Journal: Breast Cancer Research

Article Title: Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family

doi: 10.1186/bcr1650

Figure Lengend Snippet: MELK involvement in the apoptosis cascade through Bcl-G L . HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-G L (Flag-Bcl-G L ) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-G L proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-G L expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t -test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-G L expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).

Article Snippet: Human breast cancer cell lines HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SKBR3, T47D, YMB1, BSY-1 and BT-20, human cervical adenocarcinoma cell line, HeLa, and monkey kidney cells transformed with SV40 T-antigen, COS7 cells, were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Transfection, Western Blot, TUNEL Assay, Staining, Standard Deviation, Labeling, Fluorescence

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Distribution of MDA MB231 cells after exposure to IC 50 concentrations of 5-fluorouracil ( ), doxorubicin ( ), paclitaxel (▪) and untreated cells (□), in ( A ) sub-G1, ( B ) G1, ( C ) S and ( D ) G2/M phases.

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques:

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Thymidine kinase (TK) activities in MDA MB231 breast cancer cells ( A ) without, and with addition of the TK-2 inhibitors ( B ) dCTP and ( C ) KIN52. Cells were incubated with IC 50 concentrations of 5-fluorouracil ( ), doxorubicin ( ) and paclitaxel (▪), and compared with untreated cells (□).

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques: Incubation

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Relative thymidine kinase (TK)-1 activity in MDA MB231 breast cancer cells after addition of ( A ) dCTP and ( B ) KIN52. Cells were exposed to IC 50 concentrations of 5-fluorouracil ( ), doxorubicin ( ) and paclitaxel (▪), and compared with untreated cells (□). % cells are normalised to TK activity before addition of dCTP or KIN52 (set to 100%).

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques: Activity Assay

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Typical Western blot for thymidine kinase (TK)-1 levels in CEM and MDA MB231 cells after treatment with cytotoxic agents compared with untreated cells, β -actin was used as loading control.

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques: Western Blot, Control

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Mean (±s.d.) change (%) in TK-1 protein levels of MDA MB231 cells following incubation with IC 50 concentrations of 5-FU, doxorubicin and paclitaxel compared with untreated cells (set to 100%)

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques: Incubation

Journal: British Journal of Cancer

Article Title: [ 18 F]FDG and [ 18 F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

doi: 10.1038/sj.bjc.6604523

Figure Lengend Snippet: Mean (±s.d.) change (%) in FDG and FLT uptake in MDA MB231 cells after incubation with IC 50 concentrations of 5-FU, doxorubicin and paclitaxel compared with untreated cells (set to 100%)

Article Snippet: The human breast cancer cell line MDA MB231 (ATCC no. HTB-26) and CEM leukaemia cells (CEM wild type and TK-deficient CEM/TK-) were cultured in RPMI 1640 supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S), at 5% CO 2 in a humidified atmosphere at 37°C.

Techniques: Incubation

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a) LIFR mRNA levels (Minn et al . dataset) in breast tumors from patients with a poor prognosis based on Van’t Veer signature (n=38 patients with no bone metastasis, n=7 patients with bone metastasis). Student’s unpaired t-test with Welch’s Correction. (b) STAT3 mRNA levels (Minn et al . dataset) in breast tumors (n=37 good prognosis, n=45 poor prognosis). Student’s unpaired t-test. (c) Correlation of STAT3 and SOCS3 mRNA levels in The Cancer Genome Atlas (TCGA) provisional dataset, n=999 patients. Pearson and Spearman Correlation. (d) Analysis of TCGA invasive breast carcinoma (Nature 2012 dataset) patient samples for LIFR, STAT3, and SOCS3 mRNA down-regulation (n=682 patients with no mRNA down-regulation, n=55 patients with mRNA down-regulation). Logrank test. (e,f) LIFR and SOCS3 mRNA levels in TCGA invasive breast carcinoma (Nature 2012 dataset) samples stratified by PAM50 breast cancer subtype (Normal n=8, Luminal A n=230, Luminal B n=125, Basal-like n=98, HER2-enriched n=58 patients). Kruskal-Wallis test. Minima=25% percentile, maxima=75% percentile, center=median. (g) Correlation of Li et al . hypoxia gene signature with LIFR mRNA levels in TCGA invasive breast carcinoma patient dataset, as in (c). Pearson and Spearman Correlation. n=998 patients. (h) LIFR mRNA levels following 24hrs in normoxia or hypoxia. 3 technical replicates from a single experiment representative of 2 independent experiments. (i) Quantification of 3 Western blots, representative LIFR blot after 24hrs in normoxia or hypoxia. 3 technical replicates from a single experiment. (j) SOCS3 mRNA levels following 24hr culture in normoxia or hypoxia. 3 technical replicates from a single experiment representative of 2 independent experiments. (k) LIFR mRNA levels in MCF7 cells following 16hr treatment with PHD inhibitor DMOG (1mM) and LIFR Western blot representative of 3 technical replicates from a single experiment. PCR: 3 technical replicates from a single experiment. (l) SOCS3 mRNA levels in MCF7 cells following 16hr treatment with DMOG (1mM). 3 technical replicates from a single experiment. Source data for 1h–l in and unprocessed blots in . Graphs represent mean/group and error bars=SEM. *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Western Blot

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a) LIFR mRNA levels in MCF7 cells in normoxia or hypoxia for 24hrs and transfected with HIF1α or HIF2α siRNA. HIF1α and HIF2α mRNA knockdown levels shown as controls. 3 technical replicates from a single experiment representative of 3 independent experiments. (b) LIFR promoter activity of wildtype promoter (WT) or promoter with mutations in hypoxia responsive elements + reverse hypoxia element (LIFR HRE+rHRE mut) or reverse hypoxia element mutated only (LIFR rHRE mut) following culture for 48hrs in normoxia or hypoxia. Student’s unpaired t-test. n=3 biological replicates, one each from 3 independent experiments. (c) UCSC genome browser tracks for LIFR gene variants 1 and 2, H3K9me3 histone modifications by ChIP-Seq analysis from ENCODE database, and SETDB1 and HDAC2 binding sites on the LIFR gene. (d) LIFR mRNA levels in MCF7 cells in 24hrs normoxia or hypoxia and transfected with SETDB1 or HDAC2 siRNA. SETDB1 and HDAC2 mRNA knockdown levels shown as controls. 3 technical replicates from a single experiment representative of 3 independent experiments. (e) Western blot for acetylated histone H3 (AcH3) and LIFR protein levels in MCF7 cells treated with vehicle (Veh), 0.1mM, 1mM, or 10mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, for 24hrs with actin loading control. Representative of 2 independent biological replicates. (f) LIFR mRNA levels after 24hrs treatment with 0, 0.1mM, 1mM, or 10mM VPA in normoxia or hypoxia. n=3 biological replicates, each being an average from 3 independent experiments. Student’s unpaired t-test. (g) SOCS3 mRNA levels after 24hrs treatment with 0, 0.1mM, 1mM, or 10mM VPA in normoxia or hypoxia. Student’s unpaired t-test. n=3 biological replicates, each being an average from 3 independent experiments. Source data for 2a,d available in and unprocessed blots in . Graphs represent the mean/group and error bars represent standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. #p<0.05 versus 0mM Nx.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Transfection, Knockdown, Activity Assay, ChIP-sequencing, Binding Assay, Western Blot, Histone Deacetylase Assay, Control

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a,c,e,g) Western blot for LIFR, pSTAT3 (Y705), total Stat3, and β-actin (loading control) after 15 or 30 minute treatment with PBS (vehicle control), recombinant OSM (50ng/ml) or recombinant LIF (50ng/ml) in (a,c) MCF7 and SUM159 cells with low metastatic potential and (e,g) MDA-MB-231b and 4T1BM2 cells with high metastatic potential. Blots represent 3 independent biological replicates. (b,d,f,h) SOCS3 mRNA levels after 1 or 6 hour treatment with PBS, recombinant OSM (50ng/ml) or recombinant LIF (50ng/ml). b,f: Student’s unpaired t-test. d,h: Mann-Whitney test. n=3 biological replicates, each being an average from 3 independent experiments. Unprocessed blots in . Graphs represent the mean/group and error bars represent standard error of the mean (SEM). *p<0.05 and **p<0.01.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Western Blot, Control, Recombinant, MANN-WHITNEY

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a) Parathyroid hormone-related protein (PTHrP) mRNA levels in MCF7 cells overexpressing PTHrP (MCF7PTHrP). Student’s unpaired t-test. n=3 biological replicates, one each from 3 independent experiments. (b) LIFR and SOCS3 mRNA levels in MCF7PTHrP over-expressing cells. Student’s unpaired t-test. n=3 biological replicates, one each from 3 independent experiments. (c) Western blot for LIFR, pSTAT3 (Y705), total Stat3, and β-actin (loading control) in MCF7PTHrP over-expressing whole cell lysates. (d) SOCS3 mRNA levels in MCF7pcDNA and MCF7PTHrP over-expressing cells after 1 hour treatment with PBS (vehicle control) or recombinant LIF (50ng/ml). 3 technical replicates from a single experiment representative of 2 independent experiments. (e) PTHrP mRNA levels (1–139aa) inn MCF7NSC and MCF7shLIFR cells. 2 biological replicates, each being an average from 2 independent experiments. (f,g) mRNA levels of genes associated with (f) dormancy and quiescence, and (g) cancer stem cells in MCF7pcDNA (control) and MCF7PTHrP over-expressing cells. Mann-Whitney test. n=3 biological replicates, one each from 3 independent experiments. (h,i) mRNA levels of genes associated with (h) dormancy and quiescence, and (i) cancer stem cells in MCF7 cells treated for 24 hours with 0, 0.1mM, 1mM, or 10mM valproic acid (VPA). Multiple t-test with Holm-Sidak post-test. n=3 biological replicates, one each from 3 independent experiments. Source data for 5d,e available in and unprocessed blots in . Graphs represent the mean/group and error bars represent standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Expressing, Western Blot, Control, Recombinant, MANN-WHITNEY

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a) STAT3 mRNA levels in MCF7 cells infected with STAT3 lentiviral clones tested for knockdown efficiency. 3 technical replicates from a single experiment. (b) Western blot for Stat3 protein levels in MCF7 cells infected with STAT3 lentiviral clones tested for knockdown efficiency. (c) Lesion number (Total number from both tibiae and femora from each mouse) and (d) lesion area (Total area from both tibiae and femora from each mouse) in the tibiae and femora of MCF7NSC (n=9 mice) or MCF7shSTAT3 tumor-bearing mice (shSTAT3 clone 376016 n =9 mice, shSTAT3 clone 641817 n=10 mice). Student’s unpaired t-test. (e) Representative images of osteolytic bone destruction in MCF7NSC (n=9 mice), MCF7shSTAT3 376016 (n=9 mice), and MCF7shSTAT3 641817 (n=10 mice) tumor-bearing mice. Source data for 7a,c,d available in . Graphs represent the mean/group and error bars represent standard error of the mean (SEM). *p<0.05 and **p<0.01.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Infection, Clone Assay, Knockdown, Western Blot

Journal: Nature cell biology

Article Title: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow

doi: 10.1038/ncb3408

Figure Lengend Snippet: (a,b) Immunocytochemistry for basal pStat3 (Y705) protein levels in (a) MCF7NSC (2 biological replicates) and (b) MCF7shLIFR cells (2 biological replicates). Images representative of 3 independent replicates/group. Scale bar = 100μm. (c,d) mRNA levels of dormancy/quiescence and cancer stem cell-associated genes in MCF7 cells following 24 hour treatment with 5μM or 50μM of the small molecule Stat3 inhibitor ML116 (Stat3i). Multiple t-tests with Holm-Sidak method. n=3 biological replicates, one each from 3 independent experiments. (e,f) mRNA levels of dormancy/quiescence and cancer stem cell-associated genes in MCF7 cells transfected with siRNA against SOCS3 for 48 hours. Multiple t-tests with Holm-Sidak method. n=3 biological replicates, one each from 3 independent experiments. (g) Flow chart indicating hypoxia differentially regulates LIFR and PTHrP, which signal via STAT3 and SOCS3 to regulate dormancy-associated genes and thus influence bone colonization. (h) Working model for LIFR:STAT3 signaling in disseminated breast cancer cells transitioning from a dormant to invasive phenotype in strongly hypoxic regions of the bone marrow. Graphs represent the mean/group and error bars represent standard error of the mean (SEM).*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Human MCF7 and MDA-MB-231 breast cancer cells and 4T1 mouse mammary carcinoma cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).

Techniques: Immunocytochemistry, Transfection

Journal: Clinical & Translational Immunology

Article Title: SKI‐G‐801, an AXL kinase inhibitor, blocks metastasis through inducing anti‐tumor immune responses and potentiates anti‐PD‐1 therapy in mouse cancer models

doi: 10.1002/cti2.1364

Figure Lengend Snippet: SKI‐G‐801 is a novel AXL kinase inhibitor. (a) Crystal structure of SKI‐G‐801 in AXL kinase domain (left). Chemical structure of SKI‐G‐801 (right). (b) Western blot analysis of pAXL inhibition by SKI‐G‐801 and R428 in MDA‐MB‐231 and Hs578T cells. The experiment was repeated twice. (c) MDA‐MB‐231 cells were incubated with either SKI‐G‐801 or R428 at the indicated concentrations for 48 h, followed by an invasion assay. The experiment was repeated twice. (d) B16F10 cells (1 × 10 6 cells per mouse, n = 3 per group, derived from two independent experiments) were injected intravenously. SKI‐G‐801 (30 mg kg −1 day −1 ) was administered from day −1 to day +14. The mice were sacrificed to evaluate the metastasis. (e) CT26‐luciferase cells (1 × 10 4 cells per mouse, n = 5 per group, derived from two independent experiments) were implanted into the intraperitoneal cavity. SKI‐G‐801 (30 mg kg −1 , once a day) was administered from day −1 to day +15. Luciferase expression was determined on days 0, 3, 6, 9, 12 and 15 using the Xenogen in vivo imaging system.

Article Snippet: MDA‐MB‐231 (ATCC HTB‐26, RRID: CVCL_0062) and Hs578T (ATCC HTB‐126, RRID: CVCL_0332) cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Inhibition, Incubation, Invasion Assay, Derivative Assay, Injection, Luciferase, Expressing, In Vivo Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a

doi: 10.1186/s13046-017-0593-2

Figure Lengend Snippet: PDL1 is a target of miR-34a, and its functions could be inhibited by miR-34a. a The expression level of miR-34a was determined by qRT-PCR in the above cell lines. U6 snRNA was used as an internal control. b HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide, and qRT-PCR analysis demonstrated that the transfection was successful. c HCC38 and MDA-MB-231 cells were transfected as described, and the mRNA and protein expression of PDL1 was suppressed by miR-34a. d Histogram presenting cell viability based on MTS assays for HCC38 and MDA-MB-231 cells 48 h after transfection. e Transwell invasion assays demonstrated that the PDL1 3’UTR promoted cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. f The expression levels of PDL1 were determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. g The impact of miR-34a on immune cell populations in the tumor microenvironment. Flow cytometry revealed that miR-34a increased the number of CD8+ cells and CD4+ cells and reduced the number of macrophages and Tregs. All the data are shown as the mean ± s.e.m. * P < 0.05, ** P < 0.01

Article Snippet: The human mammary epithelial (HME) cell line MCF-10A and TNBC cell lines MDA-MB-453, MDA-MB-468, MDA-MB-231, BT-549, HCC38 and 4 T1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Western Blot, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a

doi: 10.1186/s13046-017-0593-2

Figure Lengend Snippet: Expression of PDL1 and LDHA is correlated and indicates a poor outcome in breast cancer . a HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide. The mRNA and protein expression of LDHA was suppressed by miR-34a. b The correlation between PDL1 and LDHA expression in 554 breast cancer patients was analyzed, and a positive correlation between them was found. c OS (left) and DFS (right) curves for breast cancer patients. d OS (left) and DFS (right) curves for TNBC patients. All the data are shown as the mean ± s.e.m. ** P < 0.01

Article Snippet: The human mammary epithelial (HME) cell line MCF-10A and TNBC cell lines MDA-MB-453, MDA-MB-468, MDA-MB-231, BT-549, HCC38 and 4 T1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Transfection

Journal: Cells

Article Title: Insights into P-Glycoprotein Inhibitors: New Inducers of Immunogenic Cell Death

doi: 10.3390/cells9041033

Figure Lengend Snippet: R -3 increases doxorubicin intracellular content by inhibiting Pgp activity. ( a ) Immunoblotting of the indicated proteins in human colon cancer HT29 cells, non-small cell lung cancer A549 cells, triple-negative breast cancer MDA-MB-231 cells, and in their doxorubicin (DX)-resistant variants. Tubulin was used as control of equal protein loading. The image is representative of 1 out of 3 experiments. ( b ) Cells were incubated for 3 h in medium containing 5 μM doxorubicin (dox), in the absence (ctrl) or presence of 75 nM Tariquidar (Tar) or 75 nM R -3. Intracellular doxorubicin content was measured fluorimetrically in duplicates ( n = 3). Data are means ± SD. * p < 0.005: treated cells vs. respective ctrl cells; ° p < 0.001: DX cells vs. parental cells. ( c ) Cells were grown for 24 h in the absence (ctrl) or presence of 75 nM Tariquidar (Tar) or 75 nM R -3. Pgp ATPase activity was measured spectrophotometrically on membrane extracts of DX variants. Data are means ± SD ( n = 3). * p < 0.001: treated cells vs. respective ctrl cells. ( d ) MDA-MB-231/DX cells were grown in the absence (ctrl) or presence of 75 nM Tariquidar (Tar) or 75 nM R -3 for 24 h, then incubated for 20 min with increasing concentrations (0–50 μM) of doxorubicin (dox). The procedure was repeated on a second series of dishes, incubated in the same experimental conditions and analyzed after 10 min. Data are means ± SD ( n = 3). The rate of doxorubicin efflux (dc/dt) was plotted versus the intracellular concentration of the drug.

Article Snippet: Human doxorubicin-sensitive colon cancer HT29 cells, non-small cell lung cancer A549 cells, and triple-negative breast cancer MDA-MB-231 cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Western Blot, Control, Incubation, Membrane, Concentration Assay

Journal: Cells

Article Title: Insights into P-Glycoprotein Inhibitors: New Inducers of Immunogenic Cell Death

doi: 10.3390/cells9041033

Figure Lengend Snippet: R -3 increases doxorubicin-induced apoptosis and cytotoxicity in Pgp-expressing cells. ( a ) Human colon cancer HT29 cells, non-small cell lung cancer A549 cells, triple-negative breast cancer MDA-MB-231 cells, and their doxorubicin (DX)-resistant variants were incubated 24 h ( a ) or 48 h ( b ), in fresh medium (ctrl), without inhibitors (- in) or with 75 nM Tariquidar (Tar) or 75 nM R -3. When indicated, 5 μM doxorubicin (dox) was added. ( a ) Activity of caspase 3 was measured fluorimetrically in duplicates ( n = 3). Data are means ± SD. * p < 0.001: treated cells vs. respective ctrl cells; ° p < 0.001: DX cells vs. parental cells. ( b ) Cell viability was measured by a chemiluminescence-based assay in quadruplicates. Data are means ± SD ( n = 3). * p < 0.001: treated cells vs. respective ctrl cells; ° p < 0.05: DX cells vs. parental cells.

Article Snippet: Human doxorubicin-sensitive colon cancer HT29 cells, non-small cell lung cancer A549 cells, and triple-negative breast cancer MDA-MB-231 cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Incubation, Activity Assay, Chemiluminescence Immunoassay

Journal: Cancer Science

Article Title: Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

doi: 10.1111/cas.13823

Figure Lengend Snippet: Forkhead box C1 ( FOXC 1) improves cell migration and invasion of basal‐like breast cancer cells. A, Real‐time PCR shows the efficiency of FOXC 1 knockdown in BT 549 cells. * P < .05, n = 3. B, Immunoblotting and quantitative analyses show the efficiency of FOXC 1 knockdown in BT 549 cells. GAPDH served as control. C, Real‐time RT ‐ PCR shows the efficiency of FOXC 1 overexpression in MDA ‐ MB ‐231 cells. D, Immunoblotting and quantitative analyses show the efficiency of FOXC 1 overexpression in MDA ‐ MB ‐231 cells. GAPDH served as control. E, Cell migration and invasion were evaluated after FOXC 1 knockdown. Inhibition of FOXC 1 expression significantly reduced cell migration and invasion compared with the control group. Three independent experiments were carried out. * P < .05. F, Overexpression of FOXC 1 significantly increased cell migration and invasion compared with the control group. * P < .05, n = 3. G, Results of colony formation assays show that FOXC 1 knockdown reduced the numbers of formed colonies. * P < .05, n = 3. H, Results of colony formation assays showed that FOXC 1 overexpression increased the numbers of formed colonies. * P < .05, n = 3

Article Snippet: The human breast cancer cell lines MDA‐MB‐231 and BT549 were obtained from ATCC (Manassas, VA, USA) and cultured according to the instructions of the manufacturer.

Techniques: Migration, Real-time Polymerase Chain Reaction, Knockdown, Western Blot, Control, Quantitative RT-PCR, Over Expression, Inhibition, Expressing

Journal: Cancer Science

Article Title: Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

doi: 10.1111/cas.13823

Figure Lengend Snippet: Forkhead box C1 ( FOXC 1) affects CXC chemokine receptor‐4 ( CXCR 4) expression. A, Real‐time RT ‐ PCR shows the CXCR 4 gene expression in BT 549 cells with downregulated FOXC 1. * P < .05, n = 3. B, Western blot and quantitative analyses show CXCR 4 expression in BT 549 cells with downregulated FOXC 1. GAPDH served as control. * P < .05, n = 3. C, Real‐time RT ‐ PCR shows the CXCR 4 gene expression in MDA ‐ MB ‐231 cells transfected FOXC 1 vector. GAPDH served as an internal control. * P < .05, n = 3. D, Western blot and quantitative analyses show the CXCR 4 expression in MDA ‐ MB ‐231 cells transfected FOXC 1 vector. GAPDH served as control. * P < .05, n = 3. E, Western blotting shows the FOXC 1 and CXCR 4 expression in clinical samples (top), and the correlation of the FOXC 1 and CXCR 4 expression was analyzed (bottom)

Article Snippet: The human breast cancer cell lines MDA‐MB‐231 and BT549 were obtained from ATCC (Manassas, VA, USA) and cultured according to the instructions of the manufacturer.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Cancer Science

Article Title: Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

doi: 10.1111/cas.13823

Figure Lengend Snippet: Forkhead box C1 ( FOXC 1) activates the transcription of CXC chemokine receptor‐4 ( CXCR 4). A, Map of the CXCR 4 promoter region with the positions of FOXC 1 potential consensus binding sites. B, Inhibition of FOXC 1 suppresses CXCR 4 promoter activity in BT 549 cells. * P < .05, n = 3. C, Overexpression of FOXC 1 activates CXCR 4 promoter activity in MDA ‐ MB ‐231 cells. * P < .05 vs control, n = 3. D, Ch IP real‐time PCR assays showed that FOXC 1 increases fold enrichment of CXCR 4 promoter. * P < .05, n = 3

Article Snippet: The human breast cancer cell lines MDA‐MB‐231 and BT549 were obtained from ATCC (Manassas, VA, USA) and cultured according to the instructions of the manufacturer.

Techniques: Binding Assay, Inhibition, Activity Assay, Over Expression, Control, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

doi: 10.1111/cas.13823

Figure Lengend Snippet: Overexpression of CXC chemokine receptor‐4 ( CXCR 4) enhanced migration and invasion of forkhead box C1 ( FOXC 1) knockdown BT 549 cells. A, Western blotting showed that transfection of CXCR 4 attenuated the reduction of FOXC 1‐induced CXCR 4 expression. B, Migration of FOXC 1 knockdown BT 549 cells with ectopic CXCR 4 expression was assessed by Transwell assays and quantification of the number of migrated cells. * P < .05, n = 3. C, Invasion of FOXC 1 knockdown BT 549 cells with ectopic CXCR 4 expression was assessed by Transwell assays and quantification of the number of invaded cells. * P < .05, n = 3. shNC, nonspecific control

Article Snippet: The human breast cancer cell lines MDA‐MB‐231 and BT549 were obtained from ATCC (Manassas, VA, USA) and cultured according to the instructions of the manufacturer.

Techniques: Over Expression, Migration, Knockdown, Western Blot, Transfection, Expressing, Control

Journal: Oncology Research

Article Title: RBMS3 Inhibits the Proliferation and Metastasis of Breast Cancer Cells

doi: 10.3727/096504017X14871200709504

Figure Lengend Snippet: RBMS3 was lowly expressed in breast cancer tissues and cell lines. (A) RBMS3 mRNA expression was determined in breast cancer tissues. (B) Western blot analysis was used to examine RBMS3 protein expression in breast cancer tissues. * p < 0.05 compared to the adjacent normal tissues. All the experiments were repeated at least three times. (C, D) The expression of RBMS3 at both mRNA and protein levels was obviously reduced in breast cancer cell lines. * p < 0.05 compared to the NBEC cell line.

Article Snippet: Human breast cancer cell lines MCF-7, MDA-MB-231, and BT-474, as well as normal human breast epithelial cell line (NBEC), were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

doi: 10.1136/jitc-2020-001128

Figure Lengend Snippet: Development of novel antipodocalyxin antibodies. (A) Flow cytometric PODO83 (blue lines) and PODO447 (red lines) binding profiles of Podxl-positive normal (HUVEC) and tumor cells (A-172, SUM149, PANC-1). (B) Flow cytometric histograms showing PODO83 (blue lines) and PODO447 (red lines) immunoreactivity to Podxl-positive (WT, solid lines) or -deficient ( PODXL -KO, dashed lines) ovarian (SKOV3), pancreatic (MIAPACA) and breast (MDA-MB-231) cancer cells. WT, wild type.

Article Snippet: HEK293, human umbilical vein endothelial (HUVEC), SKOV3, SUM149, PANC-1, A-172, MIAPACA and MDA-MB-231 cells were obtained from ATCC.

Techniques: Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

doi: 10.1136/jitc-2020-001128

Figure Lengend Snippet: PODO447 recognizes a glycomotif on Podxl’s mucin domain. (A) Flow cytometric histograms of SKOV3 wild-type (WT) cells treated with either assay buffer (control), neuraminidase (which removes the terminal sialic residues) or O-sialoglycoprotein endopeptidase (which cleaves the mucin domain). Cells were then stained with either PODO83 (blue) or PODO447 (red). (B) Flow cytometric histograms showing PODO83 (blue lines) and PODO447 (red lines) immunoreactivity to SKOV3 PODXL-KO cells re-expressing the WT podocalyxin (Podxl) (WT Podxl) protein or a Podxl-mutant lacking the mucin domain (ΔMucin Podxl).

Article Snippet: HEK293, human umbilical vein endothelial (HUVEC), SKOV3, SUM149, PANC-1, A-172, MIAPACA and MDA-MB-231 cells were obtained from ATCC.

Techniques: Control, Staining, Expressing, Mutagenesis

Journal: Journal for Immunotherapy of Cancer

Article Title: PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

doi: 10.1136/jitc-2020-001128

Figure Lengend Snippet: PODO447 antibody-drug conjugate in vitro cytotoxicity. (A) Representative image of mAb-Vedotin conjugate structure (created with BioRender.com). (B) Western blot showing PODO447 binding to lysates from OV3331, and SKOV3 WT and PODXL -KO cells. (C) In vitro cytotoxic effect of PODO447 and palivizumab–vedotin conjugates on OV3331 patient-derived ovarian high-grade serous cancer cells (p<0.001). (D) In vitro cytotoxicity of control and PODO447-ADC on SKOV3 wild-type (WT) and PODXL -KO cells (p<0.001). Representative experiments are shown, and the values indicate the mean±SD. MMAE, monomethyl auristatin E.

Article Snippet: HEK293, human umbilical vein endothelial (HUVEC), SKOV3, SUM149, PANC-1, A-172, MIAPACA and MDA-MB-231 cells were obtained from ATCC.

Techniques: In Vitro, Western Blot, Binding Assay, Derivative Assay, Control