Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Mucin 1 (MUC1) expression in human breast cancer. A , MUC1 expression levels in breast cancer (UALCAN website). B , MUC1 expression levels in different subtypes of breast cancer (UALCAN website). C , MUC1 protein expression in breast cancer (Human Protein Atlas database). D , MUC1 expression in human normal mammary epithelial cells MCF10A, TNBC cells (MDA-MB-231), and TNBC tissues (scale bar=100 μm). Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test. BRCA: breast cancer gene; TCGA: The Cancer Genome Atlas; TNBC: triple-negative breast cancer.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Expressing, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Construction and validation the Mucin 1 (MUC1) knockout cell line 231-MUC1-KO. A , Sequencing results after MUC1 knockout were confirmed by the Snapgene software to identify the monoclonal cell line 231-MUC1-KO. B , RT-qPCR for MUC1 in MDA-MB-231 and 231-MUC1-KO cells. C , Western blot for MUC1 in MDA-MB-231 and 231-MUC1-KO cells. Data are reported as means±SE. ****P<0.0001; two-tailed unpaired Student's t -test.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Biomarker Discovery, Knock-Out, Sequencing, Software, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Effect of Mucin 1 (MUC1) knockout on the proliferation, migration, and invasion of MDA-MB-231 cells. A , MDA-MB-231 cell proliferation by EDU assay (scale bar=100 μm). B , MDA-MB-231 cell proliferation by colony formation assay. C , Migration and invasion of MDA-MB-231 cells by transwell assay (scale bar=150 μm). Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Knock-Out, Migration, EdU Assay, Colony Assay, Transwell Assay, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Effect of Mucin 1 (MUC1) knockout on autophagy flow in MDA-MB-231 cells. A , Expression of LC3 I, LC3 II, and P62 proteins by western blot. B , Ad-mCherry-GFP-LC3B-labeled LC3B protein assessed the patency of intracellular autophagy flow (scale bar=100 μm). Data are reported as means±SE. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test. BafA1: Bafilomycin A1; Rapa: Rapamycin.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Knock-Out, Expressing, Western Blot, Labeling, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Effect of Mucin 1 (MUC1) knockout on lysosomes in MDA-MB-231 cells. A , Expression of SNAP29 and STXl7 by western blot. B , Expression of LAMP1 by western blot. C , pH was observed with red fluorescent probe Lysotracker Red (scale bar=100 μm). Data are reported as means±SE. **P<0.01, NS: not significant; two-tailed unpaired Student's t -test.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Knock-Out, Expressing, Western Blot, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Schematic diagram of the Mucin 1 (MUC1) oncogenic mechanism in breast cancer cell line MDA-MB-231. Knockout (KO) of MUC1 inhibits the proliferation, migration, and invasion of MDA-MB-231 cells by regulating the autophagic flow, and the possible mechanism may be related to the blocking of the autophagic flow caused by lysosomal damage.

Article Snippet: IHC data in the Human Protein Atlas database showed increased MUC1 protein expression in breast cancer tissue, but rarely expressed in normal breast tissue.

Techniques: Knock-Out, Migration, Blocking Assay

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Doxycycline inducible system for controlled expression of Muc1 or Muc1-GFP possessing 42 tandem repeats (TRs) in MCF10A epithelial cells. b , Isolation of stable cell clones with titratable expression of Muc1-GFP (1E7 clone) or Muc1 (B2 clone). c , Fluorescence and brightfield images of 1E7 cells induced at the indicated concentration of doxycycline. Scale bar, 10 μm. d , Flow cytometry analysis of 1E7 cells showing titratable Muc1-GFP levels across the indicated concentrations of doxycycline inducer. e , NK-92 cell-mediated cytotoxicity against 1E7 cells treated with control buffer or 100 nM StcE mucinase for 1 hour prior to co-culture; Muc1-GFP induction is at the indicated doxycycline concentration. Results are the mean ± s.d. of n=3 independent experimental replicates. f , NK-92 cell-mediated cytotoxicity against an MCF10A cell line expressing a Muc1-GFP construct with the TRs deleted (Muc1 0TR) under the control of a tetracycline inducible promoter at the indicated doxycycline induction level. Results are the mean ± s.d of n=3 technical replicates. g , Primary human NK cell-mediated cytotoxicity against 1E7 cells and 1E7 GNE KO cells at the indicated doxycycline concentration. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 biological replicates corresponding to three distinct human blood cell donors. h, Primary equine PBMC-mediated cytotoxicity against 1E7 cells at the indicated concentration of doxycycline; NK cell to target cell ratio is 20:1. Results are the mean ± s.d. of n=3 biological replicates corresponding to unique equine blood donors. i , NK-92 cell-mediated cytotoxicity against ZR-75-1 breast cancer cells sorted into sub-populations with low, intermediate, and high endogenous Muc1 expression. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 experimental replicates. j , NK-92 cell-mediated cytotoxicity against ZR-75-1 treated with control buffer or 100 nM StcE prior to co-culture. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 independent experimental replicates. Statistical analysis by two-tailed t tests. In panels e , g , and h , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Expressing, Isolation, Stable Transfection, Clone Assay, Fluorescence, Concentration Assay, Flow Cytometry, Control, Co-Culture Assay, Construct, Two Tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Schematic illustration of Ring-SAIM; interference of direct and reflected laser light off a silicon mirror creates standing waves of excitation light that depend on the laser incidence angle, θ, and are used to probe the glycocalyx thickness, which is measured as the gap between the substrate and fluorescently labelled membrane. b, Optical configuration for Ring-SAIM showing azimuthally scanned excitation light at varying θ; AOTF, Acousto-optic tunable filter. c , Representative widefield image and glycocalyx thickness map of live 1E7 cells expressing Muc1-GFP at the indicated doxycycline induction level. Scale bars, 10 μm. d , Glycocalyx thickness in ZR-75-1 cells before and after treatment with 100 nM StcE. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 6 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t test. e, Biosynthetic pathway for mucin-type O-glycans. f, Relative O-glycan distributions measured by the Cellular O-glycome Reporter Amplification (CORA) analysis in 1E7 wild-type, 1E7 GCNT1 overexpressing (OE), 1E7 C1GALT1 knockout (KO), and 1E7 GNE KO cells. g Glycocalyx thickness in the 1E7 cell panel at the indicated doxycycline concentration; some cellular treatments include 10 μM recombinant human galectin-1, 5 μM recombinant human galectin-3, or 10 μM TD139. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 13 cells from a representative experiment (n=3 independent experiments). Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. h , NK-92 cell-mediated cytotoxicity against the wild-type and engineered 1E7 cells at the indicated doxycycline induction level. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of at least n=3 independent experimental replicates. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. i , NK-92 cell-mediated cytotoxicity is inversely proportional to the measured glycocalyx thickness with Pearson correlation coefficient, r = −0.91; plotted data is from ( g ) and ( h ); dashed line indicates a linear fit to the data.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Membrane, Expressing, Two Tailed Test, Glycoproteomics, Amplification, Knock-Out, Concentration Assay, Recombinant

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Representative fluorescence images of endogenous GzmB-Gamillus in NK-92 cells following conjugation with Muc1-expressing B2 cells at minimal and maximal doxycycline induction of Muc1 expression; B2 cells also express cytosolic mScarlet-I as a red fluorescent marker. Scale bar, 10 μm. b , Polarity index for Gamillus-labelled cytolytic granules in NK-92 cell conjugates with B2 cells at the indicated doxycycline induction of Muc1 (0 and 1,000 ng/ml). Results are the mean ± s.d. of at least 38 cells over n=3 independent experimental replicates. Statistical analysis by two-tailed t test. c , Schematic showing structure of chimeric antigen receptor (CAR) targeting HER2 and CD19. d , Western blot analysis of HER2 in wild-type and HER2 overexpressing 1E7 cells (+ HER2 OE). e , NK-92 and HER2 CAR NK-92 cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated concentration of doxycycline. f , HER2 CAR-T cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated concentration of doxycycline. g , HER2 CAR T cell-mediated cytotoxicity against 1E7 HER2 OE cells at the indicated doxycycline induction level and target:effector ratio. h , Flow cytometry analysis of CD19 surface levels on 1E7 wild-type and CD19 overexpressing 1E7 cells (+ CD19 OE) cells at the indicated concentrations of doxycycline. i , NK-92 and CD19 CAR NK-92 cell-mediated cytotoxicity against 1E7 or CD19 OE 1E7 cells at the indicated concentration of doxycycline. n=3 independent experimental replicates. j , CD19 CAR-T cell-mediated cytotoxicity against 1E7 and 1E7 CD19 OE cells at the indicated concentration of doxycycline. k , CD19 CAR T cell-mediated cytotoxicity against 1E7 and 1E7 CD19 OE cells at the indicated doxycycline induction level and target:effector ratio. Unless otherwise indicated, effector cell to target cell ratio is 5:1. All results in e - g and i-k are shown as mean ± s.d. of n=3 independent experimental replicates. In panels e - f and i - j , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. HER2 CAR NK-92/T and CD19 CAR-T cells are second-generation CARs, while CD19 CAR NK-92 is a third-generation CAR.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Activation Assay, Fluorescence, Conjugation Assay, Expressing, Marker, Two Tailed Test, Western Blot, Concentration Assay, Flow Cytometry

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , StcE NK-92 cells prepared through tethering of StcE mucinase to the NK-92 cell surface by the native StcE X409 lectin domain. b , Surface levels of StcE and X409-deleted StcE (StcE ΔX409) on NK-92 cells following mucinase incubation at the indicated concentrations. c , ZR-75-1 cell killing by StcE NK-92 cells prepared with the indicated StcE concentration. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 technical replicates. d , Killing of 1E7 cells by NK-92, StcE NK-92, NK-92 cells tethered with catalytically dead StcE E447D, and NK-92 cells treated with StcE ΔX409. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=3 independent experimental replicates. e , Brightfield and Muc1-GFP fluorescence images of 1E7 cells conjugated with NK-92 or StcE NK-92 cells; lower images show zoomed-in regions of the NK-1E7 cell interface. Scale bar, 10 μm. f , Mean Muc1-GFP intensity (dark lines) and standard deviations (lighter bounding areas) along line profiles across the contact interface between 1E7 cell and NK-92 cells (n=6) or StcE NK-92 cells (n=10); the intensity is normalized to the mean intensity outside of the contact interface; red dotted arrows in e show representative profile lines. g , Glycocalyx thickness before and after treatment with 100 nM StcE. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 11 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t test. h, Cytotoxicity mediated by control and engineered NK-92 cells. Results are the mean ± s.d. of n=3 target cancer cell lines with individual data points indicating average cytotoxicity against a specific cell line (SKOV3, SKBR3, and 1E7 HER2 OE). NK cell to target cell ratio is 5:1 for 1E7 HER2 OE and 10:1 for SKOV3 and SKBR3. In d and h , statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. For all experiments, Muc1-GFP expression in 1E7 and 1E7 HER2 OE cells was with 1,000 ng/ml doxycycline.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Incubation, Concentration Assay, Fluorescence, Two Tailed Test, Control, Expressing

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a . Cartoon showing the design of Zip-NK-92 cells for modular surface display of glycocalyx-editing (GE) enzymes using leucine zippers. b , Flow cytometry analysis of sfGFP and ALFA-Tag on wild-type NK-92 and Zip-NK-92 cells expressing the membrane-anchored RR zipper. c , Binding isotherms for StcE-EE zipper fusion protein (StcE-EE) incubated with Zip-NK-92 cells (red) and wild-type StcE incubated with NK-92 cells (black). Representative normalized intensity plot from n=3 independent experimental replicates. d , Cytotoxicity mediated by Zip-NK-92 cells coupled with StcE-EE at the indicated concentration against Muc1-expressing B2 cells at the indicated doxycycline induction level. NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 technical replicates for each doxycycline level. e, f, Cytotoxicity mediated by Zip-NK-92 cells coupled with the indicated concentration of StcE-EE ( e ) and Sialidase-EE ( f ) against target cancer cell lines. NK cell to target cell ratio is 10:1. Results are the mean ± s.d. of n=4 or 5 target cancer cell lines (Capan-2, SKBR3, KPL-1, T47D, ZR-75-1). Statistical analysis by two-tailed t test.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Flow Cytometry, Expressing, Membrane, Binding Assay, Incubation, Concentration Assay, Two Tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a,b , Representative confocal and brightfield images of two clonally expanded MCF10A cell lines expressing Muc1-GFP with 42 tandem repeats (TRs) under the control of a tetracycline inducible promoter at the indicated doxycycline induction level. Scale bar, 10 μm. c , Representative confocal and brightfield images of Muc1 expresssing B2 clonal cells labelled with anti-Human Muc1 Janelia Fluor 549; the doxycycline induction level is indicated. Scale bar, 10 μm. d , NK-92 cell-mediated cytotoxicity against the polyclonal Muc1-GFP and three Muc1-GFP expressing clonal cell lines (1E7, 2E4, and 2G9) expressing MCF10A line from which the clonal lines were isolated, and a clonal MCF10A cell line expressing the Muc1 construct without the GFP reporter (B2 clone); the doxycycline induction levels are indicated. Results are the mean ± s.d of n=3 experimental replicates. NK cell to target cell ratio is 5:1. e . Mean fluorescence intensity of cell-surface Muc1-GFP in 1E7 cells treated with the indicated concentration of StcE mucinase; cell-surface Muc1-GFP probed with Alexa Fluor 647 conjugated GFP nanobody. Results are shown for induction of Muc1-GFP at 100 ng/ml and 1000 ng/ml doxycycline. Results are the mean ± s.d. of at least 14 cells from one representative of 3 independent experiments. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Expressing, Control, Isolation, Construct, Fluorescence, Concentration Assay

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Muc1-GFP size standards with biopolymer domains comprised of 0, 10, 21, and 42 tandem repeats (TRs). b , Western blot analysis of Muc1-GFP constructs stably expressed in MCF10A epithelial cells; arrows indicate fully glycosylated Muc1 with 10, 21, or 42 TRs; the primary antibody reacts with the Muc1 TRs and does not probe the 0TR Muc1. c , Flow cytometry analysis of cell-surface Muc1-GFP probed with Alexa Fluor 647 conjugated GFP nanobody. d , Examples of the pixelwise SAIM interferograms for Muc1-GFP constructs labelled with Alexa Fluor 647 conjugated GFP nanobody. e , Representative widefield image and glycocalyx thickness map of live MCF10A epithelial cells expressing the indicated Muc1 constructs labeled with Alexa Fluor 647 conjugated GFP nanobody; the Muc1 x42 mutant construct is comprised of 42 TRs of the mutated Muc1 tandem repeat – PD A RPAPG AT APPAHGV TA A – which has three of the five serine/threonine O-glycosylation sites mutated to alanine. Scale bars, 10 μm. f, g, Quantification of glycocalyx thickness in cells expressing the indicated Muc1 constructs (f) and the Muc1-expressing B2 clonal line induced at the indicated doxycycline levels (b). Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition include a minimum of 19 cells from a representative experiment (n=3 independent experiments). Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. h , Log of the mean glycocalyx thickness versus log of the mucin biopolymer length from ( f ); best-fit line to a power-law model with slope = 0.16 and R 2 = 0.62. i , Log of the mean glycocalyx thickness in 1E7 cells expressing Muc1-GFP at varying doxycycline induction levels versus the log of the mean GFP nanobody signal, a proportionate measure of Muc1 surface density; plotted data from Fig. 2f and Fig. 1d ; best-fit line to a power-law model has slope = 0.06 and R 2 = 0.79. j , Quantification of glycocalyx thickness in cells expressing the Muc1 x42 and the Muc1 x42 mutant construct. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 23 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t tests. Unless otherwise indicated, doxycycline induction of mucin expression is at 1,000 ng/ml.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Microscopy, Western Blot, Construct, Stable Transfection, Flow Cytometry, Expressing, Labeling, Mutagenesis, Glycoproteomics, Two Tailed Test

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Agarose gel electrophoresis of PCR-amplified and EcoRI-digested segment of C1GALT1 showing successful homozygous knockout (KO) in 1E7 cells (See 2A7 sub-clone of 1E7); the homology directed repair (HDR) template for CRISPR/Cas9 mediated C1GALT1 KO introduces a unique EcoRI site. b , Sangersequencing results of a PCR-amplified segment of C1GALT1 with insertion of EcoRI restriction site (GAATTC) and stop codon (TAA) in the 1E7 C1GALT1 KO clone, verifying homozygous KO. (Top: 1E7; bottom: 1E7 C1GALT1 KO) c , Fluorescence images of Glucosaminyl (N-Acetyl) Transferase 1 (GCNT1) in wild-type and GCNT1 overexpressing ( GCNT1 OE ) 1E7 cells with Muc1-GFP expression induced at 1,000 ng/ml of doxycycline; GCNT1 is localized in the Golgi. d , Relative composition of Core O-glycans in wild-type, 1E7 GCNT1 OE, and 1E7 GNE KO cells measured with the Cellular O-glycome Reporter (CORA) method e , Flow cytometry analysis of wild-type and glycoengineered 1E7 cells. Results for the parental MCF10A epithelial cells that do not express Muc1-GFP (-Muc1-GFP) are included as a control. f , Western blot showing the relative size of Muc1 in the wild-type and glycoengineered 1E7 cells. g , sWGA lectin blot after extended SDS-PAGE run time showing increased molecular weight of Muc1-GFP in 1E7 GCNT1 OE cells compared to wild-type 1E7 cells. h , Rate of proliferation of wild-type and glycoengineered 1E7 cells. Results are the mean of 2 independent experiment replicates. i , Flow cytometry analysis of 1E7 cells and their glycoengineered progeny induced at 0 ng/ml and 1,000 ng/ml of doxycycline.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Biomarker Discovery, Agarose Gel Electrophoresis, Amplification, Knock-Out, CRISPR, Fluorescence, Expressing, Flow Cytometry, Control, Western Blot, SDS Page, Molecular Weight

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a , Quantification of recombinant human galectin-3 (rhGal-3) binding to wild-type, C1GALT1 knockout (KO), and GCNT1 overexpressing (OE) 1E7 cells; data normalized to the Muc1 signal intensity and presented as the mean and ± s.d. of 20 cells from one representative of n=3 independent experiments. b , Same as in a , with 5,000 U/mL PNGase F treatment to selectively remove N-glycans prior to analysis. c , Quantification of endogenous cell-surface galectin-3 (Gal-3) and Muc1 before and after treatment with 200 nM StcE mucinase. Results are the mean ± s.d. of at least 11 cells from one representative of n=3 independent experiments. Statistical analysis by two-tailed t tests. d , Representative confocal images of Muc1-GFP and immuno-labelled Gal-3 in wild-type and LGALS3 overexpressing ( LGALS3 OE) 1E7 cells. Scale bars, 10 μm. e , Quantification of endogenous Gal-3 on the 1E7 cell surface following treatment with the indicated concentration of galectin inhibitor, TD139; data normalized to the Muc1 signal intensity and presented as the mean ± s.d. of 10 cells from one representative of n=3 independent experiments. f , Representative confocal images of endogenous galectin-3 on the cell membrane in presence and absence of 50μM TD139. Scale bars, 10 μm. g , Quantification of recombinant human galectin-1 (rhGal-1) binding to wild-type and glycoengineered 1E7 cells; data normalized to the Muc1 signal intensity and presented as the mean ± s.d. of 20 cells from one representative of n=3 independent experiments. h , Same as in g with 5,000 U/mL PNGase F treatment to selectively remove N-glycans prior to analysis. i , FRAP analysis of Muc1-GFP in wild-type and 1E7 GCNT1 OE cells treated with control buffer, as well as 1E7 GCNT1 OE cells treated with 10 μM rhGal-1. Results are the mean ± s.d. of intensity profiles of n=11, 10, 9 cells, respectively. j,k , Quantification of mobile fraction ( j ) and half recovery time ( k ) from ( i ). In all experiments, Muc1-GFP expression was induced with 1,000 ng/ml of doxycycline. Unless otherwise indicated, statistical analysis by one-way ANOVA with Tukey’s post-hoc tests.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Recombinant, Binding Assay, Knock-Out, Two Tailed Test, Concentration Assay, Membrane, Control, Expressing

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a, Quantification of 1E7 cell death resulting from treatment with the indicated concentrations of recombinant perforin for 30 minutes; Muc1-GFP expression induced at 1, 100, 1,000 ng/ml doxycycline. Results are the mean ± s.d. of n=3 technical replicates for one representative of three independent experiments. b , Quantification of cell viability (%) of uninduced (0 ng/ml) and doxycyclineinduced (1,000 ng/ml) 1E7 cells following treatment with indicated concentration of doxorubicin for 48 hours. Results are the mean ± s.d. of n=3 technical replicates.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Expressing, Recombinant, Concentration Assay

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: Using CRISPR/Cas9 and an appropriate homology directed repair template, the gene sequence for a flexible GGSGGSGGS linker and the pH-tolerant green fluorescent protein, Gamillus, was inserted immediately before the endogenous GZMB stop codon in NK-92 cells. a , Representative brightfield and fluorescence images of NK-92 GzmB-Gamillus cells stained with Lysotracker Deep Red. Scale bar, 10μm. b , Pearson’s coefficients of correlation between GzmB-Gamillus and Lysotracker. Results are the mean ± s.d. of 20 cells. c , d , NK-92 GzmB-Gamillus mediated cytotoxicity against 1E7 with Muc1-GFP expression induced at the indicated doxycycline concentration. Results are the mean ± s.d. of n=3 independent experimental replicates. NK cell to target cell ratio is 10:1. Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. e , Western blot showing the relative expression level of Granzyme B (GzmB) in the NK-92 cells and NK-92 GzmB-Gamillus knock-in (KI) cells. Note that the acidlabile linker between GzmB and Gamillus is expected to undergo cleavage in the low pH of lysosomes and cytolytic granules.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Biomarker Discovery, Labeling, CRISPR, Sequencing, Fluorescence, Staining, Expressing, Concentration Assay, Western Blot, Knock-In

Journal: Nature materials

Article Title: Immunoengineering can overcome the glycocalyx armor of cancer cells

doi: 10.1038/s41563-024-01808-0

Figure Lengend Snippet: a, Representative confocal images of Muc1-GFP on 1E7 cells treated with control buffer or 100 nM StcE, StcE ΔX409, StcE ΔX409 fused to the EE leucine zipper (StcE-EE); Alexa Fluor 647 conjugated GFP nanobody is used to probe cell-surface Muc1-GFP constructs. Scale bar, 10 μm. b , Viability of NK-92 and Zip-NK-92 after treatment with indicated concentration of StcE-EE. Propidium iodide (20 μg/ml) was used to detect dead cell population by flow cytometry. c , Cytotoxicity mediated by Zip-NK-92 cells coupled with the indicated concentration of Sialidase-EE zipper fusion protein (Sialidase-EE) against B2 cells at the indicated doxycycline induction level; NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n=3 technical replicates for each doxycycline concentration.

Article Snippet: For sorting, ZR-75-1 cells were labelled with anti-Human Muc1 Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals) diluted 1:200 in 0.5% BSA PBS at 4°C for 1 hour and sorted the cells with defined standardized gates for low, intermediate, and high Muc1 expression by FACS (Sony MA900 Cell Sorter).

Techniques: Control, Construct, Concentration Assay, Flow Cytometry

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The expression characteristics of MUC1 in lung adenocarcinoma (LUAD). (A) the expression of MUC1 in normal and LUAD tissues; (B) the expression of MUC1 in different stages; (C) the expression of MUC1 in different T stages; (D) the expression of MUC1 in different N stages; (E) the expression of MUC1 in different M stages; (F) the expression of MUC1 in different genders; (G) the expression of MUC1 in different smoking history; (H) the protein expression of MUC1 in normal and LUAD tissues.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The immune infiltration analysis of MUC1 expression. (A) The immune cell infiltration in high-MUC1 group and low-MUC1 group; (B) the correlation between MUC1 expression and differential immune cells, the length of the line represents the correlation, the direction of the line represents the positive or negative correlation, and the colour and size of the end point represents p -value.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The immune correlation analysis between high-MUC1 group and low-MUC1 group. (A) The effect of MUC1 copy number variation on the immune cells infiltration in LUAD; B-E) the immune score M (B), stromal score (C), tumour purity (D), and ESTIMATE score (E) in high-MUC1 group and low-MUC1 group. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques:

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The expression of MUC1, EGFR, and IL-6 in A549 and A549/TAX cells. (A) The expression of MUC1, EGFR, and IL-6 in LUAD cells (A549) and paclitaxel-resistant LUAD cells (A549/TAX); (B) the protein expression of MUC1, EGFR/p-EGFR, and IL-6 in A549 cells and A549/TAX cells; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: Knock down MUC1 reduces the expression of stem cell pluripotency factor in paclitaxel resistant LUAD cells. (A) screening of effective interference targets for knock down MUC1 in A549 cells and A549/tax cells; (B) cell Counting kit-8 assay (CCK-8) detects the cellular activity of negative control cells (A549/TAX-NC) and MUC1 interfering cells (A549/TAX-siRNA-MUC1); (C) the expression of MUC1, EGFR, and IL-6 in A549/TAX-NC and A549/TAX-siRNA-MUC1 cells; (D) the expression of Oct4, Sox2, and myc in A549/TAX-NC and A549/TAX-siRNA-MUC1 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Knockdown, Expressing, Cell Counting, CCK-8 Assay, Activity Assay, Negative Control

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The differentially expressed genes analysis of between A549/Tax-NC and A549/TAX-siRNA-MUC1. (A–B) The volcano plot (A) and heat map (B) of DEGs between A549/Tax-NC and A549/TAX-siRNA-MUC1; (C–E) the expression of ABCB1 (C), ABCC2 (D), and ABCG2 (E) between A549/Tax-NC and A549/TAX-siRNA-MUC1.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: The top 20 DEGs between A549/tax and A549/TAX-siRNA-MUC1.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques:

Journal: Annals of Medicine

Article Title: MUC1-EGFR crosstalk with IL-6 by activating NF-κB and MAPK pathways to regulate the stemness and paclitaxel-resistance of lung adenocarcinoma

doi: 10.1080/07853890.2024.2313671

Figure Lengend Snippet: GO and KEGG analysis of DEGs between A549/Tax-NC and A549/TAX-siRNA-MUC1. (A) The GO enrichment of up-regulated DEGs; (B) The KEGG pathway of up-regulated DEGs; (C) The GO enrichment of down-regulated DEGs.

Article Snippet: The protein expression of MUC1 in LUAD tissues and normal tissues were retrieved in the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Signal pathway and mechanism of MUC1-C regulating TNBC Occurrence and progression (Comprehensive arrangement according to the published articles).

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Schematic illustration of MUC1-C shRNA@Fe 3 O 4 MNPs construction and the therapeutic mechanism on TNBC. MUC1-C shRNA and Fe 3 O 4 MNPs were self-assembled into MUC1-C shRNA@ Fe 3 O 4 MNPs by electrostatic adsorption. After entering TNBC cells and under the action of AMF, on the one hand, it induces apoptosis and ferroptosis by targeting MUC1-C, on the other hand, it increases the temperature (43 °C) and the Fe 2+ concentration directly kills TNBC cells and stimulates apoptosis and ferroptosis mechanisms.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Adsorption, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( D ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ). IHC results of MUC1 in TNBC tissues, paraneoplastic tissues and normal breast tissues ( F ). Error bars represent means ± SD. **p<0.01, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: Expressing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Silence efficiency of MUC1-C shRNA on TNBC cell line HCC-70. RT-qPCR ( A ) and Western blot detected the silencing efficiency of MUC1-C shRNA ( B and C ). MUC1-C shRNA (3#) plasmid map ( D ) and target sequence (the highlighted yellow section) ( E ). Error bars represent means ± SD. ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Sequencing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Characterization of MUC1-C shRNA@Fe 3 O 4 MNPs. Appearance ( A ) and TEM image ( D ) of the prepared MUC1-C shRNA@Fe 3 O 4 MNPs. DSL Size distribution of Fe 3 O 4 MNPs ( B and C ) and MUC1-C shRNA@Fe 3 O 4 MNPs ( E and F ). Tyndall effect of MUC1-C shRNA@Fe 3 O 4 MNPs ( G ). Zeta potential of MUC1-C shRNA@Fe 3 O 4 MNPs ( H ). Encapsulation efficiency at different shRNA/MNPs ratios ( I ). TEM elemental mapping analysis of MUC1-C shRNA@Fe 3 O 4 MNPs ( J ). Raman spectrum of MUC1-C shRNA, Fe 3 O 4 MNPs, and MUC1-C shRNA@Fe 3 O 4 MNPs ( K ). Release profiles of Fe 2+ from MUC1-C shRNA@Fe 3 O 4 MNPs at different pH values with or without AMF ( L ). The heating curves of MUC1-C shRNA@Fe 3 O 4 MNPs with different concentrations (50–250μg/mL) ( M ). Hemolysis test at different concentrations (MUC1-C shRNA@Fe 3 O 4 MNPs) (n=3) ( N and O ). Hemolysis test (200μg/mL, n=3) ( P and Q ). Error bars represent means ± SD. TEM scale bar: 50nm.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Zeta Potential Analyzer, Encapsulation

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Cellular uptake of NPs. Cellular uptake of MUC1-C shRNA@Fe 3 O 4 MNPs in the HCC-70 cells was observed under a CLSM. Green fluorescence indicates MUC1-C shRNA@Fe 3 O 4 -FITC and blue fluorescence indicates the nucleus ( A ). Investigation of internalization pathway of MUC1-C shRNA@Fe 3 O 4 MNPs in HCC-70 cells in the presence of different endocytosis inhibitors (n=3) ( B ). Error bars represent means ± SD. Scale bar: 50μm. ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: MUC1-C shRNA@Fe 3 O 4 MNPs suppress the migration, invasion, and proliferation in HCC-70 cell lines. Wound healing ( A ), Transwell ( B ), CCK-8 OD 450nm ( C ), and inhibition rate based on CCK-8 ( D and E ). The protein bands ( F ) and quantitative statistical results ( G ) of Bax, cleaved-caspase3, GPX4, NRF2, and FTH1 of HCC-70 cell lines (n=3). Error bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Migration, CCK-8 Assay, Inhibition

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: The in vivo therapeutic effects of the MUC1-C shRNA@Fe 3 O 4 MNPs on TNBC. IR thermal images of TNBC-bearing C-NKG mice with local injection of MUC1-C shRNA@Fe 3 O 4 MNPs (200 µg mL −1 ), or normal saline under AMF (3Kw) ( A ). Images ( B ) and weights ( C ) of xenograft tumors harvested from the mice after various treatments on the 25th day. Tumor volumes ( D ) and Body weight ( E ) growth in mice were measured after different treatments every 5 days within 25 days (n=4). Prussian blue reaction of tumor tissue showing iron staining and tumor necrosis, labeled area was typical necrosis (200x) ( F ). Expression of Bax, cleaved-caspase 3, GPX4, NRF2, and FTH1 in tumor tissues ( G ). Quantitative statistical results according to the protein bands ( H ). Error bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: In Vivo, shRNA, Injection, Saline, Staining, Labeling, Expressing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Serum biochemistry indicators assessment of TNBC mice (n=4). Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Total bilirubin (TBIL). Blood urea nitrogen (BUN), Creatinine (CR) ( A ). Effect of MUC1-C shRNA@Fe 3 O 4 MNPs on the histopathology of heart, lung, spleen, Kidney, and liver from TNBC mice ( B ). Error bars represent means ± SD. Scale bar: 100μm.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Histopathology

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Signal pathway and mechanism of MUC1-C regulating TNBC Occurrence and progression (Comprehensive arrangement according to the published articles).

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Schematic illustration of MUC1-C shRNA@Fe 3 O 4 MNPs construction and the therapeutic mechanism on TNBC. MUC1-C shRNA and Fe 3 O 4 MNPs were self-assembled into MUC1-C shRNA@ Fe 3 O 4 MNPs by electrostatic adsorption. After entering TNBC cells and under the action of AMF, on the one hand, it induces apoptosis and ferroptosis by targeting MUC1-C, on the other hand, it increases the temperature (43 °C) and the Fe 2+ concentration directly kills TNBC cells and stimulates apoptosis and ferroptosis mechanisms.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Adsorption, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( D ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ). IHC results of MUC1 in TNBC tissues, paraneoplastic tissues and normal breast tissues ( F ). Error bars represent means ± SD. **p<0.01, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: Expressing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Silence efficiency of MUC1-C shRNA on TNBC cell line HCC-70. RT-qPCR ( A ) and Western blot detected the silencing efficiency of MUC1-C shRNA ( B and C ). MUC1-C shRNA (3#) plasmid map ( D ) and target sequence (the highlighted yellow section) ( E ). Error bars represent means ± SD. ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Sequencing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Characterization of MUC1-C shRNA@Fe 3 O 4 MNPs. Appearance ( A ) and TEM image ( D ) of the prepared MUC1-C shRNA@Fe 3 O 4 MNPs. DSL Size distribution of Fe 3 O 4 MNPs ( B and C ) and MUC1-C shRNA@Fe 3 O 4 MNPs ( E and F ). Tyndall effect of MUC1-C shRNA@Fe 3 O 4 MNPs ( G ). Zeta potential of MUC1-C shRNA@Fe 3 O 4 MNPs ( H ). Encapsulation efficiency at different shRNA/MNPs ratios ( I ). TEM elemental mapping analysis of MUC1-C shRNA@Fe 3 O 4 MNPs ( J ). Raman spectrum of MUC1-C shRNA, Fe 3 O 4 MNPs, and MUC1-C shRNA@Fe 3 O 4 MNPs ( K ). Release profiles of Fe 2+ from MUC1-C shRNA@Fe 3 O 4 MNPs at different pH values with or without AMF ( L ). The heating curves of MUC1-C shRNA@Fe 3 O 4 MNPs with different concentrations (50–250μg/mL) ( M ). Hemolysis test at different concentrations (MUC1-C shRNA@Fe 3 O 4 MNPs) (n=3) ( N and O ). Hemolysis test (200μg/mL, n=3) ( P and Q ). Error bars represent means ± SD. TEM scale bar: 50nm.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Zeta Potential Analyzer, Encapsulation

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Cellular uptake of NPs. Cellular uptake of MUC1-C shRNA@Fe 3 O 4 MNPs in the HCC-70 cells was observed under a CLSM. Green fluorescence indicates MUC1-C shRNA@Fe 3 O 4 -FITC and blue fluorescence indicates the nucleus ( A ). Investigation of internalization pathway of MUC1-C shRNA@Fe 3 O 4 MNPs in HCC-70 cells in the presence of different endocytosis inhibitors (n=3) ( B ). Error bars represent means ± SD. Scale bar: 50μm. ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: MUC1-C shRNA@Fe 3 O 4 MNPs suppress the migration, invasion, and proliferation in HCC-70 cell lines. Wound healing ( A ), Transwell ( B ), CCK-8 OD 450nm ( C ), and inhibition rate based on CCK-8 ( D and E ). The protein bands ( F ) and quantitative statistical results ( G ) of Bax, cleaved-caspase3, GPX4, NRF2, and FTH1 of HCC-70 cell lines (n=3). Error bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Migration, CCK-8 Assay, Inhibition

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: The in vivo therapeutic effects of the MUC1-C shRNA@Fe 3 O 4 MNPs on TNBC. IR thermal images of TNBC-bearing C-NKG mice with local injection of MUC1-C shRNA@Fe 3 O 4 MNPs (200 µg mL −1 ), or normal saline under AMF (3Kw) ( A ). Images ( B ) and weights ( C ) of xenograft tumors harvested from the mice after various treatments on the 25th day. Tumor volumes ( D ) and Body weight ( E ) growth in mice were measured after different treatments every 5 days within 25 days (n=4). Prussian blue reaction of tumor tissue showing iron staining and tumor necrosis, labeled area was typical necrosis (200x) ( F ). Expression of Bax, cleaved-caspase 3, GPX4, NRF2, and FTH1 in tumor tissues ( G ). Quantitative statistical results according to the protein bands ( H ). Error bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: In Vivo, shRNA, Injection, Saline, Staining, Labeling, Expressing

Journal: International Journal of Nanomedicine

Article Title: The Therapeutic Effects of MUC1-C shRNA@Fe 3 O 4 Magnetic Nanoparticles in Alternating Magnetic Fields on Triple-Negative Breast Cancer

doi: 10.2147/IJN.S426849

Figure Lengend Snippet: Serum biochemistry indicators assessment of TNBC mice (n=4). Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Total bilirubin (TBIL). Blood urea nitrogen (BUN), Creatinine (CR) ( A ). Effect of MUC1-C shRNA@Fe 3 O 4 MNPs on the histopathology of heart, lung, spleen, Kidney, and liver from TNBC mice ( B ). Error bars represent means ± SD. Scale bar: 100μm.

Article Snippet: Figure 1 MUC1 and its expressions in various types of tumors (from UCSC genome browser on human, the red pentagram indicates breast invasive carcinoma) ( A ), MUC1 protein expression in different tumor cell lines ( B ) and BRCA cell lines ( C ) (based on datasets of Human Protein Atlas), MUC1 expression between BRCA and normal tissue ( ) (based on datasets of The Cancer Genome Atlas), MUC1 expression in normal human breast cell (MCF-10A) and TNBC cell lines (BT-20, MDA-MB-231, MDA-MB-468, HCC-70) ( E ).

Techniques: shRNA, Histopathology

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 4 Activated EGFR upregulates MUC1-CT expression. a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1- CT in EGFR TL (T790M; L858R)-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL-induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox- impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL-induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL-induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test (c, d, e, g); one-way ANOVA (a, b); Brown-Forsythe and Welch ANOVA (f, h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1- CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c, d Growth of PDX (c) and transgenic mouse EGFR TL- induced lung tumour (d)-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA (b, c, d). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Blocking Assay, Transgenic Assay, Derivative Assay