FIGURE 2.

Overexpression of sXBP1 enhances miR-346 levels in HeLa cells. A, overexpression of sXBP1 and uXBP1 constructs in HeLa cells. HeLa cells were transfected with empty vector (EV), sXBP1, and uXBP1 constructs. To assess transfection efficiency, XBP1 mRNA levels were measured by qRT-PCR (n = 3). B, immunofluorescence of XBP1 protein expression. These experiments were performed to confirm that functional sXBP1 is translated following transfection with the sXBP1 construct. In control cells, XBP1 expression cannot be seen (top left panel, nuclei are stained with DAPI). Induction of ER stress (TM) results in XBP1 mRNA splicing, sXBP1 translation, and nuclear targeting (top right panel, DAPI and anti-XBP1 antibody staining). Transfected cells (EV, sXBP1, or uXBP1) were stained with ER-Tracker (red), and XBP1 protein was detected with an anti-XBP1 antibody that recognizes both the sXBP1 and the uXBP1 proteins. Both sXBP1 and uXBP1 proteins enter the nucleus (green). In cells expressing sXBP1, the ER is expanded (red, middle panel) as expected. C, measurement of miR-346 levels. Using the same samples as in A, miR-346 levels were also determined. Results are plotted as -fold increase over EV-transfected, control samples (n = 4). A significant increase in miR-346 levels was observed in cells transfected with sXBP1.