Sex Hormone Binding Globulin Modifies Testosterone Action and Metabolism in Prostate Cancer Cells

Figure 1

(a) Internalisation of Alexa Fluor 546 labeled SHBG (Alexa-SHBG) by LNCaP cells using a DeltaVision Deconvolution Microscope. Cells were incubated with (1) serum free RPMI medium, (2) Alexa 546 dye, and (3) Alexa Fluor 546 labeled SHBG (red) in serum-free medium for 1 hour and then costained with Alexa Fluor 488 phalloidin (green) to outline the actin cytoskeleton and DAPI (blue) to localise nuclei. Bar = 10 μm. (b) To confirm Alexa labeled SHBG protein internalisation, cells were incubated with Alexa-SHBG for 1 hour and then costained with mouse anti-SHBG monoclonal antibody conjugated with anti-mouse Alexa Fluor 488 secondary antibody (green). (1) Internalised Alexa-SHBG (red) was detected inside the cells. (2) Intracellular SHBG was detected by anti-SHBG antibody (green). (3) Merged images (1) and (2) show internalised Alexa-SHBG in orange and endogenous SHBG in green. Bar = 10 μm. (c) Alexa-SHBG uptake by LNCaP cells is dose dependent. The cells were incubated with 5 nM (closed circle), 25 nM (open square), 50 nM (open triangle), and 100 nM (cross) Alexa-SHBG for 30 hours. Data presented as mean number of red objects counted per image. Imaged using IncuCyte ZOOM Live Cell Imaging System. (d) Quantitation of Alexa-SHBG uptake by LNCaP cells using the IncuCyte ZOOM Live Cell Imaging System. Untreated LNCaP cells (control, closed diamond) and LNCaP cells treated with Alexa-546 dye only (dye, open square) were used as negative controls. Cells were starved with serum-free RPMI for 1 hour and treated with 50 nM Alexa546-SHBG only (closed triangle) and with 10 nM testosterone (Alexa-SHBG + Te, closed circle). Data presented as mean number of red objects counted per image.

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