1. Products > MCF-7 Transfection Reagent (Breast Cancer, HTB-22) Altogen Biosystems offers the MCF-7 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The MCF-7 Transfection Reagent is an advanced formulation of a cationic lipids based reagent, and it has been developed to provide high transfection efficiency with the MCF-7 cell line. This cell line is a good host for studying human breast cancer. When cultured in vitro, MCF-7 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 MCF-7 Transfection Kit at www.Altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

2. The MCF-7 cell line is a breast cancer cell line that is widely used and characterized as one of the best cell lines among breast cancer cell lines. It originates from the cells of a 69-year old Caucasian female isolated in 1970. The cell line was isolated at the Michigan Cancer Foundation, hence the name “MCF.” Prior to the discovery of MCF-7, there were no mammary cell lines that progressed beyond a few months. As a result, MCF-7 is considered to be the best model for studying malignant progression. MCF-7 displays characteristics associated with mammary epithelium. These include the ability to form domes as well as the ability to process estradiol via cytoplasmic estrogen receptors. Research shows that TNF alpha can inhibit MCF-7 growth and that the application of anti-estrogens will transform insulin-like growth factor protein secretion. MCF-7 Cell Line Description and Applications Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com MCF-7 cells Altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)

3. MCF-7 Transfection Protocol 1. Plate 10,000 - 15,000 MCF-7 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 MCF-7 cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO 2 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 MCF-7 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 Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)

4. 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 Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)

5. MCF-7 Transfection Kit Product Details Proprietary cationic lipids formulation High transfection efficiency of small RNA (siRNA, shRNA, miRNA), mRNA, pDNA Effective and robust intracellular delivery Kit includes Transfection Enhancer reagent Produces consistent results, lot-to-lot, plate-to-plate, and well-to-well Work in the presence of serum A proven reagent for establishing stable cell lines Optimized transfection protocols are adapted for use with both standard & reverse transfection methods 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 > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)

6. Data Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in the MCF-7 cells transfected with siRNAs targeting GAPD or non-silencing siRNA. Forty-eight hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA expression levels. Data were normalized against the 18S rRNA signal. Control samples were either mock-transfected or untreated. 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 > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)

7. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MCF-7 Transfection Reagent (Breast Cancer, HTB-22) Figure 2. Protein expression of GAPDH in MCF-7 cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into MCF-7 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.

8. Pre-optimized transfection protocol for MCF-7 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 MCF-7 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 MCF-7 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide MCF-7 Transfection Kit Benefits 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 > MCF-7 Transfection Reagent (Breast Cancer, HTB-22)