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Fig. 5 | BMC Cancer

Fig. 5

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

Fig. 5

The IGF-1-mTORC1-SRPK2 axis mediates FASN regulation through SRSF-1 A MDA-MB-231 and MCF-7 cells were grown in 8-well chamber slides and co-transfected with pSRSF-1-EGFP and either control or SRPK2 siRNA for 24 hours prior to treatment with vehicle or rapamycin. Cells were exposed with or without IGF-1 (100 ng/mL) for 6 hours before being fixed for subsequent fluorescence microscopy analysis. Fixed cells were incubated with anti-SRPK2 or anti-P-SRPK2(Ser474) primary antibodies followed by Cy3 conjugated secondary antibodies (Red). Nuclei were stained with DAPI nuclear stain (Blue) that is present in the mounting media. Images were obtained on a Leica confocal microscope. Trials were repeated at least 2 times. B MCF-7 and MDA-MB-231 cells were co-transfected with SRSF-1-eGFP plasmid with either control siRNA or SRPK2 siRNA for 48 hours. Cells were treated with vehicle or (100 nM) rapamycin for 16 hours prior to being exposed with or without IGF-1 for an additional 6 hours. Fixed cells were incubated with either SRPK2 or p-SRPK2 antibody followed by CY3 conjugated secondary and mounted with a DAPI nuclear stain containing medium. Nuclear speckle area was measured using image J and standardized to total nuclear area. Graphs are representative of at least 20 speckles from 3 independent trials. Average nuclear speckle/total nuclear area arbitrary units were used to construct box plots below [15, 16]. Statistical analysis was performed using one-way ANOVA followed by a Tukey’s post hoc analysis. Bars above box plots represent the significance between the respected conditions. Codes for significance between groups are as follows: p < .05 = * p < .01 = **, p < .001 = ***. C MDA-MB-231 transfected cells were exposed with (+) or without (−) IGF-1 for 6 hours followed by RNA-IP with SRSF-1 or control (IgG) antibodies. RNA was eluted from the antibodies and expressed as % of antibody bound transcript. Bars represent an average of three independent trials (n = 3) ± SD. Student’s T test were performed for statistical analysis. D MCF-7 and MDA-MB-231 cells were transfected with control or SRSF-1 siRNA for 48 hours, serum starved for 16 hours in the presence of 100 ng/mL of IGF-1 for an additional 6 hours. RNA was subjected to RT-qPCR. Graphs represent mean fold changes from three independent trials (n = 3). FASN, PFK, and SRSF-1 expression was standardized to actin and normalized to the control siRNA group to be expressed as a relative fold change. Students t test were performed for statistical analysis

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