Antisense oligonucleotide nomenclature.

<p>MSP, muscle-specific peptide and B - β-alanine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111079#pone.0111079-Yin3" target="_blank">[34]</a>.</p><p>Antisense oligonucleotide nomenclature.</p

Duration of exon skipping effects in <i>C57BL6</i> with different AOs by local intramuscular injection.

<p>(A) RT-PCR results for PMO, PNA and 2′Ome PS in <i>C57BL6</i> at different time-points e.g. 2 and 4 weeks after local intramuscular injection. (B) Quantification of exon 23 skipping efficiency for PMO, PNA and 2′Ome PS at different time-points (**p<0.001, n = 3).</p

Efficient exon skipping in other wild-type mice by local intramuscular injection.

<p>(A) RT-PCR results for PMO, PNA and2′Ome PS in <i>C3H</i> mice 48 hr after local intramuscular injection. The numbered Δexon23 is for exon 23 skipping. (B) Quantitative analysis of exon skipping induced by different AOs in <i>C3H</i> mice (*P<0.05, n = 3). (C) RT-PCR results for PMO, PNA and 2′Ome PS in <i>ICR</i> mice 48 hr post-injection. (D) Quantitative analysis of exon skipping efficiency by different AOs in <i>ICR</i> mice (**P<0.001, n = 3). (E) RT-PCR results for PMO, PNA and 2′Ome PS in <i>BABL/C</i> mice 48 hr post-injection. (F) Quantitative analysis of exon skipping induced by different AOs in <i>BABL/C</i> mice (*P<0.05, n = 3). (G) Cross-comparison between different wild-type and <i>mdx</i> mice per AO chemistry.</p

Effective exon skipping in <i>C57BL6</i> and <i>mdx</i> mice by local intramuscular injection of different AOs.

<p>(A) RT-PCR for detecting exon skipping at the RNA level with treated TA muscles 48 hr after intramuscular injection of 2 µg PMO, 5 µg PNA and 5 µg 2′Ome PS in <i>C57BL6</i> and <i>mdx</i> mice. The numbered Δexon23 is for exon 23 skipping. –ve stands for RT-PCR blank control and the same below unless otherwise specified. (B) Quantitative analysis of exon skipping induced by different AOs in <i>C57BL6</i> and <i>mdx</i> mice (**P<0.001; *P<0.05, n = 3). (C) Quantitative PCR validation for detecting exon skipping at the RNA level with treated TA muscles 48 hr after intramuscular injection in <i>mdx</i> mice. (D) Sequence analysis of the exon 23 skipped band in the RT-PCR products.</p

Systemic injection of Hepa1-6 cells in syngeneic <i>C57BL6</i> mice.

<p>Hepa1-6 cells (2x10⁶) suspended in PBS were injected into <i>C57BL6</i> mice intravenously. (a) Morphological examination of tumor nodules in different tissues. The results showed that tumor formation in lung, heart and ribs. (b) Histological assessment of liver tumor nodules in lung, liver and heart (scale bar = 50 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C.</p

Tumor take rates in different models.

<p>Tumor take rates in different models.</p

Intrasplenic and intrahepatic inoculation of Hepa1-6 cells in <i>C57BL6</i> mice.

<p>Hepa1-6 cells (2x10⁶) suspended in PBS were injected into <i>C57BL6</i> mice intrasplenicly or intrahepatically as described in Materials and Methods. (a) Morphological examination of tumor nodules in different tissues from orthotopic HCC mice generated by intrasplenic inoculation of Hepa1-6 cells. The results showed the tumor formation in liver and spleen. (b) Histological assessment of liver tumor nodules in spleen, liver and lung (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C. (c) MRI analysis of the progression of liver tumors after intrahepatic inoculation of Hepa1-6 cells at different time-points. Arrows point to the tumor nodules. (d) Morphological examination of tumor nodules in liver from orthotopic HCC mice via intrahepatic injection of Hepa1-6 cells. The results showed both solitary and multinodular tumors formed in liver. (e) Histological assessment of liver tumor nodules in liver and lung (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C.</p

Intrahepatic tissue implantation in <i>C57BL6</i> mice.

<p>Hepa1-6 cells (3x10⁶) were suspended in 50μl PBS and injected into the left axilla of <i>C57BL6</i> mice subcutaneously with 1 ml syringe. Subsequently, tumor tissues were cut into about 1mm³ pieces. Tumor pieces with a size about 1mm³ were implanted in the left liver lobe of <i>C57BL6</i> mice. (a) MRI analysis of the progression of liver tumors after tissue implantation at different time-points. Arrows point to the tumor nodules. (b) Measurement of liver tumor sizes at different time-points after implantation. The data represents as mean±sem and significant difference was detected between different time-points (*p<0.05; **p<0.01, n = 10 for 2 week time points; n = 20 for 3 week time point; n = 19 for 7 week time point after implantation). (c) Assessment of survival rates of orthotopic HCC mice generated by intrahepatic tissue implantation (n = 19). (d) Morphological examination of tumor nodules in different tissues. The results showed the tumor formation in liver, peritoneum, mesenterium and diaphragm. (e) Histological assessment of liver tumor nodules in lung and liver (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C. (f) Immunohistochemistry of CD3+ and Foxp3+ regulatory T cells in liver sections from orthotopic HCC mice and HCC patients to determine the extent of T cell infiltration (scale bar = 100 μm). Arrowhead points to CD3+ or Foxp3+ T cells. TI represents Tissue Implantation; CI denotes as Cell Inoculation. (g) Measurement of immune cytokines including IFN-γ and IL-10 in serum from orthotopic HCC mice (n = 5, *P<0.05; **P<0.01). The comparison was conducted between CI, TI and normal controls. TI represents Tissue Implantation; CI means Cell Inoculation.</p