14 and WKYMVM. E Molecular docking demonstrated that WKYMVM binds to the METTL3 binding domain in METTL14. F For the binding of WKYMVM, significant METTL14-induced fluorescence changes were observed in the MST assay; thus, the resulting dose‒response curves were fitted to a one-site binding model. G A schematic diagram showing the construction of the METTL14 truncation mutants. The wild type contains the full-length amino acid sequence of METTL14. The deletion type 1 and deletion type 2 constructs were designed for the deletion of aa 245–260 and 245–298, respectively, in METTL14. These three sequences were tagged with GST. H WKYMVM tagged with His was incubated with each of the three forms of METTL14 tagged with GST in Glutathione Agarose Resin, and the eluate was analyzed by dot blot, where the protein samples applied onto a nitrocellulose (NC) membrane, followed by incubation with antibodies, and then carrying out a chemiluminescence reaction. I Immunoprecipitation (IP) with an anti-HA antibody (METTL3) followed by immunoprecipitation with an anti-FLAG antibody (METTL14) and subsequent immunoblotting (IB) showed that METTL3 pulled down less METTL14 after WKYMVM treatment (1 μM, 48 h). The data are presented as the means ± SDs. ns P > 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: CCK-8 Assay, Dot Blot, Control, Binding Assay, Fluorescence, Sequencing, Construct, Incubation, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: The effectiveness of CDK4/6i treatment was augmented by the METTL14 inhibitor WKYMVM in vitro and in vivo. A The present study aimed to investigate the proliferative capacity of T47D-Par cells treated with CDK4/6i, as well as T47D-Par cells overexpressing METTL14 treated with CDK4/6i, CDK4/6i + PBS, and the combination of the CDK4/6i (1 μM, 48 h) and WKYMVM (1 μM, 48 h). This was accomplished through colony formation assays, and the resulting colony numbers were subsequently summarized (one-way ANOVA test). B The proliferative ability of T47D-Par cells with and without stable METTL14 overexpression treated with a CDK4/6i, CDK4/6i + PBS, or the combination of the CDK4/6i (1 μM) and WKYMVM (1 μM) was investigated via a CCK-8 assay at 48 h (one-way ANOVA test). C MeRIP-qPCR analysis of alterations in the m6A modification of E2F1 mRNA in T47D-Res cells with or without WKYMVM treatment (1 μM) ( t test). D Changes in E2F1 mRNA expression in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) were confirmed using qPCR ( t test). E Changes in E2F1 protein expression in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) were confirmed via western blotting. F Decay rate of E2F1 mRNA after treatment with actinomycin D (5 g/ml) in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) ( t test). G The proliferation of T47D-Res cells treated with WKYMVM (1 μM, 48 h) alone, a CDK4/6i (1 μM, 48 h) alone, or the combination of WKYMVM and the CDK4/6i was assessed using an EdU incorporation assay. Representative images of the cells are displayed. PBS was utilized as the control treatment. Scale bar = 25 μm. H – K Mice bearing CDK4/6i-resistant PDXs were treated with control, WKYMVM, a CDK4/6i, or the combination of the CDK4/6i and WKYMVM (CDK4/6i, 90 mg/kg, p.o., one injection per day for 2 weeks; WKYMVM, 50 mg/kg, i.p., one injection every two days for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. H Schematic diagram illustrating the workflow for constructing CDK4/6i-resistant PDX models. I The size of the tumors was measured at the indicated time points (one-way ANOVA). J The weights of tumors from the indicated groups were measured (one-way ANOVA). K The appearance of the tumors in the indicated groups. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Over Expression, CCK-8 Assay, Modification, Expressing, Western Blot, Control, Injection

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Targeted delivery of a CDK4/6i combined with WKYMVM sensitized breast cancer cells to CDK4/6i treatment. A A drug delivery system incorporating a CDK4/6i in combination with WKYMVM was developed to target breast cancer cells. Liposomes were designed to contain both a CDK4/6i and WKYMVM, and folate was conjugated to the liposome membrane surface, allowing the liposomes to target breast cancer cells with high folate receptor expression. The CDK4/6i was located between the phospholipid bilayers, while the peptide WKYMVM was located inside the hydrophilic interior of the liposome. B Transmission electron micrograph showing the microstructure of the liposomes in each group. Lipo-V, liposomes containing vehicle. Lipo-I, liposomes containing only the CDK4/6i. Lipo-W, liposomes containing only WKYMVM. Lipo-C, liposomes containing both the CDK4/6i and WKYMVM. C-D The average hydrodynamic size and average zeta potential of the liposomes in each group. E T47D cells were scanned after coculture with Cy3-labeled liposomes (1 µg or 10 µg) for 12 h. F-actin (green) indicates the cytoskeleton, DAPI (blue) indicates the nucleus, and Cy3 (red) indicates the Cy3-labeled liposome. F T47D cells were scanned after coculture with Cy3-labeled liposomes (1 µg) for 1 h with or without a folate receptor blocking antibody (FR Ab). F-actin (green) indicates the cytoskeleton, DAPI (blue) indicates the nucleus, and Cy3 (red) indicates the Cy3-labeled liposome. G The proliferative ability of T47D-Res cells was investigated via colony formation assays, and the numbers of colonies were summarized (one-way ANOVA). H The proliferation of T47D-Res cells treated with different liposomes was tested by a CCK-8 assay at 48 h (one-way ANOVA). I – L Mice bearing CDK4/6i-resistant PDXs were treated with liposomes. Lipo-V, liposomes containing vehicle. Lipo-I, liposomes containing CDK4/6i alone. Lipo-W, liposomes containing WKYMVM alone. Lipo-C-Liposomes containing both CDK4/6i and WKYMVM (all liposomes, 1 g/kg, i.v., one injection every three days for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. I The timeline of treatment with liposomes and tumor measurements in PDX model mice. J The size of the tumors was measured at the indicated time points (one-way ANOVA). K The weights of tumors from the indicated groups were measured (one-way ANOVA). L Representative images of IHC and HE staining of tumor sections from the indicated groups. Scale bar = 25 μm. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Liposomes, Membrane, Expressing, Transmission Assay, Zeta Potential Analyzer, Labeling, Blocking Assay, CCK-8 Assay, Injection, Staining

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Characterization of ZR-75-1 as an HR + /HER2-low breast cancer cell line. ( A , B ) Heat maps of gene expression in PAM50 cells of different breast cancer subtypes and heat maps of ERBB2 expression in HR + /HER2- breast cancer cells. ( C ) HER2 expression in HR + /HER2-0 and HR + /HER2-low breast cancer subtypes by cellular immunofluorescence assay. ( D ) HER2 levels in HR + /HER2-0 and HR + /HER2-low breast cancer subtypes measured with ImageJ, P = 0.029. ( E ) HER2 expression in HR + /HER2-0 and HR + /HER2-low breast cancer subtypes by fluorescence quantitative PCR assay, P = 0.015, * P < 0.05; n = 3.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Immunofluorescence, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Efficacy of CDK4/6 inhibitor combined with endocrine therapy in the ZR-75-1 cell line in vitro and in vivo. ( A , B ) CCK-8 and colony formation assays were performed to detect the effects of various concentrations of CDK4/6 inhibitor (palbociclib) combined with endocrine therapy (100 nM, fulvestrant) on the proliferation of HR + /HER2-low breast cancer cells. The effect was not as significant as in HR + /HER2-0 breast cancer. For 200 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.003. For 400 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. For 800 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. For 100 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. ( C – E ) CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) combined with endocrine therapy (fulvestrant, 100 mg/kg, i.m.) inhibited tumor growth in HR + /HER2-low breast cancer xenografted mice, but the effect was not as significant as in the HR + /HER2-0 subtype. For HER2-low tumor volume on day 21: normal saline vs. ET + CDK4/6i, P = 0.001. For HER2-0 tumor volume on day 21: normal saline vs. ET + CDK4/6i, P = 0.0001. For tumor inhibition rate on day 21: HER2-low vs. HER2-0, P = 0.0001. ( F ) ER, PR, and HER2 expression levels in tumor tissues of HR + /HER2-0 and HR + /HER2-low breast cancer xenografted mice, measured by immunohistochemistry, the arrow points to the positive area and scale bar = 100 μm. ** P < 0.01; *** P < 0.001; n = 6. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, ER : estrogen receptor, PR progesterone receptor.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, In Vivo, CCK-8 Assay, Saline, Inhibition, Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Role of HER2 in the poor efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR + /HER2-low breast cancer. ( A , B ) Stable knockdown of HER2 in human HR + /HER2-low breast cancer cells by lentiviral shRNAs (shHER2 #1 and shHER2 #2). Knockdown effects were verified by western blot and quantitative PCR. shNC vs. shHER2 #1, P = 0.0009; shNC vs. shHER2 #2, P = 0.0019. ( C , D ) Knockdown of HER2 enhanced the effect of CDK4/6 inhibitor (250 nM, palbociclib) combined with endocrine therapy (100 nM, fulvestrant) in inhibiting HR + /HER2-low breast cancer cell proliferation as examined by the CCK-8 and colony formation experiments assays. For 100 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.004. For 200 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.045. For 400 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.019. For 800 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.001. For 1600 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.016. ( E – H ) In cell cycle assay, western blot, and apoptosis assay, knockdown of HER2 promoted G1 phase arrest and reduced Cyclin D1, CDK4 and EGFR expression. For HER2-low cells G1 phase: shNC vs. shHER2, P = 0.001. ** P < 0.01; *** P < 0.001; n = 3. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Cell Cycle Assay, Apoptosis Assay, Expressing

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR + /HER2-low breast cancer in vitro. ( A , B ) CCK8 and colony formation assays were performed to assess the proliferation of HR + /HER2-low breast cancer cells administered various concentrations of neratinib and CDK4/6 inhibitor, respectively, combined with endocrine therapy. For HER2-low cells: ET + 100 nM CDK4/6i + 125 nM neratinib, CI = 0.34; ET + 200 nM CDK4/6i + 250 nM neratinib, CI = 0.34; ET + 400 nM CDK4/6i + 500 nM neratinib, CI = 0.23; ET + 800 nM CDK4/6i + 1000 nM neratinib, CI = 0.46; ET + 1600 nM CDK4/6i + 2000 nM neratinib, CI = 0.29. ( C ) The cell cycle assay was performed in HR + /HER2-low breast cancer cells administered the triple combination of 125 nM neratinib, CDK4/6 inhibitor (100 nM, palbociclib), and endocrine therapy (100 nM, ET, fulvestrant). For HER2-low cells’ G1 phase: DMSO vs. ET + CDK4/6i, P = 0.024; DMSO vs. ET + CDK4/6i + neratinib, P = 0.0001. ( D ) Cyclin D1, CDK4, HER2, and EGFR protein expression levels in HR + /HER2-0 and HR + /HER2-low breast cancer cells, measured by western blot. * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3. ET : endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner Neratinib.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Cell Cycle Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR + /HER2-low breast cancer in vivo. ( A ) Tumor photographs in animals treated with normal saline (control group), CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) combined with endocrine therapy (fulvestrant, 100 mg/kg, i.m.), and triple therapy consisting of standard dose of neratinib group (40 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy. ( B ) Weight changes in each group of HR + /HER2-low tumor-bearing mice. For HER2-low weight on day 14: normal saline vs. ET + CDK4/6i, P = 0.217; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.0052. ( C ) Tumor growth curves and ( D ) tumor inhibition rates of HR + /HER2-low tumor-bearing mice in various groups. For HER2-low tumor volume on day 14: normal saline vs. ET + CDK4/6i, P = 0.009; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.0003. For HER2-low tumor inhibition rate on day 14: normal saline vs. ET + CDK4/6i, P = 0.0055. ( E ) Tumor HER2 mRNA expression of in each group of HR + /HER2-low tumor-bearing mice. For HER2-low tumor HER2 mRNA expression on day 14: normal saline vs. ET + CDK4/6i, P = 0.381; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.001. ( F ) HER2 expression in mouse HR + /HER2-low breast cancer tissue by IHC staining. ( G ) Cyclin D1, CDK4 and EGFR protein expression levels in HR + /HER2-low breast cancer, measured by western blot. * P < 0.05; ** P < 0.01; *** P < 0.001; n = 5. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Saline, Control, Inhibition, Expressing, Immunohistochemistry, Western Blot

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy by reducing HER2 mRNA stability through the interaction of HER2’s 3’-UTR region with hsa-miR-23a-5p. ( A , B ) After knockdown of HER2, the effect of the triple combination of neratinib, CDK4/6 inhibitor, and endocrine therapy on HR + /HER2-low breast cancer cell proliferation was assessed by the CCK-8 and colony formation assays. ( C , D ) In cell cycle analysis and western blot, neratinib was shown to promote G1 phase arrest through the HER2 pathway, to downregulate Cyclin D1 and CDK4, and to improve the effect of CDK4/6 inhibitor combined with endocrine therapy in HR + /HER2-low breast cancer. ( E ) Effects of CDK4/6 inhibitor + endocrine therapy and triple combination treatment (neratinib, CDK4/6 inhibitor and endocrine therapy) on HER2 mRNA stability. MicroRNA sequencing ( F ) revealed the effect of neratinib on miRNA changes, and compared with CDK4/6 inhibitor + endocrine therapy group, the triple combination treatment group administered neratinib, CDK4/6 inhibitor, and endocrine therapy had significantly upregulated hsa-miR-23a-5p ( G ), which highly interacted with HER2’s 3’-UTR region ( H ), and significantly lowered HER2 mRNA stability ( I ) and downregulated HER2 and EGFR protein expression ( J ). * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, CCK-8 Assay, Cell Cycle Assay, Western Blot, Sequencing, Expressing

Journal: Scientific Reports

Article Title: Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR + /HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

doi: 10.1038/s41598-024-82137-9

Figure Lengend Snippet: Optimization of neratinib dosing for enhanced CDK4/6 inhibitor combined with endocrine therapy efficacy. ( A ) Tumor photographs in animals treated with normal saline (control group), CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) + endocrine therapy (fulvestrant, 100 mg/kg, i.m.), medium-dose neratinib (20 mg/kg) alone, a triple therapy consisting of medium-dose neratinib group (20 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy, and a triple therapy consisting of low-dose neratinib (10 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy. ( C ) Weight changes in various groups of HR + /HER2-low tumor-bearing mice. ( B ) Tumor inhibition rates and ( D ) tumor growth curves of HR + /HER2-low tumor-bearing mice in various groups. For HER2-low tumor volume on day 21: normal saline vs. medium-dose ET + CDK4/6i + Ner, P = 0.0001; normal saline vs. low-dose ET + CDK4/6i + Ner, P = 0.0017; normal saline vs. ET + CDK4/6i, P = 0.003; ET + CDK4/6i vs. medium-dose ET + CDK4/6i + Ner, P = 0.0021. For HER2-low tumor inhibition rate on day 21: normal saline vs. medium-dose ET + CDK4/6i + Ner, P = 0.0217; normal saline vs. low-dose ET + CDK4/6i + Ner, P = 0.1271. ** P < 0.01; *** P < 0.001; n = 5. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.

Article Snippet: HR + /HER2 + breast cancer BT474, HR + /HER2-low breast cancer ZR-75-1, and HR + /HER2-0 breast cancer T47D cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Saline, Control, Inhibition

Journal: Journal of Proteome Research

Article Title: Use and Comparison of Machine Learning Techniques to Discern the Protein Patterns of Autoantibodies Present in Women with and without Breast Pathology

doi: 10.1021/acs.jproteome.4c00759

Figure Lengend Snippet: Western Blot image. Image containing the nitrocellulose membrane strips with the expression of bands obtained from the Western Blot of the total protein lysate of the T47D cell line (ductal carcinoma of the breast). The blue numbers at the top of the strips are the patient identifiers. The red number at the top of the strips is the control patient. The gray numbers at the bottom of the strips indicate the sequence in which the strips were arranged to form the image. The strip on the left is the molecular weight marker. The blue bands represent molecular weights from 10 kDa to 250 kDa, pink from 25 kDa to 75 kDa, green from 37 kDa.

Article Snippet: The breast cancer cell line T47D (ATCC; Manassas, VA, USA) was cultured in phenol-free RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mU/ml streptomycin, and 250 ng/mL amphotericin B in plastic culture plates (Costar, Cambridge, UK) under atmospheric conditions of 95% humidity and 5% CO2 at 37 °C.

Techniques: Western Blot, Membrane, Expressing, Control, Sequencing, Stripping Membranes, Molecular Weight, Marker

Journal: Marine Drugs

Article Title: Composition of Triterpene Glycosides of the Far Eastern Sea Cucumber Cucumaria conicospermium Levin et Stepanov; Structure Elucidation of Five Minor Conicospermiumosides A 3 -1, A 3 -2, A 3 -3, A 7 -1, and A 7 -2; Cytotoxicity of the Glycosides Against Human Breast Cancer Cell Lines; Structure–Activity Relationships

doi: 10.3390/md22120560

Figure Lengend Snippet: The cytotoxic activities of glycosides of C. conicospermium against human erythrocytes (okhotoside A 1 -1 ( 7 ) is positive control) and the MCF-10A, MCF-7, T-47D, and MDA-MB-231 human cell lines (cisplatin is positive control).

Article Snippet: Human mammary epithelial cell line MCF-10A CRL-10317, human breast cancer cell lines T-47D HTB-133, MCF-7 HTB-22, and MDA-MB-231 CRM-HTB-26 were received from ATCC (Manassas, VA, USA).

Techniques: Positive Control, Control

Journal: Pharmaceuticals

Article Title: Ganoderma lucidum (Curtis) P. Karst. Immunomodulatory Protein Has the Potential to Improve the Prognosis of Breast Cancer Through the Regulation of Key Prognosis-Related Genes

doi: 10.3390/ph17121695

Figure Lengend Snippet: RNA levels of risk genes SRGAP2, SLC35A2, FAM114A1, TP53I11, TMEM63C, and PIGR regulated by FIP-glu in breast cancer T-47D ( A ) and MCF-7 ( B ) cells.

Article Snippet: Breast cancer T-47D and MCF-7 cells were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques:

Journal: Pharmaceuticals

Article Title: Ganoderma lucidum (Curtis) P. Karst. Immunomodulatory Protein Has the Potential to Improve the Prognosis of Breast Cancer Through the Regulation of Key Prognosis-Related Genes

doi: 10.3390/ph17121695

Figure Lengend Snippet: Effect of TMEM63C overexpression on cell growth and proliferation. ( A ) CCK-8 assay revealed the enhanced viability by TMEM63C overexpression in both breast cancer T-47D and MCF-7 cells. Data are presented as mean ± SD ( n = 6). ( B ) Clone formation assay showed the increase of clone formation ability by TMEM63C overexpression in both breast cancer T-47D and MCF-7 cells. Data are presented as mean ± SD ( n = 3).

Article Snippet: Breast cancer T-47D and MCF-7 cells were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Over Expression, CCK-8 Assay, Tube Formation Assay

Journal: Pharmaceuticals

Article Title: Ganoderma lucidum (Curtis) P. Karst. Immunomodulatory Protein Has the Potential to Improve the Prognosis of Breast Cancer Through the Regulation of Key Prognosis-Related Genes

doi: 10.3390/ph17121695

Figure Lengend Snippet: FIP-glu alleviated TMEM63C-mediated pro-proliferative effects. ( A ) CCK-8 assay revealed that FIP-glu inhibited the TMEM63C-induced enhanced viability in T-47D and MCF-7 cells. Data are presented as mean ± SD ( n = 6). ( B ) Clone formation assay revealed that FIP-glu inhibited the TMEM63C-induced increase of clone formation ability in T-47D and MCF-7 cells. Data are presented as mean ± SD ( n = 3).

Article Snippet: Breast cancer T-47D and MCF-7 cells were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: CCK-8 Assay, Tube Formation Assay

Journal: Biomedicines

Article Title: Obesity-Related Inflammation Reduces Treatment Sensitivity and Promotes Aggressiveness in Luminal Breast Cancer Modulating Oxidative Stress and Mitochondria

doi: 10.3390/biomedicines12122813

Figure Lengend Snippet: Obesity-related inflammation induces aggressiveness in luminal breast cancer. Mammospheres of T47D, MCF7, and MDA-MB-231 cell lines obtained after exposure to vehicle (CTRL) or ELIT cocktail ( A ). Mammosphere forming efficiency (MFE) of T47D, MCF7, and MDA-MB-231 cell lines under CTRL or ELIT conditions ( B ). mRNA expression levels of CDH1 in mammospheres in CTRL or ELIT condition ( C ). Data are presented as mean ± SEM. Statistical significance was analyzed by Student’s t -test and set at * p ≤ 0.05. Enrichment analysis of Gene Sets related to aggressiveness in obese vs. lean luminal breast cancer patients from GSE189757 database ( D , E ). BMI: Body Mass Index; GSEA: Gene Set Enrichment Analysis.

Article Snippet: Human breast cancer cell lines T47D and MCF7 (luminal) and MDA-MB-231 (triple-negative) were obtained from American Type Culture Collection ATCC (Manassas, VA, USA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Obesity-Related Inflammation Reduces Treatment Sensitivity and Promotes Aggressiveness in Luminal Breast Cancer Modulating Oxidative Stress and Mitochondria

doi: 10.3390/biomedicines12122813

Figure Lengend Snippet: ELIT exposure modifies antioxidant, mitochondrial markers in luminal breast cancer mammospheres with high ESR2 mRNA expression. mRNA expression levels of oxidative stress-related genes ( A ), mitochondrial markers ( B ), and ESR1 and ESR2 ( C ) in T47D and MCF7 mammospheres under CTRL or ELIT condition. Data are presented as mean ± SEM. Statistical significance was analyzed by Student’s t -test and set at * p ≤ 0.05 and # p ≤ 0.1. UD: Undetected values.

Article Snippet: Human breast cancer cell lines T47D and MCF7 (luminal) and MDA-MB-231 (triple-negative) were obtained from American Type Culture Collection ATCC (Manassas, VA, USA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Obesity-Related Inflammation Reduces Treatment Sensitivity and Promotes Aggressiveness in Luminal Breast Cancer Modulating Oxidative Stress and Mitochondria

doi: 10.3390/biomedicines12122813

Figure Lengend Snippet: ELIT exposure decreases drug sensitivity of T47D 3D-derived cells. Experimental design for T47D 3D-derived cells analysis ( A ). Cell viability ( B , C ) and H 2 O 2 production ( D , E ) in T47D 3D-derived cells under CTRL or ELIT condition after treatment with Tamoxifen or Paclitaxel, respectively. Enrichment analysis of Gene Sets related to drug response in obese compared to lean luminal breast cancer patients from GSE189757 database ( F ). ANOVA analysis was carried out, where E means ELIT effect; T means tamoxifen effect; P means Paclitaxel effect; and E*T or E*P mean interactive effect of ELIT with Tamoxifen or Paclitaxel, respectively. Data are presented as mean ± SEM. * Significant difference between cells treated with Tamoxifen or Paclitaxel and untreated cells ( p ≤ 0.05). $ Significant differences between CTRL and ELIT-exposed cells ( p ≤ 0.05).

Article Snippet: Human breast cancer cell lines T47D and MCF7 (luminal) and MDA-MB-231 (triple-negative) were obtained from American Type Culture Collection ATCC (Manassas, VA, USA).

Techniques: Derivative Assay