Article Figures & Data
Figures
- Figure 1
RNAi modeling and annealing. (A) An idealized model of RNA folding for IL-10 based on enthalpy and free-energy calculations. Brackets indicate the location of the mouse IL-10 RNAi employed in these experiments. The sequence begins at nucleotide 317 and appears exposed in a hairpin. (B) Acrylamide gel of RNA stained with ethidium bromide. Lane 1 is dH2O as a negative control, lane 2 is dsRNAi, lane 3 is ssRNAi strand 1, and lane 4 is ssRNAi strand 2. The size of the ladder on the far left (y-axis) is given in nucleotides. The arrow indicates the band for dsRNAi.
- Figure 2
RNAi inhibits cellular growth in a time-dependent manner. The graph shows the cell count over 72 hr of no treatment (dashed line), treatment with 2 µM scrambled IL-10 dsRNA (dsScr, dotted line), and treatment with 2 µM RNAi IL-10 (RNAi, solid line). The x-axis corresponds to the number of days of treatment and the y-axis to the cell number. The graph is representative of triplicate IL-10 RNAi treatments.
- Figure 3
RNAi effectively limits cellular proliferation in a dose-dependent manner. Columns corresponds to treatment groups, with the mean OD values for MTT analysis plotted on the y-axis and the bars corresponding to the standard error of the mean. (A) Cells treated for 16.5 hr with varying concentrations of IL-10 RNAi (RNAi) or scrambled IL-10 dsRNA (dsScr) (0.2-2.0 µM): Gray bar, 0.2 µM ; horizontal stripe, 0.5 µM; diamond bar, 1.0 µM; hatched bar, 2.0 µM. Error bars are the SEM of triplicate results. Asterisks indicate P < 0.05 for a Student's t-test comparing dsScr to RNAi. (B) Cells treated for 48 hr with 2 µM RNAi or dsScr. Error bars are the SEM of triplicate results. Asterisks indicate P < 0.05 for a Student's t-test comparing dsScr to RNAi.
- Figure 4
RNAi induces a G2/M block and apoptosis. Histograms showing PI staining of DNA content, with the x-axis reflecting DNA content and the y-axis the cell number. Arrows indicate sub-G1 cells (apoptotic). Flow cytometric determination of cell cycle distribution of LNC cells following 24 hr (A-C) or 42 hr (D-F) of treatment: Control (A) and (D), scrambled IL-10 dsScr (B) and (E), RNAi IL-10 (RNAi) (C) and (F). The distribution of cells in each phase of the cell cycle following treatment was summed to give the percentage of cycling cells. The percentage of apoptotic cells is expressed as a percentage of the whole gated population.
- Figure 5
IL-10 protein analysis by ELISA. The amount of IL-10 protein in LNC supernatants was measured by ELISA. Columns correspond to groups treated for 72 hr with either 2 µM IL-10 RNAi (hatched bar), 2 µM dsScr (black bar), or untreated control (gray bar). Error bars are the SEM of triplicate results. Asterisks indicate P < 0.05 for a Student's t-test comparing dsScr to RNAi.
- Figure 6
IL-10 message analysis by real-time RT-PCR. Fluorescence of Taqman™ reporter dyes for IL-10 and ribosomal 18S are labeled. Black curves represent results from LNC cells treated for 72 hr with 2 µM IL-10 RNAi. Gray curves are untreated control. Ribosomal 18S was used to normalize samples. The graph is representative of three real-time PCR reactions.
- Figure 7
Semiquantitative RT-PCR analysis of IL-10 and critical cell cycle components. (A) Graphic analysis of semiquantitative RT-PCR for IL-10, cdc25C, p27, IL-10 R1, IL-10 R2 and TNF-alpha RNA levels after 48 hours of treatment with either 2 µM IL-10 RNAi (hatched bars), 2 µM dsSCR (gray bars) as compared to an untreated control. Numbers correspond to the percentage of control values from triplicate gels and error bars to the SEM. Asterisks indicate P < 0.05 for a Student's t-test comparing dsScr to RNAi. (B) Representative PCR gels stained with ethidium bromide: Control (lane 1), 2 µM dsSCR (lane 2) and 2 µM IL-10 RNAi (lane 3).
- Figure 8
Semiquantitative RT-PCR analysis of apoptosis regulators. (A) Graphic analysis of semiquantitative RT-PCR for Bcl2, Bcl7C, and Bax RNA levels after 48 hr of treatment with either 2 µM IL-10 RNAi (hatched bars) or 2 µM dsScr (gray bars) when compared to an untreated control. Numbers represent percent of control values from triplicate gels. Error bars are SEM. Asterisks indicate P < 0.05 for a Students t-test comparing dsScr to RNAi. (B) Representative PCR gels stained with ethidium bromide. Lane 1 is control, lane 2 is 2 µM dsScr and lane 3 is 2 µM IL-10 RNAi.
Tables
- Table 1
Genes, PCR primers and products.
Gene PCR Primer Sequences PCR Product Name GI No. Size (bp) Nucleotides Spanned IL-10 6754317 5': CGGGAAGACAATAACTG 186 148-333 3': CATTTCCGATAAGGCTTGG IFN-gamma 28501526 5': AACGCTACACACTGCATCTTG 237 112-348 3': GACTTCAAAGAGTCTGAGG TNF-alpha 7305584 5': AGTGGTGCCAGCCGATGGGTTGT 253 537-789 3': GCTGAGTTGGTCCCCCTTCTCCAG Bax 17390521 5': CCAAGAAGCTGAGCGAGTGTCTC 146 235-381 3': AGTTGCCATCAGCAAACATGTCA Bcl2 6753167 5': CAGCTGCACCTGACG 303 343-641 3': AGAGACAGCCAGGAG Bcl7C 13542972 5': GGCGACCATCGAGAAGGTCCG 318 305-622 3': GTTGGTGATCCAGGGCGGC cdc25C 13435584 5': CACCAGTTTAAAGGCATTGG 230 519-748 3': GACTTAAGCCCTTGGCGTCC IL-10R1 6680388 5': CTGAGCCTAGAATTCATTGCATAC 388 122-509 3': CATAGATGATGCCGTCCATTGCTT IL-10R2 17646387 5': CCACCCCCTGAGAAGGTC 599 80-678 3': CAGGAAGGGGTTATTTCG p27 17939614 5': GGCTCTGCTCCATTTGACTG 393 342-734 3': CCTGCCATTCGTATCTGCCC IL-19 28482156 5': CCGGATCCCTCAGTTCATATCTACAGTCTTAG 477 166-643 3': TTGAATTCAGGCTGCAGGAGTTTCCAGATG IL-22 8393604 5': AAATGCGCTGCCCGTCAACAC 192 146-337 3': ACCTGCTTCATCAGGTAGCAC IFN-lambda 27261790 5': GCGGTACCATGACTGGGGACTGCACGCCAGTG 595 9-603 3': CGGAATTCAGGTGGACTCAGGGTGGGTTGA HPRT 13435620 5': GTTGGATACAGGCCAGACTTTGTTG 162 575-737 3': GATTCAACTTGCGCTCATCTTAGGC
Volume 4, Issue 1
