Fig. 3

BMP4 knockdown suppresses cancer cell growth, migration, invasion, tumorigenesis, and metastasis. a qRT-PCR of Bmp4 in 344SQ transfected with Bmp4 shRNAs (KD #2 and #3) or nontargeting control (NTC) vector. The expression levels were normalized to that of NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. b Western blot of BMP4, phospho-SMAD1/5 (pSMAD1/5), and total SMAD1/5 in 344SQ-Bmp4-KD cells. Actin was used as a loading control. (C) Cell numbers of 344SQ-Bmp4-KD and NTC cells over 7 days. Mean ± SD, n = 4; p, two-tailed Student’s t-test. (D) Diameters of 3-D acini of 344SQ-Bmp4-KD and NTC cells cultured on Matrigel for 10 days. Mean ± SD, n = 15; p, two-tailed Student’s t-test. e, f Migrated (e) and invaded (f) cells in Boyden chambers were photographed and counted. Mitomycin C (1 μg/mL) was added to the culture media to block cell proliferation. Mean + SD, n = 3; p, two-tailed Student’s t-test. g Primary tumor weight (left) and lung metastases (right) in syngeneic mice injected with 344SQ-Bmp4-KD and NTC cells. Box-and-whisker plots denote median and upper/lower quartiles + 1.5 × IQR (interquartile range). p, two-tailed Student’s t-test