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Fig. 2 | BMC Cancer

Fig. 2

From: An IGF-1R-mTORC1-SRPK2 signaling Axis contributes to FASN regulation in breast cancer

Fig. 2

(A-D). IGF-1 contributes to SRPK2 nuclear localization and phosphorylation through mTORC1. A MCF-7 and 231 cells were serum starved 16 hours in the presence of DMSO or rapamycin (100 nM) followed by exposure to full growth media with or without IGF-1 (100 ng/mL) for various time points. Cells were fixed, permeabilized, and stained for SRPK2 (1:100) followed by anti-mouse Cy3 (Red) (1:1200) antibody. Cells were mounted using mounting medium containing DAPI nuclear stain (Blue). White arrows indicate where the image was zoomed in. Image channels were merged using Image J NIH software and quantified. B Quantification of nuclear or nuclear and cytoplasmic SRPK2 was performed using FIJI (Image J) software by analyzing the merged channel images for SRPK2 and DAPI. A threshold was set for the colocalization of DAPI and SRPK2 and cells that were over the threshold were counted. Nuclear positive SRPK2 cells were expressed as a percentage of the total amount of cells present in the image. Experiments were repeated three times and at least 200 cells were counted for each condition. C MCF-7 and MDA-MB-231 cells were serum starved overnight (16 hours) with DMSO or rapamycin (100 nM) followed by either full growth media alone or with 100 ng/mL of IGF-1 for 6 hours. Cells were harvested and analyzed by western blot for p-SRPK2, SRPK2, and GAPDH. All blots for fig. 2 were cropped to provide a concise representation of the data. Full blots can be found in Fig. S1. D MCF-7 and MDA-MB-231 were serum starved 16 hours with DMSO or rapamycin (100 nM) followed by 6 hours of full media exposure with 100 ng/mL of IGF-1. Cells were harvested and fractionated into both cytosolic and nuclear fractions. Fractions were analyzed by western blot. GAPDH served as the loading control for cytosolic fractions and HDAC1 served as the nuclear fraction loading control. The blot is a representation of 1 trial. Numbers under p-SRPK2 represent an average band intensity from three independent trials (n = 3). Band intensity was standardized to respected loading controls, GAPDH (cytoplasm) or HDAC1 (nucleus)

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