Altogen labs Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) Altogen Biosystems offers the HMEC Cell Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The HMEC Cell Transfection Reagent is a lipid-mediated formulation, and it has been developed to provide high transfection efficiency with HMEC. This cell line is a good host for studying breast cancer and oncogenesis. When cultured in vitro, HMEC cells are a monolayer and adhere to the surface of the culture flask. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase HMEC Transfection Kit at www.Altogen.com Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

HMEC Cell Line Description and Applications Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) HMEC Cell Line Description and Applications The HMEC cell line originates from human mammary epithelial cancer cells. These cells were surgically removed from glandular tissue from a human female patient with breast cancer. This cell line displays cuboidal morphology with a characteristic pattern of swirls. Research studies regarding breast tissue indicate that cell cultures derived from breast cancer tissue are ideal for studying therapeutics or oncogenesis. The primary application of the HMEC Transfection Kit is with human mammary epithelial cancer cells and researching breast cancer. Other uses of the HMEC cell line include carcinogen screening and three dimensional culturing. HMEC cells Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

HMEC Transfection Protocol Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) HMEC Transfection Protocol 1. Plate 10,000 - 15,000 HMEC cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection 2. Wash with 1xPBS and add 0.5 ml of fresh growth medium 3. Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and • 750 ng DNA (or mRNA), or • 30 nM - 50 nM of siRNA (or microRNA) *Referred to a final volume including growth medium 4. Incubate transfection complexes at RT for 15 - 30 minutes 5. Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however, it may increase cell toxicity 6. Add prepared transfection complexes to 0.5 ml of complete growth medium with HMEC cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection Optional: Transfection efficiency can be increased by addition of Transfection Enhancer reagent. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection If the viability of HMEC cells being transfected is affected at 16 - 24 hours post-transfection, the level of cytotoxicity can be decreased by changing the growth medium and eliminating redundant exposure of cells to transfectant Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

General Lipoplex-mediated Transfection Mechanism of Action Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) General Lipoplex-mediated Transfection Mechanism of Action Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

HMEC Transfection Kit Product Details Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) HMEC Transfection Kit Product Details Two-component formulation enhances lipid-mediated transfection efficiency Optimized easy-to-use transfection protocol provided for transfection of siRNA, DNA, mRNA, and microRNA Kit includes Transfection Enhancer reagent and recommended transfection protocol High transfection efficacy in the presence of serum Expand your RNAi application with a reagent optimized for delivery of both siRNA and plasmid Reproducible transfection results Works well for standard reverse transfection and high-throughput applications Developed and manufactured by Altogen Biosystems (www.Altogen.com) Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) Data Figure 1. SiRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into HMEC cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by qRT-PCR for gene expression levels. 18S rRNA levels were used to normalize the Lamin A/C data. Values are normalized to untreated sample. Data are means ± SD (n=3).

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) Data Figure 2. Protein expression of Lamin A/C in HMEC cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into HMEC cells following Altogen Biosystems transfection protocol. At 72 hours post-transfection the cells were analyzed by Western Blot for protein expression levels (normalized by total protein, 10 µg of total protein loaded per each well). Untreated cells used as a negative control.

HMEC Transfection Kit Benefits Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HMEC Transfection Reagent (Mammary Epithelial, Breast Cancer Cells) HMEC Transfection Kit Benefits Pre-optimized transfection protocol for HMEC cell line Compatible with DNA, small RNA (siRNA, shRNA, miRNA), mRNA, and small protein complexing Free of serum and protein of animal origin Compatible with standard and reverse transfection methods (both protocols provided in the kit manual) Easy to use HMEC transfection protocol ensures great performance with expedited experimental timeline Equally efficient for single or multiple transfections Can be used for transient transfection and development of stable HMEC cell lines Bio-degradable after endocytosis Used for preclinical research worldwide Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com