Figure 5.
Figure 5. miR-125b upregulates VEGFA production and signaling to promote leukemia cell expansion. (A) Cytokine microarray analyses were performed for the culture supernatants from AF9+Ri125b AML cells in +Dox or −Dox conditions. The names of the 16 different cytokines are indicated. The fluorescence intensities from the cytokine microarray assay are plotted (n = 3; *P < .05; **P < .001). (B) The absolute concentrations of VEGFA in the culture supernatants from AF9+Ri125b AML cells in +Dox or −Dox conditions were determined by recombinant VEGFA standards (n = 3; *P < .05). (C) VEGFA and VEGFC messenger RNA (mRNA) expressions were determined by quantitative reverse transcription polymerase chain reaction in AF9+Ri125b+Dox and AF9+Ri125b−Dox cells on day 6 after Dox withdrawal. 18S ribosomal RNA was used as a control (n = 3; **P < .01). (D) Primary AF9+Ri125b leukemia cells were cultured in the presence or absence of Dox. Recombinant VEGFA (500 pg/mL) was added to AF9+Ri125b−Dox cells. Cell numbers were counted on indicated days (n = 3; **P < .001). (E) AF9+Ri125b+Dox cells were transduced with control shRNA (shCtrl) or shRNAs against VEGFA (shVEGFA_1, shVEGFA_2). VEGFA RNA levels were determined by quantitative reverse transcription polymerase chain reaction after 3 days (n = 3; *P < .05). Dox was withdrawn from shRNA-transduced AF9+Ri125b AML cells for 5 days, and AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were then plated. Cell numbers were counted 2.5 days after plating (n = 3; *P < .05; **P < .01). (F) AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were treated with dimethyl sulfoxide (DMSO; vehicle) or 3 nM SU1498. Cell numbers were plotted on indicated days (n = 3; *P < .05; **P < .001). (G) VEGFR2 downstream proteins Bcl2, Bax, phosphorylated ERK (pERK), and total ERK were measured in AF9+Ri125b leukemia cells cultured with or without Dox for 6 days. β-actin was used as a loading control. (H) AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were treated with DMSO (vehicle) or 3 nM SU1498. Protein expression levels were determined by western blot after 3 days. Representative blots are shown. For all panels, error bars represent standard deviations.

miR-125b upregulates VEGFA production and signaling to promote leukemia cell expansion. (A) Cytokine microarray analyses were performed for the culture supernatants from AF9+Ri125b AML cells in +Dox or −Dox conditions. The names of the 16 different cytokines are indicated. The fluorescence intensities from the cytokine microarray assay are plotted (n = 3; *P < .05; **P < .001). (B) The absolute concentrations of VEGFA in the culture supernatants from AF9+Ri125b AML cells in +Dox or −Dox conditions were determined by recombinant VEGFA standards (n = 3; *P < .05). (C) VEGFA and VEGFC messenger RNA (mRNA) expressions were determined by quantitative reverse transcription polymerase chain reaction in AF9+Ri125b+Dox and AF9+Ri125b−Dox cells on day 6 after Dox withdrawal. 18S ribosomal RNA was used as a control (n = 3; **P < .01). (D) Primary AF9+Ri125b leukemia cells were cultured in the presence or absence of Dox. Recombinant VEGFA (500 pg/mL) was added to AF9+Ri125b−Dox cells. Cell numbers were counted on indicated days (n = 3; **P < .001). (E) AF9+Ri125b+Dox cells were transduced with control shRNA (shCtrl) or shRNAs against VEGFA (shVEGFA_1, shVEGFA_2). VEGFA RNA levels were determined by quantitative reverse transcription polymerase chain reaction after 3 days (n = 3; *P < .05). Dox was withdrawn from shRNA-transduced AF9+Ri125b AML cells for 5 days, and AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were then plated. Cell numbers were counted 2.5 days after plating (n = 3; *P < .05; **P < .01). (F) AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were treated with dimethyl sulfoxide (DMSO; vehicle) or 3 nM SU1498. Cell numbers were plotted on indicated days (n = 3; *P < .05; **P < .001). (G) VEGFR2 downstream proteins Bcl2, Bax, phosphorylated ERK (pERK), and total ERK were measured in AF9+Ri125b leukemia cells cultured with or without Dox for 6 days. β-actin was used as a loading control. (H) AF9+Ri125b+Dox and AF9+Ri125b−Dox cells were treated with DMSO (vehicle) or 3 nM SU1498. Protein expression levels were determined by western blot after 3 days. Representative blots are shown. For all panels, error bars represent standard deviations.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal