Journal: Oncogenesis

Article Title: Protein tyrosine kinase 6 regulates mammary gland tumorigenesis in mouse models

doi: 10.1038/oncsis.2013.43

Figure Lengend Snippet: Generation of MMTV-PTK6 transgenic mice. ( a ) A schematic diagram of the MMTV-PTK6 construct is shown. A 2.2 kb human PTK6 complementary DNA (gray region) was inserted into the third exon of the rabbit β-globin gene under the control of the MMTV LTR (striped region). ( b ) Expression of the MMTV-PTK6 construct transfected into NMuMG cells stimulated with dexamethasone. Transgenic PTK6 protein levels (NMuMG +Tg) are comparable to that produced in human breast cancer cell lines MCF7, MDA-MB-231 and MDA-MB-453. PTK6 was not detected in the MDA-MB-435 cell line. Expression of β-actin was examined as a loading control. ( c ) Ribonuclease protection assays were performed with RNAs prepared from mammary glands of three transgenic lines (B28, B33 and B35) and nontransgenic control mice (NT). PTK6 mRNA was detectable in the three transgenic lines but not in NT animals. Mouse cyclophilin was used as loading control. ( d ) Ectopic PTK6 expression was detected in transgenic mammary glands by immunoblotting. Levels of ectopic PTK6 protein expression correlated with the levels of PTK6 mRNA shown in c . ( e ). Immunohistochemistry demonstrates expression of ectopic human PTK6 in the transgenic mammary gland epithelial cells, as shown in the virgin animals (B28, B33 and B35) and pregnant animals (B33.Pg). The nontransgenic mammary gland stained negative for PTK6. Size bar=20 μm.

Article Snippet: The human breast cancer cell lines MCF7 (HTB-22), MDA-MB-231 (HTB-26) and MDA-MB-453 (HTB-131), human melanoma cell line MDA-MB-435S (HTB-129) and the mouse mammary epithelial cell line NMuMG (CRL-1636) were obtained from ATCC (American Type Culture Collection) and cultured according to the ATCC guidelines.

Techniques: Transgenic Assay, Construct, Control, Expressing, Transfection, Produced, Western Blot, Immunohistochemistry, Staining

Journal: The Journal of biological chemistry

Article Title: Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. Isolation, genomic structure, and functional expression of the receptors.

doi: 10.1074/jbc.270.50.30102

Figure Lengend Snippet: FIG. 8. Northern blot analysis of the NPY-Y1a and NPY-Y1b receptor mRNAs. Expression of the NPY-Y1a (a) and NPY-Y1b re- ceptor mRNA (b) in various mouse tissues and embryo. Each lane contains 2 mg of poly(A)1 RNA. In c, b-actin probe was used as an internal control. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis; lane 9, embryo (7 days); lane 10, embryo (11 days); lane 11, embryo (15 days); and lane 12, embryo (17 days).

Article Snippet: Northern Blot Analysis—Mouse Multiple Tissue Northern blot and Mouse Embryo Multiple Tissue Northern blot were purchased from Clontech that contained 2 mg of poly(A)1 RNA from various sources.

Techniques: Northern Blot, Expressing, Control

Journal: Oncotarget

Article Title: Apoptosis-linked gene 2 promotes breast cancer growth and metastasis by regulating the cytoskeleton

doi: 10.18632/oncotarget.13740

Figure Lengend Snippet: ( A and D ) Immunoblot analysis of ALG-2 and β-actin in MDA-MB-231 (A) and BT549 (D) cells. ( B and E ) Silencing of ALG-2 inhibits the proliferation of MDA-MB-231 (B) and BT549 (E) cells as determined by MTT and SRB assays. ( C and F ) Colony formation by control or ALG-2 siRNA-transfected MDA-MB-231 (C) and BT549 (F) cells cultured for 2 weeks. ( G and J ) Flow cytometric analysis of apoptosis in MDA-MB-231 (G) and BT549 (J) cells 72 hours after transfection with control or ALG-2 siRNAs. ( H and I ) Experiments were performed as in G, and the percentages of early apoptotic cells (annexin V-FITC+, propidium iodide-) and late apoptotic cells (annexin V-FITC+, propidium iodide+) were quantified. For each group, 1 × 10 5 cells were counted. ( K and L ) Experiments were performed as in J, and the percentages of early and late apoptotic cells were quantified. For each group, 1 × 10 5 cells were counted. Error bars indicate means ± SEM. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: MDA-MB-231 and BT549 human breast cancer cells, MCF-10A immortalized human breast epithelial cells, and HEK293T human embryonic kidney epithelial cells were purchased from the American Type Culture Collection.

Techniques: Western Blot, Control, Transfection, Cell Culture

Journal: Oncotarget

Article Title: Apoptosis-linked gene 2 promotes breast cancer growth and metastasis by regulating the cytoskeleton

doi: 10.18632/oncotarget.13740

Figure Lengend Snippet: ( A ) Wound healing assays using MDA-MB-231 and BT549 cells transfected with control or ALG-2 siRNAs. Wound margins were imaged 16 hours after wounding. ( B ) Experiments were performed as in A, and the percentage of wound closure was quantified. ( C ) Wound healing assays using 4T1-luc cells transfected with control or mALG-2 siRNAs. ( D ) Experiments were performed as in C, and the percentage of wound closure was quantified. ( E ) Wound healing assays using BT549 cells transfected with RFP-V or RFP-ALG-2. ( F ) Experiments were performed as in E, and the percentage of wound closure was quantified. ( G ) Transwell migration assays using MDA-MB-231 and BT549 cells transfected with control or ALG-2 siRNAs. ( H ) Experiments were performed as in G, and the extent of transwell migration was quantified. ( I ) Transwell migration assays using 4T1-luc cells transfected with control or mALG-2 siRNAs. ( J ) Experiments were performed as in I, and the extent of transwell migration was quantified. ( K – M ) Analysis of random migration of MDA-MB-231 cells. Cell movement paths were tracked (K), and the velocity of cell movement (L) and the accumulated distance were analyzed (M). Error bars indicate means ± SEM. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: MDA-MB-231 and BT549 human breast cancer cells, MCF-10A immortalized human breast epithelial cells, and HEK293T human embryonic kidney epithelial cells were purchased from the American Type Culture Collection.

Techniques: Transfection, Control, Migration

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal T47D cells with mdm2 shRNA or control vector were treated with or without 4μg/ml doxycycline (dox) for 3 days, followed by 10nM estrogen for 5 days in the presence or absence of dox. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, p53 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. The graph represents an average of three independent experiments with standard deviation in inducible clonal T47D cells with mdm2 shRNA or control vector. (C) A constitutive pool of T47D cells with mdm2 shRNA or control vector were grown with or without 10nM estrogen for 5 days. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, total Rb and Actin protein levels from 50μg whole cell protein extract is shown. (D) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. Graph represents average of three independent experiments with standard deviation in constitutive pool of T47D cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, *** represents a p-value ≤ 0.001. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D.sh mdm2 cells were treated with 10nM estrogen (lane 1) and either had MDM2 knockdown (lane 2) or 10μM fulvestrant treatment (lane 3), or both (lane 4) for 5 days. A representative western blot analysis of MDM2, phosphoRb and Actin protein levels from 50μg whole cell protein extract is shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to Actin from three independent experiments. (B) MTT assay was performed in T47D.control vector and inducible T47D.sh mdm2 clonal cell lines after treatments. Percentage mitochondrial activity represents an average of 2 independent experiments. (A) & (B) * represents a p-value ≤ 0.05, *** represents a p-value ≤ 0.001, **** represents a p-value ≤ 0.0001. The p-value was determined by 2-tailed Student t-test. (C) A representative live cell image by confocal microscopy with 20X objective of T47D.vector and inducible clonal T47D.sh mdm2 clonal cell lines after treatments. Red fluorescence represents staining with propidium iodide. Blue fluorescence represents staining of nuclear DNA. (D) MDM2 drives phosphorylation of Rb in ER+ breast cancer cells. In vitro kinase assay was performed to detect phosphorylation of Rb with or without overnight estrogen treatment in either presence or absence of bacterially expressed and purified MDM2 (1μl or 2μl). A representative image of Western blot analysis of MDM2, phospho Rb and total Rb protein level from nuclear extract of MCF7 (left) and T47D (right) cells are shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to lamin A from three independent experiments, when 1μl of purified MDM2 was added to the nuclear extracts of MCF7 (left) and T47D (right) cells. The p-value for MCF7 cells with overnight estrogen treatment and addition of purified MDM2 (compare lane 4 to lane 5) was statistically significant. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Western Blot, MTT Assay, Control, Plasmid Preparation, Activity Assay, Confocal Microscopy, Fluorescence, Staining, Phospho-proteomics, In Vitro, Kinase Assay, Purification

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Number of large colonies (50μm or larger) determined by counting the colonies of MCF7 cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA induction (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (160, 158, 175), control vector + shRNA induction (153, 151, 197), mdm2 shRNA –shRNA induction (140, 137, 200) and mdm2 shRNA + shRNA induction (17, 11, 8). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.002. (B) Number of large colonies (100μm or larger) determined by counting the colonies of T47D cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (220, 191, 70), control vector + shRNA induction (202, 195, 33), mdm2 shRNA –shRNA induction (166, 175, 69) and mdm2 shRNA + shRNA induction (27,20, 9). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.02. (C) Representative images of colonies that T47D cells formed in soft agar in the presence or absence of MDM2 knockdown. (D) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in presence or absence of 4μg/ml doxycycline, were fixed and stained with propidium iodide. Colony masses were categorized in 5 different groups and the number of masses in each group were counted and presented as percentages in the total population. This is an average of two independent experiments. The total number of masses scored for 2 independent experiments were control vector –shRNA induction (20, 15), control vector + shRNA induction (25, 27), mdm2 shRNA –shRNA induction (93, 95) and mdm2 shRNA + shRNA induction (86, 83). The p-value was determined by 2-tailed Student t-test. The p-value for large and small colonies for comparisons with and without MDM2 knockdown were p-value=0.03 and p-value=0.05 respectively. Two independent scorers counted the numbers of colonies for each independent experiment.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Fluorescence, Microscopy, Control, Plasmid Preparation, Knockdown, Staining

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in the presence or absence of 4 μg/ml dox, were fixed, stained with F-Actin and mounted with DAPI containing mounting media. Confocal z-stack images were acquired. Masses with lumen were counted and presented as percent of total number of masses grown in 3D matrigel. An average of two independent experiments are shown. The number of masses counted for 2 independent experiments were control vector -shRNA induction (21, 41), control vector +shRNA induction (21, 47), mdm2 shRNA -shRNA induction (31, 46) and mdm2 shRNA +shRNA induction (31, 62). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.01. Two independent scorers counted the numbers of masses for each independent experiment. (B) A representative image from confocal immunofluorescence microscopy showing a single slice from z-stack of DAPI, GFP and F-Actin of estrogen treated inducible clonal T47D.sh mdm2 cells grown in 3D-matrigel in the presence or absence of 4μg/ml doxycycline (dox) for 3 weeks. The top and middle rows show hollow lumen and ductal lumen respectively in the presence of shRNA expression to mdm2 ; the GFP (green) indicates shRNA induction to mdm2 . The third row shows mass structure (disruption of normal mammary glandular architecture) in the absence of shRNA expression to mdm2 .

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Control, Plasmid Preparation, shRNA, Knockdown, Immunofluorescence, Microscopy, Expressing, Disruption

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) & (B) T47D cells ((A) inducible sh mdm2 clonal and vector pool; (B) constitutive sh mdm2 and vector pool) grown in the presence of estrogen in 3D matrigel for 3.5 weeks. The cells were fixed, permeabilized, blocked, stained with phospho-histoneH3 antibody and mounted with DAPI containing mounting media. Images were taken with confocal microscope. Quantitative analysis of phospho-histone H3 positive cells were performed by capturing optical Z-stack sections of masses and dividing the number of positive phospho-histone H3 cells by the total number of masses. (A) The number of masses counted in each group for 2 independent experiments were control vector -shRNA induction (29, 30), control vector +shRNA induction (30, 30), mdm2 shRNA -shRNA induction (29, 30) and mdm2 shRNA +shRNA induction (30, 30). (B) The number of masses counted in each group for 2 independent experiments were control vector (49, 60) and sh mdm2 (48, 60). Average of two independent experiments are shown for each knockdown method. The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.001 (A) and p-value=0.009 (B). Two independent scorers counted the numbers of masses for each independent experiment. (C) Representative confocal Z-stack image (single slice) showing DAPI, phospho-histone H3 and GFP in estrogen-treated inducible clonal T47D.sh mdm2 cells in the presence and absence of 4μg/ml doxycycline. (D) Cell cycle analysis by FACS (Fluorescence Activated Cell Sorting). T47D cells were harvested, fixed and stained with propidium iodide and subjected to cell cycle analysis by FACS. Data are presented as percent of cells in S phase in a total population of 10,000 cells and analyzed by FACS in each group. Average of 4 independent experiments are shown. The p-value was determined by 2-tailed Student t-test and * represents a p-value ≤ 0.05

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Plasmid Preparation, Staining, Microscopy, Control, shRNA, Knockdown, Cell Cycle Assay, Fluorescence, FACS

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal MCF7 cells with mdm2 shRNA or control vector were treated with and without 2μg/ml doxycycline(dox) for 3 days to induce shRNA expression, followed by 10nM estrogen for 5 days in the presence and absence of dox. A representative image of western blot analysis of phospho Rb, Total Rb, E2F1, MDM2 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for phospho Rb, E2F1 and MDM2 protein levels normalized to Actin. Graph represents average of four independent experiments with standard deviation in inducible clonal of MCF7 cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Expressing, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: Model showing that MDM2 is a central hub in estrogen signaling and works through an Rb-E2F1 pathway to promote proliferation. Breast cancer cells harboring SNP309 have increased binding of the transcription factor Sp1, which causes elevated MDM2 protein levels. Fulvestrant blocks the MDM2 pathway and the Rb-E2F1 pathway.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca 2+ Imbalance

doi: 10.3390/ijms23052648

Figure Lengend Snippet: Targeting PSMD14 induces cell death accompanied by vacuolation in breast cancer cells but not in normal breast cells. ( A , B ) MDA-MB 435S cells were transfected with the siRNA against PSMD14. ( A ) Cellular viability was assessed, as described in Materials and Methods, and knockdown of PSMD14 was confirmed by Western blotting. * p < 0.001 vs. siNC-transfected cells. ( B ) Cells were observed by phase-contrast microscopy. Bars, 20 μm. ( C , D ) Cells were treated with the indicated concentrations of CZM for 12 h or 8-TQ for 24 h. ( C ) Cellular viability was assessed using IncuCyte, as described in Materials and Methods. * p < 0.001 vs. untreated cells. ( D ) Cells were observed by phase-contrast microscopy. Bars, 20 μm. ( E ) Cells were treated with the indicated concentrations of CZM for 24 h, and cellular viability was assessed using IncuCyte. * p < 0.001 vs. control. ( F ) Cells were treated with 5 μM CZM for 12 h and observed by phase-contrast microscopy. Bars, 20 μm. ( G , H ) MDA-MB 435S cells pretreated with the indicated inhibitors of various cell death modes were further treated with 5 μM CZM for 24 h ( G ) or 12 h ( H ). ( G ) Cellular viability was assessed as described above. * p < 0.001 vs. control, # p < 0.001 vs. CZM-treated cells. ( H ) Cells were observed by phase-contrast microscopy. Bars, 20 μm. ( I ) MDA-MB 435S cells were transfected with the siRNA against PSMD14 or treated with 5 μM CZM or 200 ng/mL TRAIL for the indicated time points. Western blotting of caspase-3 and PARP was performed using β-actin as a loading control.

Article Snippet: MDA-MB 435S, MDA-MB 231, MDA-MB 468, and MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Knockdown, Western Blot, Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca 2+ Imbalance

doi: 10.3390/ijms23052648

Figure Lengend Snippet: Targeting PSMD14 induces paraptosis in MDA-MB 435S cells. ( A , B ) YFP-ER cells were treated with 5 μM CZM for the indicated time durations ( A ) or transfected with siRNA against PSMD14 for 36 h ( B ) and stained with MitoTracker-Red. Cells were observed under the confocal microscope. Bars, 10 μm. ( C ) MDA-MB 435S cells treated with 5 μM CZM for 12 h were subjected to electron microscopy. Bars, 5 μm. ( D ) YFP-ER cells pretreated with or without 1 μM CHX were further treated with 5 μM CZM for 12 h, stained with MitoTracker-Red, and observed by confocal microscopy. Bars, 10 μm.

Article Snippet: MDA-MB 435S, MDA-MB 231, MDA-MB 468, and MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Staining, Microscopy, Electron Microscopy, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca 2+ Imbalance

doi: 10.3390/ijms23052648

Figure Lengend Snippet: Proteasome inhibition alone is not sufficient to explain the anticancer effect of PSMD14. ( A ) Cell extracts were prepared from MDA-MB 435S cells treated with 5 μM CZM or transfected with the siRNA against PSMD14 for the indicated time durations. Western blotting of the indicated proteins was performed using β-actin as a loading control. ( B ) MDA-MB 435S cells transfected with Ub G76V -GFP were treated with the indicated concentrations of CZM or Bz for 12 h, and the fluorescence intensity was assessed as described in Materials and Methods. ( C ) Viability was assessed using IncuCyte in MDA-MB 435S cells treated with CZM or Bz at the indicated concentrations for 24 h. * p < 0.001 vs. untreated cells. ( D ) MDA-MB 435S cells were treated with 5 μM CZM or 5 nM Bz for 12 h and observed by phase-contrast microscopy. Bars, 20 μm ( E ). MDA-MB 435S cells were treated with 5 μM CZM or 5 nM Bz for the indicated time durations, and Western blotting of the indicated proteins was performed using β-actin as a loading control. ( F , G ) MDA-MB 435S cells were treated with 5 μM CZM or 5 nM Bz for 12 h, and immunocytochemistry of the indicated proteins was performed. Bars, 10 μm ( F ) or 5 μm ( G ).

Article Snippet: MDA-MB 435S, MDA-MB 231, MDA-MB 468, and MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Transfection, Western Blot, Control, Fluorescence, Microscopy, Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca 2+ Imbalance

doi: 10.3390/ijms23052648

Figure Lengend Snippet: PSMD14 inhibition induces intracellular Ca 2+ imbalance in MDA-MB 435S cells. ( A , B ) MDA-MB 435S cells were treated with 5 μM CZM for the indicated time durations, stained with Fluo-3 ( A ) or Rhod-2 ( B ) and processed for FACS analysis. The results of FACS analysis after treatment with 5 μM CZM for 8 h are depicted in the graphs. ( C ) MDA-MB 435S cells treated with the indicated chemicals were observed under the confocal microscope. Bars, 10 μm. ( D ) YFP-Mito cells treated with the indicated chemicals were stained with Rhod-2 and observed under the confocal microscope. Celastrol was used as a positive control to confirm the increase in Ca 2+ in the cytosol and mitochondria. Bars, 10 μm. ( E ) MDA-MB 435S cells transfected with the plasmids encoding G-CEPIA1er and cyto-RCaMP1h or cells transfected with those G-CEPIA1er and mito-RCaMP1h were treated with 5 μM CZM for the indicated time durations. Subcellular Ca 2+ dynamics were monitored under the confocal microscope, and representative images were displayed. Bars, 10 μm. Time course Ca 2+ signal in the ER, cytosol, and mitochondria in CZM-treated cells was analyzed using Image J/Fiji software. ( F ) YFP-ER cells pretreated with 10 μM BAPTA-AM or 20 μM Ru360 were further treated with 5 μM CZM for 8 h and stained with Rhod-2. Cells were observed under the confocal microscope. Bars, 20 μm.

Article Snippet: MDA-MB 435S, MDA-MB 231, MDA-MB 468, and MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Staining, Microscopy, Positive Control, Transfection, Software

Journal: International Journal of Molecular Sciences

Article Title: PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca 2+ Imbalance

doi: 10.3390/ijms23052648

Figure Lengend Snippet: Disruption of both proteostasis and Ca 2+ homeostasis is required for the paraptosis induced by PSMD14 inhibition in MDA-MB 435S cells. ( A , B ) MDA-MB 435S cells pretreated with 10 μM BAPTA-AM or 20 μM Ru360 were further treated with 5 μM CZM for 24 h or 12 h. ( A ) Cellular viability was assessed using IncuCyte, as described above * p < 0.001 vs. untreated cells, # p < 0.001 vs. CZM-treated cells. ( B ) Cells were observed by phase-contrast microscopy. Bars, 20 μm. ( C ) YFP-ER cells were pretreated with 10 μM BAPTA-AM or 20 μM Ru360 were further treated with 5 μM CZM for 8 h and stained with MitoTracker-Red. Cells were observed under the confocal microscope. Bars, 10 μm. ( D , E ) Cells pretreated with 5 μM BAPTA-AM or 1 μM CHX were further treated with 5 μM CZM for the indicated time durations or 8 h. ( D ) Western blotting of the indicated proteins was performed using β-actin as a loading control. ( E ) Cells were stained with Fluo-3 and observed by confocal microscopy. Bars, 20 μm.

Article Snippet: MDA-MB 435S, MDA-MB 231, MDA-MB 468, and MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Disruption, Inhibition, Microscopy, Staining, Western Blot, Control, Confocal Microscopy

Journal: Translational Oncology

Article Title: Extracellular vesicles extracted from bone marrow mesenchymal stem cells carrying MicroRNA-342-3p inhibit the INHBA/IL13Rα2 axis to suppress the growth and metastasis of breast cancer

doi: 10.1016/j.tranon.2021.101333

Figure Lengend Snippet: The effect of BMSC-EVs carrying miR-342-3p on the proliferation, migration, invasion, and apoptosis of MCF-7 cells. A, The expression of miR-342-3p in the microarray GSE35412. B, The enrichment of miR-342-3p in various tissues determined by EVmiRNA. C, The expression of miR-342-3p in breast cancer cells MCF-7, SKBR3, T47D, MDA-MB-231, and human normal breast epithelial cell line MCF10A determined by RT-qPCR, ** p < 0.01 in comparison with MCF10A cells. D, The entering of PKH67-labeled EVs carrying Cy3-labeled miR-342-3p into MCF-7 cells observed by a confocal microscope. E, The expression of miR-342-3p in MCF-7 cells incubated with BMSC-EVs determined by RT-qPCR. F, MCF-7 cell proliferation determined by CCK-8 assay. G, MCF-7 cell apoptosis detected by flow cytometry. H, Scratch test of MCF-7 cell migration. I, MCF-7 cell invasion detected by Transwell assay. J, The expression of E-cadherin, N-cadherin and Vimentin in MCF-7 cells determined by Western blot analysis. In panel E-J, ** p < 0.01 in comparison with MCF-7 cells incubated with EVs-inhibitor-NC, ## p < 0.01 in comparison with MCF-7 cells incubated with EVs-mimic NC. Cell experiments were conducted three times independently.

Article Snippet: Breast cancer cell lines including MCF-7, SKBR3, T47D and MDA-MB-231, and human normal breast epithelial cell line of MCF10A were purchased from ATCC (Manassas, VA).

Techniques: Migration, Expressing, Microarray, Quantitative RT-PCR, Comparison, Labeling, Microscopy, Incubation, CCK-8 Assay, Flow Cytometry, Transwell Assay, Western Blot

Journal: Translational Oncology

Article Title: Extracellular vesicles extracted from bone marrow mesenchymal stem cells carrying MicroRNA-342-3p inhibit the INHBA/IL13Rα2 axis to suppress the growth and metastasis of breast cancer

doi: 10.1016/j.tranon.2021.101333

Figure Lengend Snippet: The effect of BMSC-EVs-miR-342-3p on the expression of INHBA and IL13Rα2 in MCF-7 cells. A, Venn map of miR-342-3p downstream genes predicted by the mirDIP, DIANA TOOLS, RNA22, TargetScan, and miRWalk databases. B, The top 20 genes interacting of MRFAP1 and INHBA predicted by GeneMANIA. C, The expression data of MRFAP1 in breast cancer obtained from the TCGA database analyzed by GEPIA. D, The expression data of INHBA in breast cancer obtained from the TCGA database analyzed by GEPIA, * p < 0.05. E, The expression of INHBA in breast cancer cells MCF-7, SKBR3, T47D, MDA-MB-231, and human normal breast epithelial cell line MCF10A measured by RT-qPCR (** p < 0.01 in comparison with MCF10A cells). F, The relationship between miR-342-3p and INHBA predicted by TargetScan database and verified by dual luciferase reporter assay in MCF-7 cells, ** p < 0.01 in comparison with mimic-NC. G, The relationship between INHBA and IL13Rα2 detected by MEM analysis. H, Binding between INHBA and IL13Rα2 in MCF7 assessed by RIP. ** p < 0.01 in comparison with MCF7 cells treated with sh-NC. I, IL13Rα2 protein pulled down by INHBA in MCF-7 cells detected by RNA pull-down assay. ** p < 0.01. J, The stability of IL13Rα2 protein in MCF7 cells detected by Western blot analysis. * p < 0.01 in comparison with Control MCF7 cells. K, The expression of INHBA and IL13Rα2 in MCF7 cells determined by Western blot analysis, ** p < 0.01 in comparison with MCF7 cells transfected with mimic-NC. L, The expression of INHBA and IL13Rα2 in MCF7 cells determined by Western blot analysis, ** p < 0.01 in comparison with MCF7 cells treated with EVs-mimic-NC. Cell experiments were conducted three times independently.

Article Snippet: Breast cancer cell lines including MCF-7, SKBR3, T47D and MDA-MB-231, and human normal breast epithelial cell line of MCF10A were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Luciferase, Reporter Assay, Binding Assay, Pull Down Assay, Western Blot, Control, Transfection