Figure 1
Figure 1. Myc-repressed transcripts are enriched for miR-17∼92 targets and BCR signaling components. (A) mRNA profiling was performed on P493-6 cells grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline for 48 hours. Log2 gene expression levels were plotted for MYCHIGH vs MycLOW. Genes repressed or stimulated >1.5-fold by Myc are denoted. (B) SigTerms analysis comparing the enrichment of predicted miRNA seed sequences in Myc-stimulated vs Myc-repressed genes. Predictions are based on the TargetScan algorithm. One hundred random gene sets of the same sample size were used as a control. The average and SD of those random gene sets are plotted. (C) SigTerms analysis (as in panel B) comparing genes repressed by Myc >1.5-fold, >2.5-fold, and >5-fold. (D) P493-6 cells grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline were treated with control mimic or miR-17∼92 mimic mix for 48 hours. RNA was isolated and qPCR was performed to quantitate changes in miRNA levels. RNU6B was used as an endogenous control and values are relative to MYC high/CTRL mimic. The average of 4 repetitions is reported. Error bars represent the SD. (E) mRNA profiling was performed on the cells from (D). GSEA was performed comparing the MYCLOW/CTRL mimic-treated cells to the “REST” (MYCLOW/miR-17∼92 mimic, MYCHIGH/CTRL mimic, and MYCHIGH /miR-17∼92 mimic). MYCLOW/CTRL mimic-treated cells are enriched for components of the BCR signaling pathway. The GSEA enrichment score, normalized enrichment score, P value, and FDR are indicated. For panels F through H, P493-6 cells were grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline for 48 hours prior to various treatments with anti-IgM. Western blotting was performed for P-spleen tyrosine kinase (P-SYK), total-SYK, P-B–cell linker protein (BLNK), total-BLNK, and either GAPDH or actin. (F) P493-6 cells were treated with increasing amounts of soluble anti-IgM for 15 minutes to ligate the BCR. (G) P493-6 cells were treated with 10 μg/mL soluble anti-IgM to ligate the BCR. Samples were harvested at times indicated. (H) P493-6 cells were treated with immobilized anti-IgM (imm-α-IgM) or immobilized isotype control (imm-isotype-CTRL) for 6 or 24 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative PCR.

Myc-repressed transcripts are enriched for miR-17∼92 targets and BCR signaling components. (A) mRNA profiling was performed on P493-6 cells grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline for 48 hours. Log2 gene expression levels were plotted for MYCHIGH vs MycLOW. Genes repressed or stimulated >1.5-fold by Myc are denoted. (B) SigTerms analysis comparing the enrichment of predicted miRNA seed sequences in Myc-stimulated vs Myc-repressed genes. Predictions are based on the TargetScan algorithm. One hundred random gene sets of the same sample size were used as a control. The average and SD of those random gene sets are plotted. (C) SigTerms analysis (as in panel B) comparing genes repressed by Myc >1.5-fold, >2.5-fold, and >5-fold. (D) P493-6 cells grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline were treated with control mimic or miR-17∼92 mimic mix for 48 hours. RNA was isolated and qPCR was performed to quantitate changes in miRNA levels. RNU6B was used as an endogenous control and values are relative to MYC high/CTRL mimic. The average of 4 repetitions is reported. Error bars represent the SD. (E) mRNA profiling was performed on the cells from (D). GSEA was performed comparing the MYCLOW/CTRL mimic-treated cells to the “REST” (MYCLOW/miR-17∼92 mimic, MYCHIGH/CTRL mimic, and MYCHIGH /miR-17∼92 mimic). MYCLOW/CTRL mimic-treated cells are enriched for components of the BCR signaling pathway. The GSEA enrichment score, normalized enrichment score, P value, and FDR are indicated. For panels F through H, P493-6 cells were grown in the absence (MYCHIGH) or presence (MYCLOW) of 1μM doxycycline for 48 hours prior to various treatments with anti-IgM. Western blotting was performed for P-spleen tyrosine kinase (P-SYK), total-SYK, P-B–cell linker protein (BLNK), total-BLNK, and either GAPDH or actin. (F) P493-6 cells were treated with increasing amounts of soluble anti-IgM for 15 minutes to ligate the BCR. (G) P493-6 cells were treated with 10 μg/mL soluble anti-IgM to ligate the BCR. Samples were harvested at times indicated. (H) P493-6 cells were treated with immobilized anti-IgM (imm-α-IgM) or immobilized isotype control (imm-isotype-CTRL) for 6 or 24 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative PCR.

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