Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: Expression of SMC1 in breast cancer and non-tumorigenic breast epithelial cells.

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: Expression of SMC1 was determined at the gene (RT-PCR) and protein level (western blot). For SMC1 gene expression, total RNA was purified using Trizol reagent (Invitrogen) as detailed in the methods section and expression of SMC1 mRNA was evaluated by RT-PCR analysis ( Panel A ) using gene specific primers [307–326 bp (upstream primer) and 730–750 bp (downstream primer)]. ß-actin was used as an internal control. SMC1 protein expression was evaluated by western blot analysis. Briefly, crude cell homogenate was prepared in RIPA buffer as described in method section and crude protein (50 µg) was subjected to SDS-PAGE and western blots using rabbit-anti-human SMC1 IgG ( Panel B ). Western blot was developed by ECL- reagent (Cell Signaling). GAPDH was used as an internal control. The level of SMC1 gene and protein was quantified by densitometry and was plotted as a fold change normalized to non-tumorigenic mammary epithelial breast cell, MCF10a ( Panel C ).

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Purification, SDS Page

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: Immunocytological localization SMC1 was performed on MDA-MB-231 fixed cells by method described previously with slight modifications , , , . Cells were grown on glass cover slips and fixed with ice-cold methanol and acetic acid (3∶1). Nonspecific antibody interactions were minimized by pre-treating the cells with 10% goat serum in PBS for 60 min at room temperature. The cells were subjected to immuno-cytochemistry using anti-SMC1 IgG (raised in rabbit) as a primary antibody and goat-anti-rabbit Rhodamine red-x-conjugated secondary antibody. DAPI (4′, 6-Diamidino-2-phenylindole) was used as a nuclear counter-stain. Slides were analyzed by confocal laser microscope (Zeiss LSM510 META, Germany) at 40× magnification ( Panel A ). Surface localization of SMC1 was determined by flow cytometry using indirect flow cytometry protocol . Briefly, MDA-MB-231 cells were harvested and resuspended to approximately 1×10 6 cell/ml in ice-cold PBS, 10% FBA and 1% sodium azide. Cells were incubated with 1 µg/ml anti-SMC1 IgG in 3% BSA/PBS solution and incubated at 4°C for 2 hour followed by washing with PBS and incubated with FITC-conjugated secondary antibody for 30 min at room temperature in dark. Cells were washed with PBS 3–5 times and resuspended in ice-cold PBS containing 3% BSA and 1% sodium azide and analyze by flow-cytometry ( Panel B ). Subcellular distribution of SMC1 was determined in the MDA-MB-231 cells using subcellular protein fractionation kit (Thermo Scientific) as detailed in methods section. Immuno-precipitation was performed in all the 3 fractions using anti-SMC1 IgG using the protocol as described in method section. All the immune-precipitates were characterized by western blot using anti-SMC1 and anti-SMC3 IgG ( Panel C ).

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Immunocytochemistry, Staining, Microscopy, Flow Cytometry, Incubation, Fractionation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: The MDA-MB-231 cells were transfected with the eukaryotic expression vector alone (pcDNA3.1) and SMC1/pcDNA3.1, using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection of scrambled and SMC1 siRNA was performed with Lipofectamine RNAiMax kit (Invitrogen) following manufactures instructions. Total RNA was purified using Trizol reagent and quantified using a nano-drop spectrophotometer (Thermo Scientific). Expression of SMC1 mRNA was evaluated by RT-PCR analysis using gene specific primers [nt 307–326 bp (upstream primer) and nt 730–750 bp (downstream primer)] and β-actin was used as a control. Equal amount of DNA was loaded on 1% agarose gel; lane 1, pcDNA3.1 (control vector); lane 2, pcDNA3.1/SMC1; lane 3, scrambled siRNA; and lane 4 SMC1 siRNA to check for the overexpression and silencing of SMC1( Panel A ). For SMC1-liposomes, SMC1 was purified by DNP-SG affinity chromatography and reconstituted into liposomes using our established procedure , . Viability of colonies was determined by colony forming assay, performed in untreated, control-liposomes, SMC1-liposomes, scrambled and SMC1 siRNA, pcDNA3.1 and SMC1/pcDNA3.1 transfected MDA-MB-231 cells (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium. After 10 days, cells were stained with 0.5% methylene blue for 30 min and colonies were counted using Innotech Alpha Imager. The results shown are normalized to control untreated cells. Values are means ± S.D. of three separate experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected) and suppression (SMC1 siRNA) was also checked on the cell apoptosis by using TUNEL apoptosis kit (Promega). Briefly, approximately 0.1×10 6 MDA-MB-231 cells were grown on the cover slips in 12 well plate and transfected with SMC1/pcDNA3.1 and SMC1 siRNA as described in method section. TUNEL apoptosis assay was performed using the Promega Apoptosis Detection Kit according to manufactures instructions. The slides were analyzed by fluorescence microscope (Olympus IX81 automated Inverted) using a standard fluorescein filter set to view the green fluorescence at 520 nm, and blue fluorescence at >340 nm. Photographs taken at identical exposure at 20× magnification are presented. Apoptotic cells showed green fluorescence and characteristic cell shrinkage ( Panel C ). MDA-MB-231 cells transfected with scrambled shRNA and SMC1 shRNA were also tested for the anchorage-independent growth on soft agar as described in the methods section and colonies were counted after 21 days and plotted ( Panel D ).

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Transfection, Expressing, Plasmid Preparation, Purification, Spectrophotometry, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Over Expression, Affinity Chromatography, Staining, TUNEL Assay, Apoptosis Assay, Fluorescence, Microscopy, shRNA

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: Scratch wound assays were performed in MDA-MB-231 cells transfected with scrambled siRNA (scr siRNA) or siRNA against SMC1, control vector (pcDNA3.1), and SMC1/pcDNA3.1. After 24 hours, confluent monolayers of cells were wounded, and healing of the wound by cell migration was monitored for 24 hour. Images were taken at 0 and 24 hours ( Panel A ). Migrated cells in the wound area at 24 hour were counted from five different fields and expressed as the means ± S.E. of three independent experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected, lane 2) and suppression (SMC1 siRNA, lane 3) was also determined by checking the expression of vimentin and E-cadherin, markers of angiogenesis and metastasis by western blot analysis ( Panel C ). As can be seen in ( Panel C ), there was overexpression of vimentin in SMC1 transfected MDA-MB-231 cells and suppression of E-cadherin as compared to control while on SMC1 suppression, there was suppression of vimentin while no significant change in the expression of E-cadherin.

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Transfection, Plasmid Preparation, Migration, Over Expression, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: IC 50 (half maximal inhibitory concentration) of the PARP-inhibitor, ABT-888 combined scrambled (scr) or SMC1 siRNA was tested in normal (HUVEC) and TNBC basal-like (MDA-MB-468, HCC1937) cell lines by MTT assay as detailed in methods section. Briefly, cells were transfected with scrambled siRNA or SMC1 siRNA (50 nM) using Lipofectamine RNAiMax following manufactures instructions and after 24 hours, transfected cells were treated with a range of ABT-888 (0–100 µM) and MTT assay was performed after 72 hours ( Panel A and C ). To further check the effect of SMC1 silencing on the efficacy of ABT-888, colony propagation assay was performed as described , . Briefly, MDA-MB-468 and HCC1937 were transfected with scrambled or SMC1 siRNA (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium containing 20 µM ABT-888 (MDA-MB-468) and 5 µM (HCC1937). The medium was changed every 2 days and after 10 days, the cells were stained with 0.5% methylene blue and colonies were counted by Alpha Innotech Imager ( Panel B and D ). These results showed that SMC1 siRNA sensitized basal like TNBC cells irrespective of their BRCA1 mutation status.

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Concentration Assay, MTT Assay, Transfection, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: The effect of SMC1 siRNA was also tested on the mesenchymal stem-like TNBC cell subtype (MDA-MB-231, MDA-MB-436) which constitute the minor proportion of the total population of TNBC to explore the effect of SMC1 suppression on the sensitivity of the heterogeneous TNBC cells population towards PARP inhibitor. MDA-MB-231 ( BRCA1 wild type) and MDA-MB-436 ( BRCA1 mutated) cells were transfected with scrambled (scr) siRNA or SMC1 siRNA as described in the method section and after 24 hours, were treated with a range of ABT-888 (0–100 µM) and incubated at 37°C in CO 2 incubator for 72 hours and survival of cells were determined by MTT assay. IC 50 was calculated and the dose at which there was 50% cell death was used to determine the cell propagation capacity in presence of SMC1 siRNA by colony forming assay. Briefly, MDA-MB-231 and MDA-MB-436 cells were transfected with scrambled or SMC1 siRNA (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium containing 20 µM ABT-888. The medium was changed every 2 days and after 10 days, the cells were stained with 0.5% methylene blue and colonies were counted by Alpha Innotech Imager. These results showed that SMC1 siRNA sensitized both BRCA1 functional and mutated mesenchymal stem-like TNBC cells toward ABT-888.

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: Transfection, Incubation, MTT Assay, Staining, Functional Assay

Journal: PLoS ONE

Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

doi: 10.1371/journal.pone.0064338

Figure Lengend Snippet: Targets of PARP inhibitor, ABT-888; PARP is depicted along with the BRCA1 and SMC1. DSB, double-strand break; SSB, single-strand break; PARP, poly (ADP)-ribose polymerase; BRCA1 , breast cancer 1, early onset; SMC1, structural maintenance of chromosome 1.

Article Snippet: Polyclonal rabbit-anti-human SMC1 IgG was purchased from Bethyl Laboratories (Montgomery, TX).

Techniques: