1. Products > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555) Altogen Biosystems offers the A431 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. A431 Transfection Reagent is an advanced formulation of a cationic lipid-based reagent, and it is developed to provide high transfection efficiency with A431 and other skin cancer cell lines. This cell line is a good host for studying skin cancer and other cancer types. When cultured in vitro, A431 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 A431 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 A431 cell line originates from the epidermoid carcinoma skin cells of an 85 year old female patient. These cells are a tool for studying several types of skin cancer and therapeutics for these cancers. The A431 cell line expresses elevated levels of Epidermal Growth Factor Receptor (EGFR) and as a result can also be used to study the cell cycle, cell signaling pathways associated with cancerous cells and can be used as a positive control for EGFR expression. The cell line is susceptible to mitogenic stimuli. A431 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 High Density Image of A431 Cells Atcc.org Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

3. A431 Transfection Protocol 1. Plate 10,000 - 15,000 A431 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 A431 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 A431 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 > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

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 > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

5. A431 Transfection Kit Product Details Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com 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) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

6. Data Figure 1. siRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into A431 cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by qRT- PCR for Lamin A/C 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=4). Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

7. Data Figure 2. Protein expression of Lamin A/C in A431 cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into A431 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. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)

8. Pre-optimized transfection protocol for A431 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 A431 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 A431 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide A431 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 > A431 Transfection Reagent (Epidermoid Carcinoma Cells, CRL-1555)