Inhibitory effect of commercial rhIGFBP-3 on human MCF-7 breast cancer cells and HT-29 colon cancer cells.

<p>Different concentrations of commercial rhIGFBP-3 ranged from 9.375 ng/ml to 300 ng/ml were used to treat MCF-7 and HT-29 cells. Data are shown as means ± SD.</p

Inhibitory effect of different concentration of rice-produced rhIGFBP-3 on HT-29 human colon cancer cells.

<p>Different concentration of seed proteins (ranged from 1.042 to 15.625 mg) from WT and transgenic SBK-66 and SB-57 lines were used to treat HT-29 cells. Data are shown as means ± SD. *<i>p</i> < 0.05 and ** <i>p</i> < 0.01 denote statistically significant and very significant differences, respectively, between transgenic lines and WT.</p

Glycoprotein staining of transgenic lines and WT line in rice seeds.

<p>Total protein was extracted from mature dehulled seeds of SB-57 and SBK-66 transgenic lines and WT. RNase B was used as the glycoprotein control. To evaluate endoglycosidase digestion efficiency, RNase B was digested by PNGase F and Endo H. Lane 1: marker; lane 2: WT; lane 3: SBK-66; lane 4: SK-57; lane 5: RNase B control without digestion; lane 6: RNase B digested by PNGase F; and lane 7: RNase B digested by Endo H. Band 1: PNGase F; band 2: RNase B; band 3: RNase B with Endo H digestion, and band 4: RNase B with PNGase F digestion.</p

Western blot analysis of transgenic rice grains.

<p>Total protein was extracted from mature rice grains of different transformants. Recombinant hIGFBP-3 protein was used as positive control. (A) Lanes 1-5: five independent pSB130/Gt1/hIGFBP-3 transformants (B1-5); lane 6: WT; lane 7: positive control (+ve) - commercial rhIGFBP-3 protein. (B) Lanes 1-5: five independent pSB130/Gt1/SP/hIGFBP-3 transformants (SB1-5); lane 6: WT; lane 7: +ve - commercial rhIGFBP-3 protein. (C) Lanes 1-5: five independent pSB130/Gt1/SP/hIGFBP-3::KDEL transformants (SBK1-5); lane 6: WT; lane 7: +ve - commercial rhIGFBP-3 protein.</p

Western blot analysis of rhIGFBP-3 digested by Endo H in transgenic rice seeds.

<p>Total protein was extracted from mature dehulled seeds of SB-57 and SBK-66 transgenic lines and digested by Endo H. Lane 1: SB-57; lane 2: SB-57 digested by Endo H; lane 3: marker; lane 4: SBK-66; and lane 5: SBK-66 digested by Endo H.</p

Transgenic integration of modified hIGFBP-3 in rice.

<p>Genomic DNA extracted from rice leaves of independent transformants was digested with BamHI and separated on 0.8% agarose gel, blotted on nylon membrane and hybridized with DIG-labeled hIGFBP-3 probe. Lanes 1-3: three independent pSB130/Gt1/hIGFBP-3 transformants (B1 to B3); lane 4: wild type (WT) rice plant; lanes 5-6: two independent pSB130/Gt1/SP/hIGFBP-3 transformants (SB1 to SB2); lanes 7-9: three independent pSB130/Gt1/SP/hIGFBP-3::KDEL transformants (SBK1 to SBK3).</p

Inhibitory effect of rice-produced rhIGFBP-3 on MCF-7 human breast cancer cells.

<p>(A) Equal amount (3.125 mg seed) of WT, SB and SBK total seed protein extracts were added to the MCF-7 cells separately. Data are shown as means ± SD. *<i>p</i> < 0.05 and ** <i>p</i> < 0.01 denote statistically significant and very significant differences, respectively, between transgenic lines and WT.</p