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 CCK8 assay kit (TargetMol, C0005) was used to evaluate cell viability at various time points, and the absorbance of the cells was measured at a wavelength of 450 nm.

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

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 CCK8 assay kit (TargetMol, C0005) was used to evaluate cell viability at various time points, and the absorbance of the cells was measured at a wavelength of 450 nm.

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

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 CCK8 assay kit (TargetMol, C0005) was used to evaluate cell viability at various time points, and the absorbance of the cells was measured at a wavelength of 450 nm.

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

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 CCK8 assay kit (TargetMol, C0005) was used to evaluate cell viability at various time points, and the absorbance of the cells was measured at a wavelength of 450 nm.

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