Fig. 6.

TRIM44 expression enhances HIF-1α stability and increases its downstream gene expression. a and b TRIM44^OE-CON, TRIM44^OE, TRIM44^KD-CON and TRIM44^KD U266 cells (a) or RPMI (b) were cultured in hypoxia conditions (1% O₂) for the indicated hours. HIF-1α levels were determined using immunoblotting, which was normalized using β-actin. c TRIM44^OE-CON and TRIM44^OE 293T cells were transfected with HA-HIF-1α. After 48 h, the cells were treated with 50 μg/ml cycloheximide (CHX) for the indicated hours. HIF-1α levels were determined by immunoblotting and were normalized by β-actin. d TRIM44^OE-CON, TRIM44^OE, TRIM44^KD-CON, and TRIM44^KD RPMI MM cells were incubated in hypoxia conditions for 4 h. Subsequently, cycloheximide (CHX, 50 μg/ml) was added for 0, 10, 30, 60, and 120 min under normoxia or hypoxia conditions. Cell lysates were then subjected to immunoblotting analyses using HIF-1α. Quantitation of HIF-1α expression was normalized by β-actin and shown in Supplementary Figure S4d