Figure 3
Figure 3. Endogenous prNL levels and regulation 6, miR 92-1. (A) CLL B cells express a low level of pVHL. CLL B-cell lysates (n = 6) were analyzed for endogenous expression of VHL by Western blot using an antibody to pVHL. Purified CD19+ B cells were used as control for comparison. Similarly, HIF-1α expression in these CLL B-cell lysates was also examined using a specific antibody to HIF-1α. Actin was used as a loading control. (B) CLL B cells overexpress miR-92-1. Expression levels of mature miR-92-1 in CLL B cells were measured by real-time RT-PCR using specific primers. Total RNA was extracted from the same primary CLL B cells (n = 6) used for the Western blot analysis of pVHL as indicated, and purified CD19+ (> 98%) normal B lymphocytes. The single-tube TaqMan microRNA assays were performed to quantify mature miR-92-1 and normalization was performed with RNU6B in triplicate. Relative expression (fold) was calculated using the comparative Ct method, where the expression of miR-92-1 in normal B cells was arbitrarily chosen as one. For comparison of the microRNA fold levels with the VHL protein expression in these samples, densitometric values of VHL from the CLL B-cell Western blot (panel A) were calculated based on the values from normal B cells (arbitrarily chosen as 100%) and are presented below the miR92-1 fold levels. (C) VHL is a target of miR-92-1. The 3′UTR of VHL enables miR-92-1 regulation. (i) The complementarity between VHL cDNA and miR-92-1 is conserved in human and mouse. (ii) Relative repression of firefly luciferase expression standardized to a transfection control, renilla luciferase. As expected, the mutant completely abolished the detected interaction between miR-92-1 and the 3′UTR of VHL as shown in the last column. Experiments were performed twice in triplicate (N = 6). Data are presented as mean ± 1 SE. (D) Introduction of miR-92-1 reduces pVHL level. 293T cells transfected with increasing amounts of miR-92-1 were analyzed for the expression of VHL by Western blot. Actin was used as the loading control. (E) Inhibitor of miR-92-1 up-regulates pVHL. Primary CLL B cells (n = 3) transfected with the antisense inhibitor of miR-92-1 were analyzed for pVHL by Western blot. Actin was used as the loading control. PBMCs from different patients with CLL are marked by numbers.

Endogenous prNL levels and regulation 6, miR 92-1. (A) CLL B cells express a low level of pVHL. CLL B-cell lysates (n = 6) were analyzed for endogenous expression of VHL by Western blot using an antibody to pVHL. Purified CD19+ B cells were used as control for comparison. Similarly, HIF-1α expression in these CLL B-cell lysates was also examined using a specific antibody to HIF-1α. Actin was used as a loading control. (B) CLL B cells overexpress miR-92-1. Expression levels of mature miR-92-1 in CLL B cells were measured by real-time RT-PCR using specific primers. Total RNA was extracted from the same primary CLL B cells (n = 6) used for the Western blot analysis of pVHL as indicated, and purified CD19+ (> 98%) normal B lymphocytes. The single-tube TaqMan microRNA assays were performed to quantify mature miR-92-1 and normalization was performed with RNU6B in triplicate. Relative expression (fold) was calculated using the comparative Ct method, where the expression of miR-92-1 in normal B cells was arbitrarily chosen as one. For comparison of the microRNA fold levels with the VHL protein expression in these samples, densitometric values of VHL from the CLL B-cell Western blot (panel A) were calculated based on the values from normal B cells (arbitrarily chosen as 100%) and are presented below the miR92-1 fold levels. (C) VHL is a target of miR-92-1. The 3′UTR of VHL enables miR-92-1 regulation. (i) The complementarity between VHL cDNA and miR-92-1 is conserved in human and mouse. (ii) Relative repression of firefly luciferase expression standardized to a transfection control, renilla luciferase. As expected, the mutant completely abolished the detected interaction between miR-92-1 and the 3′UTR of VHL as shown in the last column. Experiments were performed twice in triplicate (N = 6). Data are presented as mean ± 1 SE. (D) Introduction of miR-92-1 reduces pVHL level. 293T cells transfected with increasing amounts of miR-92-1 were analyzed for the expression of VHL by Western blot. Actin was used as the loading control. (E) Inhibitor of miR-92-1 up-regulates pVHL. Primary CLL B cells (n = 3) transfected with the antisense inhibitor of miR-92-1 were analyzed for pVHL by Western blot. Actin was used as the loading control. PBMCs from different patients with CLL are marked by numbers.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal