Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: A schematic presentation of the CellCollector. Straight ( a ) and spiraled ( b ) medical wire covered with anti-EpCAM (blue) and/or anti-MUC1 (black) antibody.

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques:

Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: The expression analysis of the renal normal and tumor tissues. ( a ) The transcriptome microarray analysis with a detailed description of the differentially expressed surface proteins; FPKM—Fragments Per Kilobase Million; ( b ) the protein expression of EpCAM, MUC1 and cytokeratin (green) in the clear cell renal tumor (pT3b) and corresponding control tissues. The nuclei were stained with Hoechst (blue).

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques: Expressing, Microarray, Control, Staining

Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: The surface markers in the primary ccRCC cell lines. ( a ) The transcriptome microarray analysis with a detailed description of EpCAM and MUC1 in ccRCC31, ccRCC45, ccRCC122 and ccRCC162; FPKM—Fragments Per Kilobase Million, ( b ) the localization of EpCAM (green) and MUC1 (red) proteins on the surface of the ccRCC45 separate cells and merged cells to visualize the colocalization. The images show protein localization. The nuclei were stained with Hoechst (blue).

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques: Microarray, Staining

Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: The statistically significant differential expression of EpCAM and MUC1 was determined with a microarray. All analyzed cell lines were compared with RCC45.

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques: Quantitative Proteomics, Microarray

Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: The CTCs captured by the spiraled wire. Both anti-EpCAM and anti-EpCAM/MUC1 antibody-covered CellCollector system wires were applied to the blood samples: G3 chromophiles (chRCC) and clear cells (pT3apNxpM1G2) (ccRCC). The attached cells were stained with antibodies against pan cytokeratin CK8, CK18 and CK19 (green) and CD45 (red). The nuclei were stained with Hoechst (blue).

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques: Staining

Journal: Life

Article Title: Potential Use of CTCs as Biomarkers in Renal Cancer Patients

doi: 10.3390/life12010089

Figure Lengend Snippet: The comparison of the CellCollector wires functionalized with either anti-EpCAM or anti-EpCAM/anti-MUC1 antibodies. ( a ) The number of CTCs captured by anti-EpCAM and anti-EpCAM/anti-MUC1 antibody-covered wires in the matched paired patient samples. Green line—lung metastasis, blue line—other metastases. ( b ) The presentation of CTC detection by both wires with a median of 4 (range 0–15) for anti-EpCAM-coated wires and a median of 6 (range 1–18) for anti-EpCAM/MUC1-coated wires. The mean is displayed as “+” and the whiskers represent the min and max values.

Article Snippet: Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C.

Techniques: Comparison

Journal: Cancer cell international

Article Title: Therapeutic effect of a MUC1-specific monoclonal antibody-drug conjugates against pancreatic cancer model.

doi: 10.1186/s12935-022-02839-w

Figure Lengend Snippet: Fig. 1 HzMUC1 antibody binds to MUC1 on the cell surface of pancreatic cancer cells. A Lysates from SW1990, Mia-PaCa-2, PATU-8988, PANC-1, CFPAC-1, and Capan-2 pancreatic cancer cell lines, and hTERT-HPNE (a normal epithelial pancreatic cell line) were resolved by SDS-PAGE and immunoblotted with the commercially available anti-MUC1 N-terminal (NT) antibody and reprobed with anti-β-actin antibody as loading control. B Cell lysates from Capan-2, CFPAC-1, and SW1990 cells were immunoprecipitated with human IgG or HzMUC1 antibody and immunoblotted with the anti-MUC1-NT antibody or anti-MUC1-CT antibody. C HzMUC1 antibody binds to MUC1 protein on the cell surface. Capan-2, CFPAC-1 and SW1990 cells grown on coverslips were washed with PBS, incubated with HzMUC1 antibody (1 µg/well) on ice, and subsequently with Alexa 488-conjugated secondary antibody, and examined with fluorescent (upper panels) and phase contrast (lower panels) confocal microscopy. Scale bars, 20 μm

Article Snippet: Anti-MUC1 mouse monoclonal antibody to MUC1-N (clone GP1.4; Catalog No. AM32842PU-S at 1:2000) was obtained from OriGene (Rockville, USA).

Techniques: SDS Page, Control, Immunoprecipitation, Incubation, Confocal Microscopy

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 4 ch5E6 treatment leads to a substantial decrease in the growth of organoids derived from PC patients or pancreatic and lung cancer genetically engineered mouse models. a PC patient and b KPC mouse organoid staining with ch5E6 indicating MUC16 expression (green) and specific binding of mAb compared to no binding with isotype control mAb huIgG1. The binding of ch5E6 to the ductal cells (yellow) was illustrated by colocalization with CK-19 (red) staining in human and mouse pancreatic tumor organoids. Scale bar 10 µm.The representative images for ch5E6 treated. c Human PC patient. d KPC and (e) KPA mouse-derived organoids obtained by real-time kinetics using Incucyte live cell imaging system. The data were quantitated for organoid counts using essence incucyte software, plotted as a graph of change in organoid counts or area over time (3–5 days) for both ch5E6 and huIgG1 treatments, and is shown in parallel. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Derivative Assay, Staining, Expressing, Binding Assay, Control, Live Cell Imaging, Software

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 6 Inhibition of EMT by ch5E6 is validated in PC and NSCLC cell line-derived xenografts. a Immunofluorescence analysis showing a decrease in pFAK(Y397) and N-cadherin expression in xenograft tumors of SW1990 cells treated with ch5E6 compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: three animals). The data was plotted for changes in fluorescence intensity using GraphPad Prism 9 and is shown in parallel. Nuclei were stained with DAPI. b Immunofluorescence analysis of ch5E6 treated SW1573 cell line-derived xenografts showing a reduction in pFAK(Y397) and N-cadherin levels compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: 3 animals). Scale bar, 10 µm; magnified images, 2 µm. No significant changes in the intensity of MUC16 were seen in the ch5E6 treated versus isotype control tumors derived from both cancers. c, d Immunoblot analysis of ch5E6 treated PDAC and NSCLC tumor lysates showing a substantial decrease in phosphorylated levels of FAK(Y397), p70S6K(T389) and N-cadherin as compared to huIgG1 treatment. Error bars indicate SEM. Scale bar, 400 μm; magnified images, 100 μm; *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Inhibition, Derivative Assay, Expressing, Control, Staining, Western Blot

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 7 MUC16 and N-cadherin are clinically correlated in patient tumors. a, b Representative images and quantitative illustration of immunohistochemical analyses demonstrate a strong positive correlation between MUC16 and N-cadherin (R = 0.84) in both primary PC tumors (n = 10) and liver metastasis (R = 0.99) samples (n = 8). Scale bar, 400 µm; magnified images, 100 µm. *P < 0.05; **P < 0.01. c Representative images of immunofluorescence analysis showing coexpression of MUC16 (green) and N-cadherin (red) in primary PDAC tumors compared to no MUC16 and N-cadherin in normal pancreatic sections. Scale bar, 20 µm; magnified images, 5 µm. d Schematic diagram representing ch5E6 induced downregulation of MUC16 mediated EMT resulting in its anti-tumor potential in PC and NSCLC. Overall, anti- MUC16 chimeric mAb5E6 (ch5E6) binds to the cell surface-tethered domain of MUC16, interferes with oncogenic pFAK/p70S6K/N-cadherin signaling associated with MUC16-mediated EMT, and reduces tumor burden in both PC and NSCLC models.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A , C Western blotting was performed to detect proteins as indicated in MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells treated with DMSO, 5 μM CCCP (24 h) or 5 μM CCCP (24 h) + 10 μM Mdivi-1 (24 h). Quantification of mitochondrial proteins was calculated. B , D Western blotting was performed to detect proteins as indicated in BT549/g-CTL and BT549/g-MUC1 cells treated with DMSO, 10 μM CCCP (24 h) or 10 μM CCCP (24 h) + 10 μM Mdivi-1 (24 h). Quantification of mitochondrial proteins was calculated. E After treatment with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h), western blotting was performed to detect proteins with the indicated antibodies in HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1 cells treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. Quantification of mitochondrial proteins was calculated. F The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells treated with DMSO, 5 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). G The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in BT549/g-CTL and BT549/g-MUC1 cells treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). H The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1 cells treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. HeLa229 cells were treated with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h). I , J Average length of mitochondria per cell ( I ) and the number of mitochondria per cell ( J ) in TEM micrographs from Fig. S . More than 16 cells in each graph were analyzed. K Representative fluorescence images from (Fig. S ) with Mtphagy Dye (Red), Lysosome Dye (Green), and MitoBright (Purple) staining in MDA-MB-468 cells stimulated with DMSO, 5 μM CCCP and 500 nM rapamycin. The arrow indicates colocalization pots. Rapa: Rapamycin. Bars: 20 μm. L HeLa229/g-CTL and HeLa229/g-MUC1 cells were treated with 30 μM CCCP for the indicated times, and western blotting was performed to detect pSer65-Ub and total-Ub. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated by a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Western Blot, Expressing, Negative Control, Fluorescence, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A Proteins of HeLa229 cells precipitated by anti-MUC1-C or IgG antibody were analyzed by mass spectrometry and co-IP. The proteins from the co-IP assay were separated by SDS-PAGE and stained with Coomassie brilliant blue. Screening of proteins localized in mitochondria, including ATAD3A. B – D Co-IP assay to detect the interaction of endogenous MUC1 and ATAD3A. Anti-MUC1-C immunoprecipitates from MDA-MB-468 ( B ), BT549 ( C ) and HeLa229 ( D ) cells were analyzed by western blotting with the indicated antibodies (left). Anti-ATAD3A immunoprecipitates from MDA-MB-468 ( B ), BT549 ( C ), and HeLa229 ( D ) cells were analyzed by western blotting with the indicated antibodies (right). E The schematic shows GFP fused with different truncations of MUC1-CD (upper). HEK293T cells were transfected with GFP-MUC1-CD truncated mutant plasmids, subjected to a co-IP assay with an anti-ATAD3A antibody, and then analyzed by western blotting using anti-GFP and anti-ATAD3A antibodies. F Co-IP assay of MUC1-CD and ATAD3A in HEK293T cells. HEK293T cells were cotransfected with ATAD3A and pEGFP-MUC1-CD or pEGFP-MUC1-CD-AQA. Cell lysates were subjected to denatured co-IP. G Purified recombinant GST, GST-MUC1-CD, or GST-MUC1-AQA was incubated with ATAD3A. The precipitated beads were detected by western blot using anti-ATAD3A and anti-GST antibodies. H Co-IP of endogenous MUC1 and ATAD3A in HeLa229 cells treated with 30 μM CCCP (2 h) or 30 μM Mdivi-1 (2 h). After IP with IgG or anti-ATAD3A, the immunoprecipitates were analyzed with anti-MUC1-C and anti-ATAD3A antibodies. I HeLa229/g-MUC1 cells were transfected with vector, MUC1-CD-WT and MUC1-CD-AQA plasmids and then treated with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h). Western blotting was performed using antibodies as indicated. Quantification of mitochondrial proteins was calculated. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated using a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, SDS Page, Staining, Western Blot, Transfection, Mutagenesis, Purification, Recombinant, Incubation, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A , B Western blotting was performed to detect ATAD3A protein levels in MDA-MB-468 cells ( A ) and BT549 cells ( B ). C Western blotting was performed to detect ATAD3A protein levels in HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1 cells treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. D After treatment with CHX (10 μg/ml) for different times, the degradation rates of ATAD3A proteins were measured in HeLa229/g-CTL and HeLa229/g-MUC1 cells. The relative quantification of ATAD3A protein levels after CHX treatments was analyzed, and the half-life curve of ATAD3A was generated. E Western blotting was performed to detect ATAD3A protein levels in HeLa229/g-CTL and HeLa229/g-MUC1 cells after treatment with proteasome (50 nM BTZ (8 h) or 10 μM MG132 (8 h)) or autolysosome (100 μM CQ (8 h) or 20 mM NH 4 Cl (8 h)) inhibitors, respectively. The relative quantification of ATAD3A protein levels was analyzed. F HeLa229/g-MUC1 cells were transfected with vector, MUC1-CD-WT or MUC1-CD-AQA plasmids and then treated with CHX (10 μg/ml) for 0, 4, 8, and 12 h. Western blotting was performed. The relative quantification of ATAD3A protein levels after CHX treatments was analyzed, and the half-life curve of ATAD3A was generated. G HeLa229/g-MUC1 cells were transfected with vector, MUC1-CD-WT, and MUC1-CD-AQA plasmids for 48 h and then treated with 50 nM BTZ (8 h) or 10 μM MG132 (8 h). Western blotting was performed to detect ATAD3A protein levels. The relative quantification of ATAD3A protein levels was analyzed. H HEK293T cells were transfected with His-Ub and vector or MUC1-HA plasmids and then treated with 10 μM MG132 for 8 h. After IP with anti-ATAD3A antibody, the immunoprecipitants were analyzed with the indicated antibodies. I HeLa229/g-CTL and HeLa229/g-MUC1 cells were transfected with shCTL and shATAD3A lentiviral vectors and then treated with DMSO or 30 μM CCCP (24 h). Western blotting was performed using antibodies as indicated. Quantification of mitochondrial proteins was calculated. J BT549/g-CTL and BT549/g-MUC1 cells were transfected with vector and ATAD3A-V5 lentiviral vectors and then treated with DMSO or 10 μM CCCP (24 h). Western blotting was performed using the indicated antibodies. Quantification of mitochondrial proteins was calculated. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated by a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Western Blot, Expressing, Negative Control, Quantitative Proteomics, Generated, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A Western blotting was performed to detect Pink1 protein levels in four cell lines, HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1, treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. Pink1 (FL): full-length Pink1; Pink1 (Cl): cleaved Pink1. B HeLa229/g-CTL and HeLa229/g-MUC1 cells were treated with 30 μM CCCP for the indicated times. Western blotting was then performed to detect Pink1 protein levels. C HeLa229/g-CTL and HeLa229/g-MUC1 cells were transfected with shCTL and shATAD3A lentiviral vectors for 48 h and then treated with or without 30 μM CCCP. Western blotting was performed to detect Pink1 protein levels. D HeLa229/g-CTL and HeLa229/g-MUC1 cells were transfected with vector and ATAD3A-V5 lentiviral vectors for 48 h and then treated with or without 30 μM CCCP. Western blotting was performed to detect Pink1 protein levels. E HeLa229/g-CTL and HeLa229/g-MUC1 cells were transfected with shCTL and shPink1 lentiviral vectors and then treated with DMSO or 30 μM CCCP (24 h). Western blotting was performed using antibodies as indicated. The relative quantification of protein levels was analyzed. F HeLa229/g-CTL and HeLa229/g-MUC1 cells were transfected with vector and Parkin-HA plasmids for 48 h and then treated with DMSO, 30 μM CCCP (24 h) or a combination of 30 μM CCCP (24 h) and 30 μM Mdivi-1 (24 h). Western blotting was performed using the indicated antibodies. The relative quantification of protein levels was analyzed. CP + Md: CCCP + Mdivi-1. G HeLa229/g-MUC1 cells were transfected with vector, MUC1-CD-WT and MUC1-CD-AQA plasmids and then treated with DMSO and 30 μM CCCP (2 h). Sediment from the mitochondrial fraction was detected by western blot. Tim23, Tom20, and ATAD3A served as mitochondrial markers, and α-tubulin was used as a cytosolic marker. H HeLa229 cells were transfected with shCTL and shATAD3A lentiviral vectors and treated with DMSO and 30 μM CCCP (2 h). Sediment from the mitochondrial fraction was detected by western blot. Tim23, Tom20, and ATAD3A were used as mitochondrial markers, and α-tubulin was used as a cytosolic marker. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated by a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Western Blot, Expressing, Negative Control, Transfection, Plasmid Preparation, Quantitative Proteomics, Marker, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were pretreated with DMSO, 5 μM CCCP (2 h), or a combination of 5 μM CCCP (2 h) and 10 μM Mdivi-1 (6 h) before being plated in medium with 10 μM Mdivi-1, and then the cells were subjected to a viability assay. CP + Md: CCCP + Mdivi-1. B BT549/g-CTL and BT549/g-MUC1 cells were pretreated with DMSO, 10 μM CCCP (2 h) or a combination of 10 μM CCCP (2 h) and 10 μM Mdivi-1 (6 h) before being plated in medium with 10 μM Mdivi-1, and then the cells were subjected to a viability assay. C MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were pretreated with DMSO, 5 μM CCCP (2 h) or a combination of 5 μM CCCP (2 h) and 10 μM Mdivi-1 (6 h) and then plated in medium with 10 μM Mdivi-1. Cells were subjected to the mammosphere formation assay. Representative images are shown. The number of mammospheres > 50 μm in diameter from MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells was calculated. Bars: 200 μm. ( D ) BT549/g-CTL and BT549/g-MUC1 cells were pretreated with DMSO, 10 μM CCCP (2 h) or a combination of 10 μM CCCP (2 h) and 10 μM Mdivi-1 (6 h) and then plated in medium with 10 μM Mdivi-1. Cells were subjected to the mammosphere formation assay. Representative images are shown. The number of mammospheres >50 μm in diameter from BT549/g-CTL and BT549/g-MUC1 cells was calculated. Bars: 200 μm. E MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with vector and ATAD3A-V5 lentiviral vectors. Then, the cells were subjected to a viability assay. F MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with vector and ATAD3A-V5 lentivirus. Then, cells were subjected to the mammosphere formation assay. Representative images are shown. Bars: 200 μm. The number of mammospheres >50 μm in diameter was calculated. The data represent the mean ± SD from three independent experiments. G – I Tumor sizes ( G , H ) and weights ( I ) in mice injected with the indicated MDA-MB-468 cells (g-CTL/g-CTL-ATAD3A, g-MUC1/g-MUC1-ATAD3A) were compared. Differences between linked groups were evaluated by a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Viability Assay, Tube Formation Assay, Transfection, Plasmid Preparation, Injection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A MDA-MB-468 cells were treated with 5 μM CP2 (48 h) and 5 μM GO203 (48 h) and then treated with DMSO, 5 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. GO-203, a cell-penetrating peptide containing a polyArg cell transduction domain linked to the CQCRRKN sequence ([R]9-CQCRRKN; all D-amino acids), can bind directly to the MUC1-C cytoplasmic domain. In contrast, CP2 is a GO203 analog (CP2: [R]9-AQARRKN) that is ineffective in inhibiting MUC1-C. CP2 acted as a control for GO203. B BT549 cells were treated with 5 μM CP2 (48 h) and 5 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. C T47D cells were treated with 10 μM CP2 (48 h) and 10 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h), or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. D SKBR3 cells were treated with 10 μM CP2 (48 h) and 10 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Quantification of mitochondrial proteins was calculated. E The cell viability assay was performed in MDA-MB-468 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. F The mammosphere formation assay was performed in MDA-MB-468 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. The number of mammospheres was calculated. GO + Md: GO203 + Mdivi-1. G The cell viability assay was performed in BT549 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. H A mammosphere formation assay was performed in BT549 cells treated with 5 μM. GO203 and 10 μM Mdivi-1 alone or in combination. The number of mammospheres was calculated. I MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with shCTL, shPink1-1, and shPink1-2 lentiviral vectors for 48 h and then treated with DMSO and 5 μM CCCP (2 h). The cell viability assay was performed. J MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with shCTL, shPink1-1, and shPink1-2 lentiviral vector for 48 h, then treated with DMSO and 5 μM CCCP (2 h). Cells were subjected to the mammospheres formation assay. The number of mammospheres >50 μm in diameter was calculated. The data represent mean ± SD from three independent experiments. K – M Tumor sizes ( K , L ) and weights ( M ) were compared in mice that received four different treatments (PBS, GO203, Mdivi-1 and G0203 + Mdivi-1). G + M: GO203 + Mdivi-1. Differences between linked groups were evaluated by a two-tailed Student’s t test. * P < 0.05, ** P < 0.01; *** P < 0.001; NS not significant.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Western Blot, Transduction, Sequencing, Control, Viability Assay, Tube Formation Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

doi: 10.1038/s41419-022-05345-z

Figure Lengend Snippet: A Representative images of IHC analysis for MUC1 and ATAD3A in 110 breast cancer specimens. Bars: 100 μm. B A total of 110 patient samples were divided into four groups, as indicated in the table. C The correlation between MUC1 and ATAD3A expression in breast cancer specimens was plotted by Pearson correlation analysis. ( D ) Overall survival study of patients with high and low MUC1 expression was analyzed in breast cancer patients from TCGA datasets ( n = 76). E Overall survival study of patients with high and low Pink1 expression was analyzed in breast cancer patients from TCGA datasets ( n = 1070). F Overall survival study of MUC1 low-ATAD3A high and MUC1 high-ATAD3A low breast cancer patients was analyzed from TCGA datasets ( n = 491). G Schematic overview of the work model. CCCP-induced mitochondrial impairment stimulates the translocation of MUC1 to mitochondria. In mitochondria, MUC1 binds to ATAD3A and promotes ATAD3A degradation through the ubiquitin-proteasome pathway, thereby protecting Pink1 from ATAD3A-mediated cleavage. Accumulation of Pink1 leads to increased pSer65-Ub and subsequently induces mitophagy to promote malignant properties of breast cancer. Targeting both MUC1 and mitophagy based on the combination of GO203 and Mdivi-1 essentially suppresses tumor growth.

Article Snippet: The mouse anti-human monoclonal ATAD3A primary antibody (Santa Cruz, sc-376185) was used at a dilution of 1:50, and the rabbit anti-human monoclonal MUC1 primary antibody (Cell Signaling Technology, 16564, United States of America) was used at a dilution of 1:200 overnight at 4 °C.

Techniques: Expressing, Translocation Assay, Ubiquitin Proteomics