Figure 6.
Evaluation of functional significance of MYC and MIR17HG in BL tumors and cell lines. (A) Comparison of average miR-17-92 expression and MYC expression in mBL tumors. miR17∼92 expression levels were inversely correlated with MYC expression in tumors (r = −0.23). (B) Whole-cell lysates were western blotted for PTEN, BIM, MYC, and β-actin expression in the noted BL cell lines expressing empty vector (C) or a vector containing mIR17-92 sponge (S). (C) BIM, PTEN, and MYC expression was normalized to β-actin, and relative expression (sponge/vector control) was quantified from 2 separate western blot experiments. Error bars represent 1 standard deviation. (D) Proliferation in BL cell lines after miR17∼92 sponge expression or MIR17HG knock out (Daudi-CRISPR) and/or ibrutinib (IBT) treatment. Line graph depicts 1 representative experiment of triplicate experiments, and points are the average of 3 biological replicates. Error bars represent 1 standard deviation. BL cell lines were treated with a specific concentration of IBT based on the 50% infective dose calculation for each line. (E) Comparison of mRNA levels of CD22 and FCGR2B in control (vector or parental) or miR17∼92 sponge or knockout (Daudi-CRISPR) cells with or without IgM stimulation. Fold change was calculated relative to the vector or parental controls. Graph depicts 1 representative experiment of 3 separate experiments. Error bars depict 1 standard deviation of 3 technical replicates. (F) Cell proliferation in BL cell lines after MYC KO and/or miR17∼92 knockdown (sponge). Points represent the mean of 3 biological replicates from 1 representative experiment of at least 3 separate experiments. Error bars represent the standard error of the mean. (G) Western blots of whole-cell lysates from control (GFP or parental) or miR17∼92 sponge-expressing or MIR17HG knock-out (Daudi-Cas-9) BL cell lines after induction of BCR signaling with anti-IgM. (H) Relative expression of phosphorylated/total SYK, BTK, BLNK, and ERK was quantified from the western blot, suggesting decreased BCR signaling in miR17∼92 sponge-expressing or KO BL cell lines.

Evaluation of functional significance of MYC and MIR17HG in BL tumors and cell lines. (A) Comparison of average miR-17-92 expression and MYC expression in mBL tumors. miR17∼92 expression levels were inversely correlated with MYC expression in tumors (r = −0.23). (B) Whole-cell lysates were western blotted for PTEN, BIM, MYC, and β-actin expression in the noted BL cell lines expressing empty vector (C) or a vector containing mIR17-92 sponge (S). (C) BIM, PTEN, and MYC expression was normalized to β-actin, and relative expression (sponge/vector control) was quantified from 2 separate western blot experiments. Error bars represent 1 standard deviation. (D) Proliferation in BL cell lines after miR17∼92 sponge expression or MIR17HG knock out (Daudi-CRISPR) and/or ibrutinib (IBT) treatment. Line graph depicts 1 representative experiment of triplicate experiments, and points are the average of 3 biological replicates. Error bars represent 1 standard deviation. BL cell lines were treated with a specific concentration of IBT based on the 50% infective dose calculation for each line. (E) Comparison of mRNA levels of CD22 and FCGR2B in control (vector or parental) or miR17∼92 sponge or knockout (Daudi-CRISPR) cells with or without IgM stimulation. Fold change was calculated relative to the vector or parental controls. Graph depicts 1 representative experiment of 3 separate experiments. Error bars depict 1 standard deviation of 3 technical replicates. (F) Cell proliferation in BL cell lines after MYC KO and/or miR17∼92 knockdown (sponge). Points represent the mean of 3 biological replicates from 1 representative experiment of at least 3 separate experiments. Error bars represent the standard error of the mean. (G) Western blots of whole-cell lysates from control (GFP or parental) or miR17∼92 sponge-expressing or MIR17HG knock-out (Daudi-Cas-9) BL cell lines after induction of BCR signaling with anti-IgM. (H) Relative expression of phosphorylated/total SYK, BTK, BLNK, and ERK was quantified from the western blot, suggesting decreased BCR signaling in miR17∼92 sponge-expressing or KO BL cell lines.

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