Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 expression is upregulated in BC tissues and cell lines. A CMTM6 expression in different types of cancers (versus non-tumor tissues) in the TCGA and GTEx datasets. B CMTM6 expression in BC tissues and matched adjacent non-tumor tissues in the TCGA-BRCA dataset. C CMTM6 expression in different subtypes of breast cancers in the TCGA-BRCA datasets. D Western blot analysis of the relative levels of CMTM6 protein expression in fresh BC tissues and matched adjacent non-tumor tissues ( n = 3). E qRT-PCR analysis of the relative levels of CMTM6 mRNA transcripts in fresh BC tissues and matched adjacent non-tumor tissues ( n = 30). *** P < 0.001

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 is upregulated in trastuzumab-resistant BC and correlates with poor prognosis. A qRT-PCR and B Western blot analyses of CMTM6 mRNA and protein expression in different BC cell lines. C Immunohistochemical analysis of CMTM6 protein expression in HER2+ BC tissues from trastuzumab-treated patients (scale bar, 50 μM). D Immunohistochemical analysis of CMTM6 protein expression in HER2+ BC tissues from trastuzumab-treated patients with or without relapse. E, F Kaplan–Meier analysis of overall survival (OS) and relapse-free survival (RFS) in trastuzumab-treated high or low CMTM6 expressing HER2+ BC patients

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemical staining

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: Association between CMTM6 expression and clinicopathological variables in HER2+ BC

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Expressing

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 promotes the survival, migration, invasion and trastuzumab resistance of BC cells in vitro . A qRT-PCR and Western blot validated CMTM6 silencing in JIMT-1 cells and CMTM6-overexpresstion in SKBR3 cells. Negative control (NC) JIMT-1 and SKBR3 cells were transduced with lentivirus for the control shRNA or transfected with the control plasmid, respectively. B, C CCK-8 assay determined the viability of the indicated BC cells following treatment with trastuzumab (0-100 μg/ml). D, E ethynyl-2′-deoxyuridine (EdU) analysis of the proliferation of CMTM6-silenced JIMT-1 cells, CMTM6 overexpressing SKBR3 cells, control JIMT-1 cells and SKBR3 cells following treatment with trastuzumab (10 μg/ml). (scale bar, 50 μM). F, G TUNEL analysis of apoptotic CMTM6-silenced JIMT-1 cells, CMTM6 overexpressing SKBR3 cells, control JIMT-1 and SKBR3 cells following treatment with trastuzumab (10 μg/ml). H, I Cell invasion assay revealed that CMTM6 silencing inhibited JIMT-1 cell invasion while CMTM6 overexpression enhanced SKBR3 cell invasion following treatment with trastuzumab (10 μg/ml). (scale bar, 50 μM). Data are representative images or expressed as the mean ± SD of each group from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Migration, In Vitro, Quantitative RT-PCR, Western Blot, Negative Control, Transduction, shRNA, Transfection, Plasmid Preparation, CCK-8 Assay, TUNEL Assay, Invasion Assay, Over Expression

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 directly interacts with HER2 and enhancing the HER2 signaling in BC cells. A IHC analysis of HER2 protein expression in BC tissues with low or high CMTM6 protein expression (scale bar, 50 μM). B Association between CMTM6 and HER2 protein expression in BC tissues. C Western blot analysis of CMTM6 and HER2 protein levels in BC tissues. D Correlation between CMTM6 and HER2 protein levels in BC tissues. Data are mean ± SEM. E Confocal microscopy analysis of the subcellular co-localization of CMTM6 (green) and HER2 (red) in JIMT-1 cells, with DAPI nuclear staining (blue) (scale bar, 10 μM). F, G Co-immunoprecipitation revealed the direct interaction between endogenous CMTM6 and HER2 proteins in JIMT-1 cells. H Western blot analysis of CMTM6, HER2, p-HER2, PI3K, AKT, MEK, ERK, N-cadherin and E-cadherin protein levels in CMTM6-silenced JIMT-1, CMTM6 overexpressing SKBR3, control JIMT-1 and SKBR3 cells. Data are representative images of each group of cells from three separate experiments

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Expressing, Western Blot, Confocal Microscopy, Staining, Immunoprecipitation

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 stabilizes the HER2 protein by inhibiting its poly-ubiquitination in BC cells. A Western blot analysis of CMTM6 and HER2 protein levels in CMTM6-silenced JIMT-1 cells after treatment with 10 μM MG132 or vehicle control for 24 h. B Western blot analysis of CMTM6 and HER2 protein levels in CMTM6-silenced JIMT-1 cells after treatment with 10 μM CHX or vehicle control for 24 h. C CMTM6 overexpression decreased HER2 poly-ubiquitination. HeLa cells were co-transfected with the FLAG-HER2 plasmid or combined with Myc-CMTM6 plasmid, together with the HA-Ub plasmid for 24 h and treated with 10 μM MG132 for another 24 h. The cell lysates were immunoprecipitated with anti-HER2 and immunoblotted with anti-HA antibody to determine HER2 poly-ubiquitination

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Western Blot, Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: CMTM6 promotes the growth of trastuzumab-resistant BC by preserving HER2 protein and relative signaling in BC tissues in vivo. Female node mice were injected with SKBR3, CMTM6-silenced JIMT-1 or JIMT-1 cells to establish xenograft tumors and when the tumors reached at 100 mm 3 , the tumor-bearing mice were randomized and treated with vehicle (Control), trastuzumab (Ttzm, 10 mg/kg body weight every 5 days for 4 times) or combination of trastuzumab and Pertuzumab (Ttzm+Ptzm, n = 4 per group). A The dynamic growth of tumors. B IHC analysis of CMTM6, HER2, Ki67, and Caspase-3 protein expression (scale bar, 50 μM). C Western blot analysis of the relative levels of CMTM6, HER2, p-HER2, PI3K, AKT, MEK, ERK protein levels in the indicated tumors. Data are presentative images of each group from at least three separate experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques: Preserving, In Vivo, Injection, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

doi: 10.1186/s12943-023-01716-y

Figure Lengend Snippet: A schematic diagram illustrates the action of CMTM6 in trastuzumab resistance of BC. CMTM6 promotes trastuzumab resistance by inhibiting HER2 ubiquitination and degradation in HER2+ BC

Article Snippet: The cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature for 1 min, and blocked with 5% BSA at 37 °C for 1 h. The cells were incubated overnight with anti-CMTM6 antibody (HPA026980, Sigma-Aldrich) and/or anti-HER2 antibody (60311-1-AP, Proteintech) at 4 °C.

Techniques:

Journal: Journal of Cancer

Article Title: Invivo Pen: A novel plasma source for in vivo cancer treatment

doi: 10.7150/jca.38613

Figure Lengend Snippet: Comparisons on invivo Pen and PAM treatments in inducing in vivo tumor apoptosis and reducing tumor migrative abilities. (A) Tumor cell apoptosis examined using TUNEL staining. (B) Tumor migrative ability examined using immunohistochemistry staining of tumor migration suppressor E-Cadherin. These assays were conducted on tumor samples from mice inoculated with TNBC cells MDBMA231.

Article Snippet: After being heated in 10 mM citrate buffer for 15 min, tissue sections were incubated with primary antibodies ER (21244-1-AP, 1:200; Proteintech, Rosemont, IL, USA), HER2 (60311-1-Ig, 1:200; Proteintech), ALDH1 (#54135, 1:200; Cell Signaling Technology, St Louis, MO, USA) and E-Cadherin (#14472, 1:200; Cell Signaling Technology) overnight at 4°C.

Techniques: In Vivo, TUNEL Assay, Staining, Immunohistochemistry, Migration

Journal: Journal of Cancer

Article Title: Invivo Pen: A novel plasma source for in vivo cancer treatment

doi: 10.7150/jca.38613

Figure Lengend Snippet: Immunohistochemistry staining of breast cancer subtyping and cancer stem cell markers in tumor samples. (A) ER and (B) HER2 are canonical breast cancer subtyping markers, and (C) ALDH1 is a typical cancer stem cell marker. The staining was conducted on tumor samples from mice inoculated with TNBC cells MDBMA231.

Article Snippet: After being heated in 10 mM citrate buffer for 15 min, tissue sections were incubated with primary antibodies ER (21244-1-AP, 1:200; Proteintech, Rosemont, IL, USA), HER2 (60311-1-Ig, 1:200; Proteintech), ALDH1 (#54135, 1:200; Cell Signaling Technology, St Louis, MO, USA) and E-Cadherin (#14472, 1:200; Cell Signaling Technology) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Marker