Western blot analysis and quantitative real-time PCR

Cell and tissue lysates were prepared and performed as previously described. Briefly, membranes were blocked with 5% nonfat dry milk in TBST for 1 h and incubated overnight at 4 °C with primary Abs against CD133 (64326S), CD44 (37259S), Oct-4 (2750S), Nanog (4903S), Sox2 (14962S), ABCG2 (42078S), Bach1 (4578), HIF-1α (14179), and GADPH (5174) (purchased from Cell Signaling Technologies). The membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary Ab (Cell Signaling Technologies), and protein bands were detected by chemiluminescence using Immobilon Forte Western HRP substrate (Millipore WBLUF0500). Total RNA was extracted from A549 or SPCA1 using TRIzol reagent (Takara Bio, Shiga, Japan). cDNA was synthesized from the isolated RNA, and quantitative PCR was performed according to the manufacturer's instructions. This quantitative assay was performed using an SYBR QPCR kit (Toyobo, Osaka, Japan). The primer sequences used for PCR were provided in Table ​Table1.1. Densitometry was performed using ImageJ software (NIH, Bethesda, MD). All experiments were performed in triplicate.

Primers used for real-time PCR

CD44

Sox2

Nanog

Oct4

CD133

GCATTGCAGTCAACAGTCGAAGA

GACAGTTACGCGCACATGAA

GTGATTTGTGGGCCTGA AGA

GGTATTCAGCCAAACGA CCA

GAACAGGGCTACTCGCAAAG

CCTTGTTCACCAAATGCACCA

TAGGTCTG CGAGCTGGTCAT

ACACAGCTGGGTGGAAGAGA

CACACTCGGACCACATCCTT

AAAGG GCAGTTGACGGAAC