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ATCC human bt549

Piperlongumine (PL) exerts inhibitory effects on the growth of triple-negative breast cancer (TNBC) cells. (A) The chemical structure of PL. (B) MDA-MB-231 and <t>BT549</t> TNBC cells were treated with various concentrations of PL (0, 1.25, 2.5, 5, 10, and 25 μ M) for 24, 48, and 72 h, and cell viability was measured by CCK8 assay. Data are presented as percentages relative to the untreated group, with values expressed as the mean ± standard deviation (SD) of three independent experiments. IC50 values were calculated. (C) Representative images of EdU-stained proliferative MDA-MB-231 and BT549 cells treated with PL for 24 h. Scale bar, 100 μ m. (D) Apoptosis of MDA-MB-231 and BT549 cells treated with PL for 24 h was detected by flow cytometry. (E) Western blot analysis of BAX and BCL2 expression (left) and cleaved-caspase 3 and cleaved-caspase 9 expression (right) in MDA-MB-231 and BT549 cells treated with PL for 24 h. Quantitative analysis was performed using ImageJ software. * p < 0.05, ** p < 0.01, and *** p < 0.001, as determined by unpaired t -tests.

Human Bt549, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human bt549 - by Bioz Stars, 2026-09

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1) Product Images from "Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells"

Article Title: Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells

Journal: Translational Oncology

doi: 10.1016/j.tranon.2026.102700

Figure Legend Snippet: Piperlongumine (PL) exerts inhibitory effects on the growth of triple-negative breast cancer (TNBC) cells. (A) The chemical structure of PL. (B) MDA-MB-231 and BT549 TNBC cells were treated with various concentrations of PL (0, 1.25, 2.5, 5, 10, and 25 μ M) for 24, 48, and 72 h, and cell viability was measured by CCK8 assay. Data are presented as percentages relative to the untreated group, with values expressed as the mean ± standard deviation (SD) of three independent experiments. IC50 values were calculated. (C) Representative images of EdU-stained proliferative MDA-MB-231 and BT549 cells treated with PL for 24 h. Scale bar, 100 μ m. (D) Apoptosis of MDA-MB-231 and BT549 cells treated with PL for 24 h was detected by flow cytometry. (E) Western blot analysis of BAX and BCL2 expression (left) and cleaved-caspase 3 and cleaved-caspase 9 expression (right) in MDA-MB-231 and BT549 cells treated with PL for 24 h. Quantitative analysis was performed using ImageJ software. * p < 0.05, ** p < 0.01, and *** p < 0.001, as determined by unpaired t -tests.

Techniques Used: CCK-8 Assay, Standard Deviation, Staining, Flow Cytometry, Western Blot, Expressing, Software

Piperlongumine (PL) exerts inhibitory effects on the invasiveness of triple-negative breast cancer (TNBC) cells. (A) Transwell migration and invasion assays of BT549, MDA-MB-231, and 4T1 TNBC cells treated with PL (0, 1, and 5 μ M) for 24 h, with representative images (left) and quantitative analysis (right). Scale bar, 50 μ m. (B) Wound-healing assay of TNBC cells treated with PL for 24 h, with quantification of wound closure by ImageJ shown on the right. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired t -test. Scale bar, 100 μ m. (C) Immunofluorescence staining of actin filaments in TNBC cells treated with PL (1 μ M) for 24 h using Alexa Fluor 488-conjugated phalloidin. Scale bar, 50 μ m. (D) Western blot analysis of epithelial–mesenchymal transition (EMT) marker expression in BT549 and MDA-MB-231 cells treated with PL for 24 h.

Figure Legend Snippet: Piperlongumine (PL) exerts inhibitory effects on the invasiveness of triple-negative breast cancer (TNBC) cells. (A) Transwell migration and invasion assays of BT549, MDA-MB-231, and 4T1 TNBC cells treated with PL (0, 1, and 5 μ M) for 24 h, with representative images (left) and quantitative analysis (right). Scale bar, 50 μ m. (B) Wound-healing assay of TNBC cells treated with PL for 24 h, with quantification of wound closure by ImageJ shown on the right. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired t -test. Scale bar, 100 μ m. (C) Immunofluorescence staining of actin filaments in TNBC cells treated with PL (1 μ M) for 24 h using Alexa Fluor 488-conjugated phalloidin. Scale bar, 50 μ m. (D) Western blot analysis of epithelial–mesenchymal transition (EMT) marker expression in BT549 and MDA-MB-231 cells treated with PL for 24 h.

Techniques Used: Migration, Wound Healing Assay, Immunofluorescence, Staining, Western Blot, Marker, Expressing

Piperlongumine (PL) inhibits YAP signaling in triple-negative breast cancer (TNBC) cells. (A) Western blot analysis of YAP protein levels in MDA-MB-231 and BT549 cells treated with PL (0, 1, and 5 μ M) for 24 h. (B) Western blot analysis of YAP and phosphorylated YAP (p-YAP) expression treated with PL in different time points. (C) Western blot analysis of phosphorylated LATS (p-LATS) and phosphorylated AMPK (p-AMPK) at different time points after PL treatment. (D) Representative immunofluorescence images of YAP expression in BT549 and MDA-MB-231 cells treated with PL for 24 h at the indicated concentrations, with quantification of nuclear YAP-positive cells. Scale bar, 50 μ m. * p < 0.05 and ** p < 0.01, as determined by unpaired t -tests.

Figure Legend Snippet: Piperlongumine (PL) inhibits YAP signaling in triple-negative breast cancer (TNBC) cells. (A) Western blot analysis of YAP protein levels in MDA-MB-231 and BT549 cells treated with PL (0, 1, and 5 μ M) for 24 h. (B) Western blot analysis of YAP and phosphorylated YAP (p-YAP) expression treated with PL in different time points. (C) Western blot analysis of phosphorylated LATS (p-LATS) and phosphorylated AMPK (p-AMPK) at different time points after PL treatment. (D) Representative immunofluorescence images of YAP expression in BT549 and MDA-MB-231 cells treated with PL for 24 h at the indicated concentrations, with quantification of nuclear YAP-positive cells. Scale bar, 50 μ m. * p < 0.05 and ** p < 0.01, as determined by unpaired t -tests.

Techniques Used: Western Blot, Expressing, Immunofluorescence

ET-1 is a downstream of YAP and is suppressed by PL. (A) Real-time PCR analysis and Western blotting analysis of ET-1 and YAP expression levels in YAP-knockdown BT549 and MDA-MB-231 cells. (B) qPCR analysis of ET-1 mRNA levels in MDA-MB-231 and BT549 cells treated with PL (5 μ M) for 24 h. (C) Western blotting analysis of YAP, p-YAP (S61), and ET-1 protein levels in BT549 and MDA-MB-231 cells treated with increasing concentrations of PL for 24 h. (D) Representative immunofluorescence images of ET-1 expression in TNBC cells treated with PL (5 μ M) for 24 h. *** p < 0.001 as determined by unpaired t -tests. Scale bar, 50 μ m.

Figure Legend Snippet: ET-1 is a downstream of YAP and is suppressed by PL. (A) Real-time PCR analysis and Western blotting analysis of ET-1 and YAP expression levels in YAP-knockdown BT549 and MDA-MB-231 cells. (B) qPCR analysis of ET-1 mRNA levels in MDA-MB-231 and BT549 cells treated with PL (5 μ M) for 24 h. (C) Western blotting analysis of YAP, p-YAP (S61), and ET-1 protein levels in BT549 and MDA-MB-231 cells treated with increasing concentrations of PL for 24 h. (D) Representative immunofluorescence images of ET-1 expression in TNBC cells treated with PL (5 μ M) for 24 h. *** p < 0.001 as determined by unpaired t -tests. Scale bar, 50 μ m.

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Knockdown, Immunofluorescence

Image Search Results

Journal: Translational Oncology

Article Title: Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells

doi: 10.1016/j.tranon.2026.102700

Figure Lengend Snippet: Piperlongumine (PL) exerts inhibitory effects on the growth of triple-negative breast cancer (TNBC) cells. (A) The chemical structure of PL. (B) MDA-MB-231 and BT549 TNBC cells were treated with various concentrations of PL (0, 1.25, 2.5, 5, 10, and 25 μ M) for 24, 48, and 72 h, and cell viability was measured by CCK8 assay. Data are presented as percentages relative to the untreated group, with values expressed as the mean ± standard deviation (SD) of three independent experiments. IC50 values were calculated. (C) Representative images of EdU-stained proliferative MDA-MB-231 and BT549 cells treated with PL for 24 h. Scale bar, 100 μ m. (D) Apoptosis of MDA-MB-231 and BT549 cells treated with PL for 24 h was detected by flow cytometry. (E) Western blot analysis of BAX and BCL2 expression (left) and cleaved-caspase 3 and cleaved-caspase 9 expression (right) in MDA-MB-231 and BT549 cells treated with PL for 24 h. Quantitative analysis was performed using ImageJ software. * p < 0.05, ** p < 0.01, and *** p < 0.001, as determined by unpaired t -tests.

Article Snippet: The human BT549 (RRID: CVCL_1092) and MDA-MB-231 (RRID: CVCL_0062) TNBC cell lines were purchased from American Type Culture Collection.

Techniques: CCK-8 Assay, Standard Deviation, Staining, Flow Cytometry, Western Blot, Expressing, Software

Journal: Translational Oncology

Article Title: Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells

doi: 10.1016/j.tranon.2026.102700

Figure Lengend Snippet: Piperlongumine (PL) exerts inhibitory effects on the invasiveness of triple-negative breast cancer (TNBC) cells. (A) Transwell migration and invasion assays of BT549, MDA-MB-231, and 4T1 TNBC cells treated with PL (0, 1, and 5 μ M) for 24 h, with representative images (left) and quantitative analysis (right). Scale bar, 50 μ m. (B) Wound-healing assay of TNBC cells treated with PL for 24 h, with quantification of wound closure by ImageJ shown on the right. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired t -test. Scale bar, 100 μ m. (C) Immunofluorescence staining of actin filaments in TNBC cells treated with PL (1 μ M) for 24 h using Alexa Fluor 488-conjugated phalloidin. Scale bar, 50 μ m. (D) Western blot analysis of epithelial–mesenchymal transition (EMT) marker expression in BT549 and MDA-MB-231 cells treated with PL for 24 h.

Article Snippet: The human BT549 (RRID: CVCL_1092) and MDA-MB-231 (RRID: CVCL_0062) TNBC cell lines were purchased from American Type Culture Collection.

Techniques: Migration, Wound Healing Assay, Immunofluorescence, Staining, Western Blot, Marker, Expressing

Journal: Translational Oncology

Article Title: Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells

doi: 10.1016/j.tranon.2026.102700

Figure Lengend Snippet: Piperlongumine (PL) inhibits YAP signaling in triple-negative breast cancer (TNBC) cells. (A) Western blot analysis of YAP protein levels in MDA-MB-231 and BT549 cells treated with PL (0, 1, and 5 μ M) for 24 h. (B) Western blot analysis of YAP and phosphorylated YAP (p-YAP) expression treated with PL in different time points. (C) Western blot analysis of phosphorylated LATS (p-LATS) and phosphorylated AMPK (p-AMPK) at different time points after PL treatment. (D) Representative immunofluorescence images of YAP expression in BT549 and MDA-MB-231 cells treated with PL for 24 h at the indicated concentrations, with quantification of nuclear YAP-positive cells. Scale bar, 50 μ m. * p < 0.05 and ** p < 0.01, as determined by unpaired t -tests.

Article Snippet: The human BT549 (RRID: CVCL_1092) and MDA-MB-231 (RRID: CVCL_0062) TNBC cell lines were purchased from American Type Culture Collection.

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Translational Oncology

Article Title: Piperlongumine suppresses YAP-ET-1-CXCL2 signaling to modulates aggressiveness of triple-negative breast cancer cells

doi: 10.1016/j.tranon.2026.102700

Figure Lengend Snippet: ET-1 is a downstream of YAP and is suppressed by PL. (A) Real-time PCR analysis and Western blotting analysis of ET-1 and YAP expression levels in YAP-knockdown BT549 and MDA-MB-231 cells. (B) qPCR analysis of ET-1 mRNA levels in MDA-MB-231 and BT549 cells treated with PL (5 μ M) for 24 h. (C) Western blotting analysis of YAP, p-YAP (S61), and ET-1 protein levels in BT549 and MDA-MB-231 cells treated with increasing concentrations of PL for 24 h. (D) Representative immunofluorescence images of ET-1 expression in TNBC cells treated with PL (5 μ M) for 24 h. *** p < 0.001 as determined by unpaired t -tests. Scale bar, 50 μ m.

Article Snippet: The human BT549 (RRID: CVCL_1092) and MDA-MB-231 (RRID: CVCL_0062) TNBC cell lines were purchased from American Type Culture Collection.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Knockdown, Immunofluorescence

A After the genes of the deubiquitinase library were knocked out in BT549 cells, the expression level of YAP downstream target gene CTGF was detected by real-time PCR. B After the gene of the deubiquitinating enzyme library was knocked down in BT549 cells, luciferase reporter gene assay was used to detect the transcriptional activity of the transcription factor TEAD4. C , D Immunohistochemical analysis of USP8 expression in triple-negative and normal breast cancer tissue specimens. Statistical analysis of USP8 expression in 60 cases of normal breast tissue and 63 breast cancer samples. Scale bars, 100 μM (10X), 400 μM (40X). E Kaplan–Meier survival analysis of recurrence-free survival in triple-negative breast cancer patients with high and low expression of USP8 in KMPLOT database. F Analysis of the TCGA database using GSEA revealed a positive link between USP8 expression and YAP-targeted genes. G Enrichment analysis of gene sets from RNA-Seq data of siControl or siUSP8 in TNBC cell lines. H The volcano plot showed the changes in the expression of downstream target genes of the Hippo signaling pathway after treatment of TNBC cell lines with siControl or siUSP8. I Heatmap analysis showed the correlation between USP8 and YAP downstream target genes in RNA-Seq data. J , K Immunohistochemistry (IHC) staining of USP8 and YAP expression in TNBC specimens. Evaluation of statistical data on the association between USP8 and YAP expression in 102 breast cancer specimens. Scale bars, 100 μM (10X), 400 μM (40X). Unpaired two-tailed Student’s t tests were used to determine all P-values. * P < 0.05; ** P < 0.01; *** P < 0.001 according to Student’s t test.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A After the genes of the deubiquitinase library were knocked out in BT549 cells, the expression level of YAP downstream target gene CTGF was detected by real-time PCR. B After the gene of the deubiquitinating enzyme library was knocked down in BT549 cells, luciferase reporter gene assay was used to detect the transcriptional activity of the transcription factor TEAD4. C , D Immunohistochemical analysis of USP8 expression in triple-negative and normal breast cancer tissue specimens. Statistical analysis of USP8 expression in 60 cases of normal breast tissue and 63 breast cancer samples. Scale bars, 100 μM (10X), 400 μM (40X). E Kaplan–Meier survival analysis of recurrence-free survival in triple-negative breast cancer patients with high and low expression of USP8 in KMPLOT database. F Analysis of the TCGA database using GSEA revealed a positive link between USP8 expression and YAP-targeted genes. G Enrichment analysis of gene sets from RNA-Seq data of siControl or siUSP8 in TNBC cell lines. H The volcano plot showed the changes in the expression of downstream target genes of the Hippo signaling pathway after treatment of TNBC cell lines with siControl or siUSP8. I Heatmap analysis showed the correlation between USP8 and YAP downstream target genes in RNA-Seq data. J , K Immunohistochemistry (IHC) staining of USP8 and YAP expression in TNBC specimens. Evaluation of statistical data on the association between USP8 and YAP expression in 102 breast cancer specimens. Scale bars, 100 μM (10X), 400 μM (40X). Unpaired two-tailed Student’s t tests were used to determine all P-values. * P < 0.05; ** P < 0.01; *** P < 0.001 according to Student’s t test.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Activity Assay, Immunohistochemical staining, RNA Sequencing, Immunohistochemistry, Two Tailed Test

A Western blot showed the expression level of USP8 protein in BT549 or MDA-MB-231 cell lines transfected with siControl or two independent siUSP8 for 48 h. For internal control purposes, β-actin was employed. B RT-qPCR results of USP8 mRNA expression levels in BT549 or MDA-MB-231 cells transfected with siControl or siUSP8 for 48 h. C CCK8 assay was employed to determine the viability of BT549 or MDA-MB-231 cells received treatment with siControl or siUSP8 for 36 h at the indicated time point. D , E Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell migration. F , G The Transwell assay was employed to assess the migration and invasion capabilities of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell migration and invasion. H , I EdU assay was used to determine the cell proliferation ability of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell proliferation. J , K Flow cytometry was used to determine the apoptosis level of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The quantitative outcomes of apoptosis are displayed in the right panel. L – N Four-week-old BALB/c female nude mice were subcutaneously injected with BT549 cells that stably expressed shControl and shUSP8. After 35 days, the mice were sacrificed, and the xenograft tumors were extracted. The images of tumors ( L ), their corresponding weight ( M ), and volume ( N ) are presented. O , P IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). The results are displayed as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A Western blot showed the expression level of USP8 protein in BT549 or MDA-MB-231 cell lines transfected with siControl or two independent siUSP8 for 48 h. For internal control purposes, β-actin was employed. B RT-qPCR results of USP8 mRNA expression levels in BT549 or MDA-MB-231 cells transfected with siControl or siUSP8 for 48 h. C CCK8 assay was employed to determine the viability of BT549 or MDA-MB-231 cells received treatment with siControl or siUSP8 for 36 h at the indicated time point. D , E Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell migration. F , G The Transwell assay was employed to assess the migration and invasion capabilities of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell migration and invasion. H , I EdU assay was used to determine the cell proliferation ability of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The right panel shows the quantitative results of cell proliferation. J , K Flow cytometry was used to determine the apoptosis level of BT549 or MDA-MB-231 cell lines treated with siControl or siUSP8 for 36 h. The quantitative outcomes of apoptosis are displayed in the right panel. L – N Four-week-old BALB/c female nude mice were subcutaneously injected with BT549 cells that stably expressed shControl and shUSP8. After 35 days, the mice were sacrificed, and the xenograft tumors were extracted. The images of tumors ( L ), their corresponding weight ( M ), and volume ( N ) are presented. O , P IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). The results are displayed as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Transfection, Control, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, EdU Assay, Flow Cytometry, Injection, Stable Transfection, Immunohistochemistry

A Western blot showed the expression level of USP8 protein in BT549 or MDA-MB-231 cell lines treated with DMSO or different concentrations of DUBs-IN-2 for 8 h. To ensure internal control, β-actin was utilized. B RT-qPCR results of USP8 mRNA expression levels in BT549 or MDA-MB-231 cell lines treated with DMSO or different concentrations of DUBs-IN-2 for 8 h. C To evaluate cell viability, the CCK8 assay was conducted on BT549 or MDA-MB-231 cell lines exposed to DMSO or DUB-IN-2 for 12 h at the designated time. D , E Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell migration. F , G Transwell assay was utilized to measure the migration and invasion proficiency of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell migration and invasion. H , I EdU assay was conducted to examine the proliferation of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell proliferation. J , K Flow cytometry was used to determine the apoptosis level of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of apoptosis. L – N BT549 cells were subcutaneously inoculated into 4-week-old BALB/c female nude mice treated with DMSO or DUB-IN-2 (1 mg/kg). Mice were euthanized 35 days post-injection, and xenograft tumors were excised. Displayed are representative tumor images ( L ), tumor weight ( M ), and tumor volume ( N ). O , P IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts treated with DUB-IN-2. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). All Data are shown as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A Western blot showed the expression level of USP8 protein in BT549 or MDA-MB-231 cell lines treated with DMSO or different concentrations of DUBs-IN-2 for 8 h. To ensure internal control, β-actin was utilized. B RT-qPCR results of USP8 mRNA expression levels in BT549 or MDA-MB-231 cell lines treated with DMSO or different concentrations of DUBs-IN-2 for 8 h. C To evaluate cell viability, the CCK8 assay was conducted on BT549 or MDA-MB-231 cell lines exposed to DMSO or DUB-IN-2 for 12 h at the designated time. D , E Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell migration. F , G Transwell assay was utilized to measure the migration and invasion proficiency of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell migration and invasion. H , I EdU assay was conducted to examine the proliferation of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of cell proliferation. J , K Flow cytometry was used to determine the apoptosis level of BT549 or MDA-MB-231 cell lines treated with DMSO or DUB-IN-2 for 12 h. The right panel shows the quantitative results of apoptosis. L – N BT549 cells were subcutaneously inoculated into 4-week-old BALB/c female nude mice treated with DMSO or DUB-IN-2 (1 mg/kg). Mice were euthanized 35 days post-injection, and xenograft tumors were excised. Displayed are representative tumor images ( L ), tumor weight ( M ), and tumor volume ( N ). O , P IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts treated with DUB-IN-2. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). All Data are shown as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, EdU Assay, Flow Cytometry, Injection, Immunohistochemistry

A , B Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with siControl or siUSP8. As an internal standard, β-actin was utilized. C – F RT-qPCR showed the mRNA expression levels of YAP and downstream target genes CTGF and CYR61 in BT549 or MDA-MB-231 cells treated with siControl or two independent siRNAs. G , H Luciferase reporter assay of TEAD transcriptional activity in BT549 or MDA-MB-231cells treated with siControl or siUSP8. I , J Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of DUB-IN-2. β-actin functioned as the internal control. K – N RT-qPCR showed the mRNA expression levels of YAP and downstream target genes CTGF and CYR61 in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of DUB-IN-2. O , P Luciferase reporter assay of TEAD transcriptional activity in BT549 or MDA-MB-231cells treated with DMSO or different concentrations of DUB-IN-2. All Data are shown as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A , B Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with siControl or siUSP8. As an internal standard, β-actin was utilized. C – F RT-qPCR showed the mRNA expression levels of YAP and downstream target genes CTGF and CYR61 in BT549 or MDA-MB-231 cells treated with siControl or two independent siRNAs. G , H Luciferase reporter assay of TEAD transcriptional activity in BT549 or MDA-MB-231cells treated with siControl or siUSP8. I , J Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of DUB-IN-2. β-actin functioned as the internal control. K – N RT-qPCR showed the mRNA expression levels of YAP and downstream target genes CTGF and CYR61 in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of DUB-IN-2. O , P Luciferase reporter assay of TEAD transcriptional activity in BT549 or MDA-MB-231cells treated with DMSO or different concentrations of DUB-IN-2. All Data are shown as mean ± SD, N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Control

A Western blot showed that the expression levels of USP8 and YAP proteins in BT549 cells transfected with Myc or Myc-YAP plasmids after treatment with siUSP8. The internal control in the experiment was β-actin. B RT-qPCR showed the mRNA expression levels of CTGF and CYR61 in BT549 cells transfected with Myc or Myc-YAP plasmid after treatment with siUSP8. C Luciferase reporter assay of the transcriptional activity of TEAD transfected with Myc or Myc-YAP plasmid after BT549 cells were treated with siUSP8. D The CCK8 assay determined the viability of BT549 cells transfected with Myc or Myc-YAP after siUSP8 treatment at a specific time. E The proliferation ability of BT549 cells transfected with Myc or Myc-YAP plasmid after siUSP8 treatment was assessed by EdU. The right panel shows the quantitative results of cell proliferation. F The migration ability of BT549 cells transfected with Myc or Myc-YAP plasmid after siUSP8 treatment was detected using a wound healing assay. The quantitative findings of cell proliferation are presented in the right panel. G The Transwell assay was employed to assess the migration and invasion capabilities of BT549 cells transfected with Myc or Myc-YAP plasmid following siUSP8 treatment. The right panel shows the quantitative results of cell migration and invasion. H – J BT549 cells stably expressing shControl and shUSP8 or YAP were subcutaneously inoculated into 4-week-old BALB/c female nude mice. Mice were euthanized 35 days post-injection, and the xenograft tumors were excised. Images of representative tumors ( H ), along with their weight ( I ) and volume ( J ), are displayed. K , L IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). Data are presented as mean ± SD, with N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A Western blot showed that the expression levels of USP8 and YAP proteins in BT549 cells transfected with Myc or Myc-YAP plasmids after treatment with siUSP8. The internal control in the experiment was β-actin. B RT-qPCR showed the mRNA expression levels of CTGF and CYR61 in BT549 cells transfected with Myc or Myc-YAP plasmid after treatment with siUSP8. C Luciferase reporter assay of the transcriptional activity of TEAD transfected with Myc or Myc-YAP plasmid after BT549 cells were treated with siUSP8. D The CCK8 assay determined the viability of BT549 cells transfected with Myc or Myc-YAP after siUSP8 treatment at a specific time. E The proliferation ability of BT549 cells transfected with Myc or Myc-YAP plasmid after siUSP8 treatment was assessed by EdU. The right panel shows the quantitative results of cell proliferation. F The migration ability of BT549 cells transfected with Myc or Myc-YAP plasmid after siUSP8 treatment was detected using a wound healing assay. The quantitative findings of cell proliferation are presented in the right panel. G The Transwell assay was employed to assess the migration and invasion capabilities of BT549 cells transfected with Myc or Myc-YAP plasmid following siUSP8 treatment. The right panel shows the quantitative results of cell migration and invasion. H – J BT549 cells stably expressing shControl and shUSP8 or YAP were subcutaneously inoculated into 4-week-old BALB/c female nude mice. Mice were euthanized 35 days post-injection, and the xenograft tumors were excised. Images of representative tumors ( H ), along with their weight ( I ) and volume ( J ), are displayed. K , L IHC staining shows the expression levels of USP8, YAP, and Ki67 in xenografts. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). Data are presented as mean ± SD, with N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Stable Transfection, Injection, Immunohistochemistry

A Immunofluorescence staining showed the localization of USP8 (green) and YAP (red) in BT549 cells. DAPI (blue) was used to stain the nuclei, and the scale bars indicate 10 μm. B An endogenous association between USP8 and YAP was demonstrated through immunoprecipitation analysis. To investigate endogenous interaction between USP8 and YAP, BT549 cell lysates were precipitated using anti-YAP or anti-USP8 antibodies, and the resulting precipitate was analyzed through immunoblotting. C Diagram illustrating the protein structure of UPS8 and its deletion mutants (residues 1–313, 314–714, 715–1118) utilized in the co-immunoprecipitation assay. D A schematic diagram of the YAP protein structure and its deletion mutants (residues 1–171, 1–292, 172–504, 293–504) used for the CO-IP assay. E Co-immunoprecipitation revealed that the interaction of the WW domain of YAP with USP8. F Co-immunoprecipitation demonstrated that YAP interaction necessitates the USP domain of USP8. G Surface diagram of the docking model and their interfacing residues between USP8 and YAP protein (YAP, blue; USP8, yellow; hydrogen bond interaction, dotted line). H The two sites of USP8 are mutated to alanine, and the table shows the predicted binding sites of the two proteins. I , L Western blot analysis showed that the expression level of YAP protein in BT549 and MDA-MB-231 cells. Cells were transfected with siControl or siUSP8 and then treated with MG132. J , K , M , N Western blotting was used to detect the protein levels of USP8 and YAP. For the specified time, BT549 and MDA-MB-231 cells received treatment with 100 μM cyclohexylamine (CHX). The YAP protein expression was measured with ImageJ software and illustrated in the right panel. O , P Evaluation of YAP half-life in BT549 cells following transfection with the specified plasmids. The right panel graphically displays the YAP protein expression as estimated by ImageJ software.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A Immunofluorescence staining showed the localization of USP8 (green) and YAP (red) in BT549 cells. DAPI (blue) was used to stain the nuclei, and the scale bars indicate 10 μm. B An endogenous association between USP8 and YAP was demonstrated through immunoprecipitation analysis. To investigate endogenous interaction between USP8 and YAP, BT549 cell lysates were precipitated using anti-YAP or anti-USP8 antibodies, and the resulting precipitate was analyzed through immunoblotting. C Diagram illustrating the protein structure of UPS8 and its deletion mutants (residues 1–313, 314–714, 715–1118) utilized in the co-immunoprecipitation assay. D A schematic diagram of the YAP protein structure and its deletion mutants (residues 1–171, 1–292, 172–504, 293–504) used for the CO-IP assay. E Co-immunoprecipitation revealed that the interaction of the WW domain of YAP with USP8. F Co-immunoprecipitation demonstrated that YAP interaction necessitates the USP domain of USP8. G Surface diagram of the docking model and their interfacing residues between USP8 and YAP protein (YAP, blue; USP8, yellow; hydrogen bond interaction, dotted line). H The two sites of USP8 are mutated to alanine, and the table shows the predicted binding sites of the two proteins. I , L Western blot analysis showed that the expression level of YAP protein in BT549 and MDA-MB-231 cells. Cells were transfected with siControl or siUSP8 and then treated with MG132. J , K , M , N Western blotting was used to detect the protein levels of USP8 and YAP. For the specified time, BT549 and MDA-MB-231 cells received treatment with 100 μM cyclohexylamine (CHX). The YAP protein expression was measured with ImageJ software and illustrated in the right panel. O , P Evaluation of YAP half-life in BT549 cells following transfection with the specified plasmids. The right panel graphically displays the YAP protein expression as estimated by ImageJ software.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Transfection, Software

A The analysis of YAP binding to the USP8 promoter region via ChIP-seq employed GEO data with accession numbers GSE61852 . B ChIP-PCR analysis in BT549 cells shows that YAP was concentrated at the USP8 promoter. C Predicted TEAD4 binding sequences and sites in the USP8 promoter according JASPAR. D Schematic of the hypothesized TEAD-binding site in the USP8 promoter and the primers employed for chromatin immunoprecipitation. The sequences shown include the wild-type (WT) and two mutated (Mut) USP8 promoter luciferase constructs. E ChIP-qPCR was performed on BT549 cells with an anti-YAP antibody and an IgG control. F The schematic diagram of YAP and TEAD4 complex binding to the USP8 promoter. G Luciferase reporter gene assay for USP8 promoter activity in WT and YAP knockdown cells transfected with wild-type or mutant TEAD binding sites. H , I Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with siControl or siYAP. The internal control in the experiment was β-Actin. J – M RT-qPCR showed the mRNA expression level of USP8, YAP and CTGF in BT549 cells or MDA-MB-231 cells treated with siControl or two independent siRNAs. N , O The binding of YAP to the USP8 promoter was detected through a ChIP assay. YAP depletion in BT549 and MDA-MB-231 cells led to decreased binding to the USP8 gene, as demonstrated by ChIP-qPCR assays. P , Q Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of Verteporfin. β-Actin served as the internal control. R – U RT-qPCR showed the mRNA expression level of USP8, YAP and CTGF in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of Verteporfin. V , W Enhanced binding of YAP to the USP8 promoter was observed in BT549 and MDA-MB-231 cells following a 6-h treatment with Verteporfin, as demonstrated by ChIP-qPCR analysis. All data are presented as mean ± SD, with N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A The analysis of YAP binding to the USP8 promoter region via ChIP-seq employed GEO data with accession numbers GSE61852 . B ChIP-PCR analysis in BT549 cells shows that YAP was concentrated at the USP8 promoter. C Predicted TEAD4 binding sequences and sites in the USP8 promoter according JASPAR. D Schematic of the hypothesized TEAD-binding site in the USP8 promoter and the primers employed for chromatin immunoprecipitation. The sequences shown include the wild-type (WT) and two mutated (Mut) USP8 promoter luciferase constructs. E ChIP-qPCR was performed on BT549 cells with an anti-YAP antibody and an IgG control. F The schematic diagram of YAP and TEAD4 complex binding to the USP8 promoter. G Luciferase reporter gene assay for USP8 promoter activity in WT and YAP knockdown cells transfected with wild-type or mutant TEAD binding sites. H , I Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with siControl or siYAP. The internal control in the experiment was β-Actin. J – M RT-qPCR showed the mRNA expression level of USP8, YAP and CTGF in BT549 cells or MDA-MB-231 cells treated with siControl or two independent siRNAs. N , O The binding of YAP to the USP8 promoter was detected through a ChIP assay. YAP depletion in BT549 and MDA-MB-231 cells led to decreased binding to the USP8 gene, as demonstrated by ChIP-qPCR assays. P , Q Western blot showed the expression of USP8 and YAP protein in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of Verteporfin. β-Actin served as the internal control. R – U RT-qPCR showed the mRNA expression level of USP8, YAP and CTGF in BT549 or MDA-MB-231 cells treated with DMSO or different concentrations of Verteporfin. V , W Enhanced binding of YAP to the USP8 promoter was observed in BT549 and MDA-MB-231 cells following a 6-h treatment with Verteporfin, as demonstrated by ChIP-qPCR analysis. All data are presented as mean ± SD, with N = 3. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: Binding Assay, ChIP-sequencing, Chromatin Immunoprecipitation, Luciferase, Construct, ChIP-qPCR, Control, Reporter Gene Assay, Activity Assay, Knockdown, Transfection, Mutagenesis, Western Blot, Expressing, Quantitative RT-PCR

A , B DUB-IN-2 can enhance the sensitivity to PTX treatment. BT549 and MDA-MB-231 cells underwent a 24-h treatment with varying concentrations of PTX, followed by an MTT assay. The IC50 values for PTX were presented for each group. C , D Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation. CCK8 assay was used to determine the viability of BT549 or MDA-MB-231 cells transfected with DMSO, PTX or DUB-IN-2 at the indicated time point. E , F Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell migration. Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 treated with DMSO, PTX or DUB-IN-2. The right panel shows the quantitative results of cell proliferation. G , H Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell migration and invasion. Transwell assay was used to detect the migration and invasion ability of BT549 or MDA-MB-231cell line treated with DMSO, PTX or DUB-IN-2. I , J Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation. The EdU assay was employed to assess the proliferation capacity of BT549 or MDA-MB-231 treated with DMSO, PTX or DUB-IN-2. The right panel shows the quantitative results of cell proliferation. K – M Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation in vivo. BT549 cells were subcutaneously inoculated into 4-week-old BALB/c female nude mice treated with DMSO and PTX or DUB-IN-2. Mice were euthanized 35 days post-injection, and the xenograft tumors were excised. Images of representative tumors ( K ), along with their weight ( L ) and volume ( M ), are displayed. N , O The expression levels of USP8, YAP, and Ki67 in xenograft models treated with PTX or DUB-IN-2 were visualized using IHC staining. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). Data are represented as the average ± SD, based on N = 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: Cell Death & Disease

Article Title: Positive feedback regulation between USP8 and Hippo/YAP axis drives triple-negative breast cancer progression

doi: 10.1038/s41419-025-08356-8

Figure Lengend Snippet: A , B DUB-IN-2 can enhance the sensitivity to PTX treatment. BT549 and MDA-MB-231 cells underwent a 24-h treatment with varying concentrations of PTX, followed by an MTT assay. The IC50 values for PTX were presented for each group. C , D Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation. CCK8 assay was used to determine the viability of BT549 or MDA-MB-231 cells transfected with DMSO, PTX or DUB-IN-2 at the indicated time point. E , F Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell migration. Wound healing assay was used to determine the migration ability of BT549 or MDA-MB-231 treated with DMSO, PTX or DUB-IN-2. The right panel shows the quantitative results of cell proliferation. G , H Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell migration and invasion. Transwell assay was used to detect the migration and invasion ability of BT549 or MDA-MB-231cell line treated with DMSO, PTX or DUB-IN-2. I , J Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation. The EdU assay was employed to assess the proliferation capacity of BT549 or MDA-MB-231 treated with DMSO, PTX or DUB-IN-2. The right panel shows the quantitative results of cell proliferation. K – M Treatment of cells with the combination of DUB-IN-2 and PTX can inhibit cell proliferation in vivo. BT549 cells were subcutaneously inoculated into 4-week-old BALB/c female nude mice treated with DMSO and PTX or DUB-IN-2. Mice were euthanized 35 days post-injection, and the xenograft tumors were excised. Images of representative tumors ( K ), along with their weight ( L ) and volume ( M ), are displayed. N , O The expression levels of USP8, YAP, and Ki67 in xenograft models treated with PTX or DUB-IN-2 were visualized using IHC staining. The panel shows the quantitative results of Ki67. Scale bars, 100 μm (10X), 400 μm (40X). Data are represented as the average ± SD, based on N = 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The Human triple negative breast cancer BT549, MDA-MB-231 and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC).

Techniques: MTT Assay, CCK-8 Assay, Transfection, Migration, Wound Healing Assay, Transwell Assay, EdU Assay, In Vivo, Injection, Expressing, Immunohistochemistry

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