2007 Program

2007 Women\u27s Basketball Program, George Fox Universit

Effect of ectopic expression of miR-125a and/or miR-99b on NF-κB activity.

<p>NF-κB activation was determined after 48 hours from transfection of Meg-01 cells with miRNA mimics and luciferase vectors. Results are relative to cells transfected with mock RNA and expressed as mean ± SEM of n = 3 independent experiments. Statistical significance: *P<0.05; ***P<0.001.</p

Effect of miR-125a inhibition on Ara-C-stimulated erythroid differentiation of K562 cells.

<p>K562 cells pre-treated with 1 µM ASO for 6 hours and treated with ASO for 48 hours were plated in MC for 4 days before being counted and collected for the corresponding assays. (<b>A</b>) Effect on cell density after 48 hours (n = 5). (<b>B</b>) Colony formation assay (n = 5). (<b>C</b>) Benzidine-positive colony count (n = 3). The proportion of erythroid-like colonies after 4 days from plating is expressed as the number of benzidine-positive colonies, normalized to the total colony number. (<b>D</b>) Expression of CD71 (TFRC) in K562 colonies. Statistical analysis represents a grouped analysis of the main effect of ASO (n = 4). (<b>A–D</b>) Data represent mean ± SEM. Statistical significance: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.</p

Relationship between the miR-99b/let-7e/miR-125a cluster members and genes from the TLR2-NF-κB pathway in MDS.

<p>“Low” and “high” expression cohorts were established based on comparison with the mean relative expression value. (<b>A–B</b>) Correlation between the relative expression of miR-99b and TLR2 or MyD88, respectively, in MDS CD34⁺ cells. In (B), one outlier removed by ROUT method. (<b>C–E</b>) Correlation between the relative expression of miR-125b and TLR2, MyD88 or JMJD3, respectively in MDS CD34⁺ cells. In (E), five outliers removed by ROUT method. (<b>F</b>) Correlation between relative expression of miR-125a and JMJD3. Seven outliers removed by ROUT method. Statistical significance: *P<0.05; ***P<0.001.</p

Expression of miR-125a and miR-125b in BM CD34⁺ cells.

<p>(<b>A–B</b>) Relative expression of miR-125a and miR-125b in CD34⁺ cells from MDS patients (N = 48/47, respectively) and healthy donors. In (<b>A</b>), one data point (Y = 386.31) is outside the axis limits. Statistical significance versus healthy donors: **P<0.01. (<b>C</b>) Correlation between relative expression of miR-125a and miR-125b in MDS CD34⁺ cells. Six data points are outside the axis limits.</p

Correlation between the relative expression of miR-99b and overall survival in MDS.

<p>Levels of miR-99b are inversely correlated with patient survival.</p

Expression of TLR7 in MDS.

<p>(<b>A</b>) Relative expression of TLR7 in BM CD34⁺ cells. mRNA levels of TLR7 in CD34⁺ cells from MDS patients (N = 42) and healthy donors were measured by qPCR. (<b>B–E</b>) Correlation between the relative expression of TLR7 and TLR2, miR-99b, miR-125b or miR-125a, respectively, in MDS CD34⁺ cells. “Low” and “high” expression cohorts were established based on comparison with the mean relative expression value. In (B), two data points are outside the axis limits; in (C), three outliers removed by ROUT method; in (D), four outliers removed by ROUT method; in (E), seven outliers removed by ROUT method. Statistical significance: *P<0.05; ***P<0.001.</p

Expression of miR-99b and let-7e in BM CD34⁺ cells.

<p>(<b>A–B</b>) Relative expression of miR-99b and let-7e in CD34⁺ cells from MDS patients (N = 48/45, respectively) and healthy donors. One outlier removed by Grubb's method (α = 0.05) in each case. (<b>C–D</b>) Correlation between the relative expression of miR-125a and miR-99b or let-7e, respectively, in MDS CD34⁺ cells. In (C) and (D), three data points are outside the axis limits.</p

Effect of the inhibition of miR-125a and TLR2-NF-κB pathway in MDS-L cells.

<p>Black bars represent cells treated with 5 µM control peptide, and striped bars represent cells treated with 5 µM MyD88-inhibitor peptide. (<b>A</b>) Effectiveness of miR-125a ASO in MDS-L cells, expressed as relative miR-125a expression levels after 48 hours of treatment with 1 µM ASO. (<b>B</b>) Cell density after 48 hours of treatment. (<b>C</b>) Colony formation assay after 7 days from plating in methylcellulose. (<b>D–F</b>) Relative expression of EPO-R, GYPA and CD71 (TFRC), respectively, measured by qPCR after a 7-day methylcellulose culture of cells previously treated for 48 hours with 1 µM ASOs. (<b>A–F</b>) Data represent mean ± SEM of n = 3 independent experiments. Statistical significance: *P<0.05.</p

Effect of TLR stimulation and miR-125a inhibition on NF-κB activity in KG1 cells.

<p>NF-κB activation was measured after 24 hours from nucleofection of KG1 cells with the luciferase vectors and treatment with ASO. Results are expressed as relative to cells transfected with mock and represent mean ± SEM of n = 4 independent experiments. Method disclosure: technical problems regarding the endogenous <i>Renilla</i> control were experienced during these luciferase assays; only one experiment out of four efficiently expressed the <i>Renilla</i> luciferase and could be properly normalized. Because normalized results were almost identical to non-normalized data, we conducted a joint statistical analysis of the four experiments. Statistical significance: ***P<0.001.</p