Figure 6.

The Golgi apparatus is fragmented into mini-Golgi structures in p125A-silenced cells. (A) HeLa cells stably expressing ManII-GFP were silenced of p125A and then double labeled using mouse anti-GM130 and rabbit anti-GT antibodies followed by viewing the distribution of trans-Golgi marked by GT, medial-Golgi marked by ManII-GFP, and cis-Golgi marked by GM130. Bar, 10 µm. (B) Exogenous expression of myc-p125A can rescue the dispersed Golgi phenotype in p125A-silenced cells. HeLa cells stably expressing GT-GFP were transfected twice with ON-Targetplus SMARTpool siRNA (Thermo Fisher Scientific) against p125A with a 24-h interval. 48 h after siRNA transfection, the cells were transfected again to express myc-p125A. At 72 h after the initial siRNA transfection, the cells were processed for indirect immunofluorescence microscopy to view GT-GFP and myc-p125A. Asterisk denotes a rescued cell expressing myc-p125A. Bar, 10 µm. (C) ER export is delayed in p125A-silenced cells. HeLa cells stably expressing GT-GFP were transfected twice either with nontarget or p125A siRNA. At 72 h after the initial transfection, the cells were treated with BFA (5 µg/ml) for 30 min to distribute GT-GFP into the ER. The cells were then washed and incubated in BFA-free warm medium for the indicated times, and samples were fixed for fluorescence microscopy to assay for ER-Golgi transport of GT-GFP. All of the images were taken at the same exposure and processed in parallel. Bar, 10 µm.