Optimization of rAAV RI.

<p>A. Transduction efficiency of different quantities of rAAV2-Gluc using 4×10⁴ BHK21 cells; the transduction efficiency is represented by Gluc activity. B. Optimization of the virus/cell ratio. Different numbers of BHK21 cells were applied to a 96-well plate pre-coated with 5×10⁸ viral genomes/well. The transduction efficiency is represented by the EGFP intensity. C. Temperature stability assessment of rAAV2-Gluc. In total, 5×10⁸ viral genomes of rAAV were applied to each well. The plates were then treated at 4, 37, 42, or 56°C for 24 h, followed by the application of 4×10⁴ BHK21 cells per well. Gluc activity was measured 24 h later.</p

Response to NaB. Black bar, without NaB; gray bar, with NaB.

<p>A. AAV1 RI-based assay. B. AAV2 RI-based assay.</p

Changes in miRNA activity induced by TPA in K562 cells.

<p>(A) Morphological changes in K562 cells induced by TPA. K562 cells were cultured in DMEM (10% FBS) containing 16 nM TPA. Twenty-four hours later, the morphology of K562 cells was altered. (B) Comparison of miRNA activity induced by TPA versus untreated K562 cells. The miRNA activity profiles with or without TPA were detected using miRNA Asensor arrays and miRNA activities with obvious changes are shown. Error bars correspond to mean±SD (n = 3). Student T test was used for statistical anylasis. * <i>P</i> values<0.05; ** <i>P</i> values<0.01.</p

Comparison of miRNA activity profiles between HEK293 and HEK293T cells.

<p>(A) miRNA activity profiles for HEK293 and HEK293T were established using the miRNA Asensor arrays. (B) Several miRNAs were selected and their activities compared. Compared to HEK293T cells, miR-17-5p, miR-18a, miR-20a, and miR-106a activities were lower in HEK293 cells, while miR-21, miR-222, let-7a, let-7b, let-7c, let-7e, and let-7f were higher. (C) miR-21 expression level was detected by QRT-PCR. The miR-21 activity was consistent with its expression level in the two cell lines. 2^ΔCt was used to indicate the miRNA expression levels. ΔCt = Ct_U6−Ct_miRNA. RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3). Student T test was used for statistical anylasis. *indicates <i>P</i> values<0.05 and ** <i>P</i> values<0.01.</p

Comparison of miRNA activities with expression levels in HEK293 cells.

<p>(A, D) The activities of members of the let-7 family were not consistent with their expression levels. (B, E) miR-221 and miR222 activities were consistent with their expression levels. (C, F) The consistency of miRNA activity with expression levels was complex in the miR-17-92 cluster. For miR-18a, the miRNA activity was not consistent with its expression level. For miR-17-3p, miR-17-5p, miR-19a, miR-19b, miR-20a and miR-92a, the miRNA activities were consistent with their expression levels. 2^ΔCt was used to indicate miRNA expression. ΔCt = Ct_U6−Ct_miRNA. RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3).</p

miRNA activity profiles for the twelve cell lines.

<p>(A) Activities of 115 miRNAs in 12 cell lines were detected. (B–G) Comparison of specific miRNA activities in 12 cell lines. miR-199a-3p activity was specifically high in BJ, C2C12, and BHK21 cells (B). miR-143 activity was specifically high in BJ and C2C12 cells (C). miR-21 activity was high in all of the cell lines excluding HEK293T (D). miR-221/222 cluster activities were high in all cell lines excluding U937 and K562 (E). miR-142-3p and miR-142-5p activities were specifically high in K562 and U937 cell lines (F). miR-122 activity was specifically high in Huh7/CD81, while miR-194 activity was high in both Huh7/CD81 and HepG2 cells (G). RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3).</p