Figure 4
Figure 4. miR-17∼92 stimulates Myc stabilization and Ca2+ flux upon ligation of the BCR. (A-B) Western blotting was performed for P-PLCγ2, total-PLCγ2, and actin. Bands were quantified and the ratio of P-PLCγ2 /total-PLCγ2 is shown. (A) P493-6 cells were grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM for 2 minutes. (B) P493-6 cells were grown in the absence (MYCHIGH) of 1μM doxycycline and treated with control short-hairpin inhibitor or miR-17∼92 short-hairpin inhibitor mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM for 2 minutes. (C-D) Following BCR crosslinking by anti-IgM, cytosolic Ca2+ levels in P493-6 cells were monitored by fluorescence microscopy using the ratiometric Ca2+ indicator dye Fura-2. Mean traces of 3 independent experiments are plotted (top); *P < .05, **P < .01. Independent experiments are plotted in supplemental Figure 2A-B. Representative images at basal cytosolic Ca2+ and maximum amplitude are displayed (bottom). (C) P493-6 cells were grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of anti-IgM. (D) P493-6 cells (MYCHIGH) were treated with control short-hairpin inhibitor or miR-17∼92 short-hairpin inhibitor mix for 48 hours prior to addition of anti-IgM. (E) P493-6 cells grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM to ligate the BCR. Samples were harvested at times indicated. Western blotting was performed for P-Akt, pan-Akt, Pten, P-GSK-3β, total-GSK-3β, myc, and actin. (F) Model of Myc-miR-17∼92-BCR signaling feed-forward loop. Ligation of the BCR leads to phosphorylation of SYK and PI3K. ITIMs in CD22 and FCGR2B recruit the Src homology region 2 domain containing phosphatase (SHP)-1 and SHIP phosphatases, which target SYK and PI3K to dampen BCR signaling. miR-17∼92 represses cd22 and fcgr2b either directly and amplifies BCR signaling. This results in increased phospho-AKT, phospho-GSK-3β, Myc, and therefore, miR-17∼92.

miR-17∼92 stimulates Myc stabilization and Ca2+flux upon ligation of the BCR. (A-B) Western blotting was performed for P-PLCγ2, total-PLCγ2, and actin. Bands were quantified and the ratio of P-PLCγ2 /total-PLCγ2 is shown. (A) P493-6 cells were grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM for 2 minutes. (B) P493-6 cells were grown in the absence (MYCHIGH) of 1μM doxycycline and treated with control short-hairpin inhibitor or miR-17∼92 short-hairpin inhibitor mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM for 2 minutes. (C-D) Following BCR crosslinking by anti-IgM, cytosolic Ca2+ levels in P493-6 cells were monitored by fluorescence microscopy using the ratiometric Ca2+ indicator dye Fura-2. Mean traces of 3 independent experiments are plotted (top); *P < .05, **P < .01. Independent experiments are plotted in supplemental Figure 2A-B. Representative images at basal cytosolic Ca2+ and maximum amplitude are displayed (bottom). (C) P493-6 cells were grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of anti-IgM. (D) P493-6 cells (MYCHIGH) were treated with control short-hairpin inhibitor or miR-17∼92 short-hairpin inhibitor mix for 48 hours prior to addition of anti-IgM. (E) P493-6 cells grown in the presence (MYCLOW) of 1μM doxycycline and treated with control mimic or miR-17∼92 mimic mix for 48 hours prior to addition of 10 μg/mL soluble anti-IgM to ligate the BCR. Samples were harvested at times indicated. Western blotting was performed for P-Akt, pan-Akt, Pten, P-GSK-3β, total-GSK-3β, myc, and actin. (F) Model of Myc-miR-17∼92-BCR signaling feed-forward loop. Ligation of the BCR leads to phosphorylation of SYK and PI3K. ITIMs in CD22 and FCGR2B recruit the Src homology region 2 domain containing phosphatase (SHP)-1 and SHIP phosphatases, which target SYK and PI3K to dampen BCR signaling. miR-17∼92 represses cd22 and fcgr2b either directly and amplifies BCR signaling. This results in increased phospho-AKT, phospho-GSK-3β, Myc, and therefore, miR-17∼92.

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