Figure 1: STK16 localizes to the Golgi complex and its depletion or kinase inhibition impairs Golgi integrity.

(a) STK16 localizes to the Golgi complex during the whole cell cycle. HeLa-STK16-GFP-FLAG overexpression cell line grown on coverslips was fixed and stained for GFP (green) and Giantin (red). (b–e) STK16 RNAi specifically knockdown STK16. HeLa, and HeLa cells stably transfected with STK16-GFP-FLAG-WT or STK16-GFP-FLAG-MUT (RNAi resistant mutant) were transfected with control or STK16 siRNAs for 72 hours before they were lysed for RT-PCR (b,c) and western blots (d,e). (f,g) STK16 depletion disrupts Golgi complex integrity. HeLa cells and HeLa cells stably transfected with STK16-GFP-FLAG-WT or STK16-GFP-FLAG-MUT (RNAi resistant mutant) were transfected with control or STK16 siRNAs for 72 hours before they were harvested for immunofluorescence. Cells were fixed and stained with anti-GFP and anti-Giantin antibodies. Representative immunofluorescence images are shown in (f) and quantification results are shown in (g). (h) The inhibitor resistant mutant (F100C) rescues STK16-IN-1-induced Golgi structure disruption. HeLa and HeLa cells stably transfected with STK16-GFP-FLAG-WT or STK16-GFP-FLAG-F100C (drug resistant) were treated with 10 μM STK16-IN-1 for 6 hours. Cells were fixed and stained with GFP (green) and Giantin (red). (i) Quantification of the percentage of cells with fragmented Golgi complex in (h). (j) E202A, the kinase-dead mutant of STK16, induces fragmented Golgi. HeLa cells were transfected with STK16-GFP-FLAG-WT or kinase-dead mutant E202A for 48 hours before they were harvested for immunofluorescence using anti-GFP (green) and anti-Giantin (red) antibodies. (k) Quantification of % of cells with fragmented Golgi in (j) are shown. Experiments were repeated three times and representative images are shown. The data were analyzed using a student’s t- test (*p < 0.05, **p < 0.01, ***p < 0.001). Data show mean ± SEM, n = 3. Scale bar, 10 μm.