Cell lines
Human breast cancer cells MCF-7, MDA-MB-231 and non-transformed mammary epithelial cells, MCF-10A, were purchased from the American Type Culture Collection (ATCC, Manassas, VA). MDA-MB-453, SUM-159, BT-549, and SKBR3 cells were a kind gift from Dr. Ratna Vadlamudi at the University of Texas Health in San Antonio. All cells were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and 1% penicillin/streptomycin. SUM-159 cell medium also contained hydrocortisone (1μg/mL). MCF-7 cells were cultured in Eagles Minimum Essential Medium supplemented with human recombinant insulin (.05 mg/ml), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagles’ Medium (DMEM) supplemented with 10% FBS, and 1% penicillin/streptomycin. MCF-10A cells were cultured in DMEM supplemented with 10% FBS, EGF (20 ng/ml), hydrocortisone (.5 mg/ml), insulin (10 μg/ml), cholera toxin (100 ng/ml), and penicillin/streptomycin (1%). All cells were kept at 37 ̊ C and supplemented with 5% CO2.
SiRNA transfections
MDA-MB-231, MDA-MB-453, SUM-159, BT-549, and SKBR3 cells were transfected with lipofectamine RNAi Max (Thermo Fisher, #13778075) transfection reagent according to manufacturer’s protocol. IGF-1R siRNA (Cell Signaling Technology (CST), #6610S) or Control siRNA (CST, #6568) were diluted in OPTIMEM reduced serum medium (Thermo Fisher, #31985062) at a concentration of 100 nM and complexed with lipofectamine RNAi reagent. MCF-7 cells were transfected with FUGENE transfection reagent (Promega, #E2311) according to manufacturer’s protocol with either 100 nM of IGF-1R or control siRNA. SiRNA and FUGENE reagents were mixed at a ratio of 5:1 FUGENE: siRNA and transfected in antibiotic free full growth media for 12 hours. MCF-7 cells were then serum starved for 12 hours and exposed to (100 ng/mL) IGF-1 for 24 hours. The total transfection time was 48 hours. For SRPK2 (Millipore Sigma, #SASI_Hs01_00057789), SRSF-1 (Ambion, #n546318), and negative control (Millipore Sigma, #SIC003) siRNAs, both MCF-7 and MDA-MB-231 cells were transfected with lipofectamine RNAi reagent (Thermo Fisher, #13778075). Briefly, siRNAs were diluted in OPTIMEM along with lipofectamine reagent and allowed to complex for 20 minutes at room temperature. The diluted mixture was then added to the cells and allowed to incubate at 37 ̊C for 6 hours. After 6 hours the medium was replaced with normal growth medium until further treatment or serum starving. The total transfection time was 48 hours.
Plasmid transfections
The pEGFP-SF2 (pEGFP-SRSF-1) plasmid vector was a kind gift from Tom Misteli (Addgene, #17990). pEGFP-SRSF-1 was transfected into MCF-7 and MDA-MB-231 cells using lipofectamine 2000 reagent (Thermo Fisher, #11668027) according to manufacturer’s instructions. Briefly, DNA complexes and lipofectamine were mixed at a 1μg:1ul ratio in OPTIMEM media and incubated at room temperature for 20 minutes before being added to cells for 3-6 hours. For the co-transfection experiment: 10 nM of control or SRPK2 siRNA were added along with .2μg of pEGFP-SRSF-1 in OPTIMEM. Lipofectamine 2000 reagent was then added and allowed to complex for 20 minutes before being added to the cells for 3 hours. Full growth media was then added until subsequent treatments and/or serum starving. All transfections were carried out for 48 hours.
RT-qPCR
RNA was extracted from MDA-MB-231, MCF-7, MCF-10A using Trizol reagent according to the manufacturer’s instructions. 2000 ng of RNA was subjected to reverse transcription using Moloney Murine leukemia virus (MLMTV)- reverse transcriptase according to manufacturer’s protocol (Invitrogen, #28025-013), random hexamers (Thermo Scientific, #00986258), RT buffer (Applied Biosystems, #4319981) and dNTPs (Fisher Bioreagents, #BP2564-1). The resulting cDNA was diluted 5-fold and subjected to qPCR analysis with Power UP SYBER green master mix (Thermo Fisher, #A25741) and designated primers (sequences in Table S1), Ct values were standardized to actin and made relative to the negative control (dCT) as a fold change (2-^ddCT).
Intron retention PCR analysis
MDA-MB-231 cells were transfected as described in previous sections with either control or SRPK2 siRNA for 24 hours. After 24 hours, cells were serum starved for 16 hours prior to the addition of IGF-1 or full growth medium alone for an additional 24 hours. After 48 total hours, RNA was extracted, diluted to 2000 ng, and subjected to reverse transcription as described under RT-qPCR section. cDNA was then subjected to qPCR with SYBR green master mix according to manufacturer’s instructions. FASN intron 4 (intron included) and exon 4 (intron excluded) primers (sequences in Table S1) were used to amplify the cDNA. Intron 4 Ct values were normalized to Exon 4 and expressed as a relative fold change to the (−) IGF-1 condition by the 2^-ddCT method. The mean of 3 independent trials (n = 3) ± S.E.M. were expressed as a relative fold change to no-IGF-1 exposure.
mRNA stability
MCF-10A, MCF-7, and MDA-MB-231 were pretreated with or without SRPN-340 (3.5 μM) in serum free media for 18 hours followed by (5 μg/mL) actinomycin-D treatment for 0, 3,5 hours in full growth media. RNA was extracted and reverse transcribed using the same protocol as the RT- qPCR gene expression listed previously. All genes were normalized to the 0 actinomycin-D condition for either Vehicle (DMSO) or SRN-340 treated.
Western blotting
Cells were treated and lysed in 6-well dishes using 100 uL of cell lysis buffer (1% SDS, 1 mM EDTA, 10 mM Tris-HCL (pH 8.0)) supplemented with Halt phosphatase and protease inhibitor cocktail kit (Thermo Fisher #787429). Lysates were sonicated to shear DNA 3 × 10 seconds with one minute rest on ice using a Misonix ultrasonic liquid processor. Lysates were then centrifuged 10,000 rpm at 4̊C for 10 minutes. The resulting supernatant was collected and 10 uL subjected to Pierce BCA protein assay (Thermo Fisher #23227). 50 μg of protein were solubilized in 4x loading buffer (8% SDS, 250 mM Tris-Hcl (PH 6.8), 20% β-mercapthoethanol, 40% glycerol, and .2% bromophenol blue) and heated to 95̊ c for 5 minutes. Lysates were resolved on an 8% SDS-PAGE and transferred to a nitrocellulose membrane for one hour. Membranes were cut and blocked in 5% milk in TBST for one hour at room temperature and washed with TBST for 5 minutes. Primary antibodies (Table S1.) in dilution buffer (SRPK 1:300, GAPDH 1:300, S6K 1:300, p-S6K 1:300, all other primary antibodies were 1:1000) were incubated at 4̊C overnight. Membranes were washed the following day with TBST once for 15 minutes followed by an additional 5 minutes wash. Secondary antibodies anti-rabbit HRP-linked IgG (CST, #7074) (1:3000) or anti-mouse HRP-linked IgG (CST, #7076) (1:5000) were diluted in 5% milk and added to the membranes for 1 hour at room temperature. Membranes were washed in TBST 3 times for 5 minutes and incubated with West ATTO chemiluminescent substrate (Thermo Fisher, #A38554) prior to being visualized with a Syngene imaging system using the gene snap technology program.
Immunofluorescence and fluorescence microscopy
MDA-MB-231 and MCF-7 cells were grown on 16-well chamber slides (Lab-Tek, #78599) and serum starved with rapamycin or DMSO for 16 hours followed by exposure to either full growth media alone or full growth media and IGF-1 (100 ng/mL) for 0, 2, 6, 12, or 18 hours. For SRSF-1 localization, cells were transfected with either pEGFP-SRSF-1 alone or co-transfected with SRPK2 siRNA or control. Transfections were followed by serum starvation with DMSO or rapamycin (100 nM) for 16 hours and IGF-1 exposure for 0, 3, and 6 hours in full growth medium. Following respective treatments, media was removed, and cells were fixed with 3.7% paraformaldehyde in PBS for 20 minutes at room temperature. Slides were then blocked for one hour in blocking buffer (5% BSA, PBS, and .3% Triton-X-100) at room temperature. Chambers were then removed, and slides washed for 5 minutes in 1XPBS. SRPK2 (BD Biosciences, #61118) was diluted in blocking buffer (1:100) and added to the slides. Slides were incubated overnight (16 hours) at 4 ̊ C. The following day, coverslips were removed, and slides were washed three times in 1xPBS for five minutes each. Secondary antibodies, Cy3 anti-mouse IgG conjugate (Jackson laboratories, ##711-095-152) (1:1200) were diluted in the same blocking buffer and added to the slides for two hours at room temperature while protected from light. Coverslips were removed and slides were washed again three times for five minutes each in 1xPBS. Slides were then allowed to dry and mounted with Prolong Gold Antifade reagent (Invitrogen, #P36966) containing DAPI. Mounted slides were sealed with nail polish and allowed to dry for 24 hours at room temperature protected from light. Images were obtained using a Leica Confocal microscope. Images were processed using Adobe photoshop and NIH Image J software. Quantification of nuclear localization was performed using a FIJI (image J) plugin as described in [21].
Cell fractionation
MDA-MB- 231 and MCF-7 cells were grown in 100 mm dishes until confluency and serum starved overnight (16 hours) with either DMSO or rapamycin (100 nM) followed by exposure to IGF-1 (100 ng/m) in full growth media with 10% FBS for 6 hours. Cell media was removed, and dishes were washed twice with cold 1XPBS after which .3 mL of lysis buffer (.33 M sucrose, 10 mM HEPES, PH 7.4, 1 mM MgCl2, 0.1% Triton-X-100) was added to the dish. Cells were then scraped and transferred to chilled 1.5 mL tubes and incubated for an additional 15 minutes on ice and centrifuged for 10 minutes, 10,000 rpm at 4 ̊ C. Cytosolic fractions were removed and kept on ice. The resulting pellets were washed twice in lysis buffer and suspended in .1 mL of nuclear lysis buffer (.45 M NaCl, 10 mM HEPES, PH 7.4, 1 mM MgCl2) for 30 minutes on ice. Lysates were spun at 14,000 rpm for 5 minutes prior to be harvested. Both cytosolic and nuclear fractions were subjected to a pierce BCA protein assay to determine protein concentration. 50 μg of lysate was solubilized in 4x SDS loading buffer and resolved on an 8% SDS-PAGE and transferred to a nitrocellulose membrane for 1 hour. The remaining western blotting was identical to the protocol listed under western blotting section. Bands were quantified in NIH Image J software and standardized to their loading control. Cytosolic p-SRPK2 band intensity was standardized to GAPDH, and nuclear p-SRPK was standardized to HDAC1. All experiments were performed in biological triplicate.
Sample preparation for GC-MS lipid analysis
MDA-MB-231 and MCF-7 cells were gown in a T75 flask for 24 hours prior to siRNA transfection with either 10 nM control siRNA (Millipore Sigma, #SIC003) or 10 nM of SRPK2 siRNA (Millipore Sigma, # SASI_Hs01_00057789) in OPTIMEM for at least 6 hours with lipofectamine RNAi transfection reagent (thermo fisher, #13778075) according to manufacturer’s instructions. After transfection, the medium was replaced full growth medium + 10% FBS until the following day. After 24 hours, glucose-free RPMI medium containing 10% charcoal stripped FBS, 4.5 g/L of [U-C13] glucose isotope (Cambridge isotopes, #.110187-42-3), and 100 ng/mL IGF-1 was used to treat the cells for an additional 48 hours. After the 48-hour treatment, cells were suspended by trypsinization, spun down, and washed with cold 1xPBS two times. The pellets were then transferred to 1.5 mL tubes and spun again at max speed. The supernatant was discarded, and pellets were flash frozen in liquid nitrogen and stored at − 80 ̊ C until further analysis.
GC-MS fatty acid analysis
Lipid metabolites were isolated through a modified Folch extraction and analyzed by GC-MS [22]. Cells and media samples were combined with 800 μL HPLC Grade isooctane (Thermo Fisher Scientific, Waltham, MA) 200 μL of HPLC Grade methanol, 100 μL of 0.9% saline, and 20 μL of 1 N HCl. Additionally, 10 μL of the antioxidant butylated hydroxytoluene dissolved in ethanol at 200 μg/mL and 20 μL of the internal standard, deuterated heptadecanoic acid (C17:0) (Cayman Chemicals; Ann Arbor, Michigan, MI) were added for a final concentration of 20 μg/mL.
This mixture was vortexed and centrifuged in a Beckman Coulter Allegra X-15r Centrifuge (Brea, CA, USA) for 20 minutes at 4 °C for polar-apolar phase separation. The top apolar phase was transferred to a new tube and dried at 4 °C using refrigerated Labconco CentriVap Concentrator attached to − 84 °C CentriVap Cold Trap (Kansas City, MO, USA). Metabolites were derivatized by combining 30 μL of trimethyl sulfonium hydroxide (Sigma-Aldrich) (20 mg/mL) and 60ul of methyl tert-butyl ether (Sigma- Aldrich) in an automated manner as described in previous publication [23]
A small volume (3 μL) of derivatized samples were injected via spitless injection into a Thermo Scientific Thermo Scientific Trace 1310 gas chromatograph loaded with a Phenomenex Zebron ZB- (Torrance, CA, USA) 1 ms fused silica capillary column (length = 30 m, I.D. = 0.25 mm, film = 0.25 μm), which was connected to a Thermo ISQ (Thermo Fisher Scientific) single quadrupole mass spectrometer. Ultra-high purity helium (Praxair) was used as the carrier gas at a flow rate of 1.10 ml/minute. Optima HPLC Grade methanol (Thermo Fisher Scientific) was used to wash the injection syringe between each sample.
Each sample underwent a heating ramp in the Trace 1310 that started at 70 °C which was maintained for 2 minutes and then ramped up at a rate of 25 °C/min until reaching 350 °C which was maintained for 5 minutes. The MS transfer line was kept at 280 °C and the ion source was maintained at 230 °C. Electron ionization at 70 eV and a scan time of 0.25 seconds over the range of 50.0-500.0 amu was sufficient for analysis. Mono-isotopic ions for each iteration of 13C labeled palmitate were extracted by through the Xcalibur version 4.4 through the ICIS peak detection algorithm, which utilized minimal smoothing and a maximum baseline of 12 scans for integration. Palmitate retention time was confirmed by standards. Total ion current peaks of different metabolites were normalized to the internal standard 3 deuterium labeled heptadecanoic acid [23].
RNA immunoprecipitation
MDA-MB-231 cells were treated with or without IGF-1 with either control siRNA or SRPK2 siRNA as described in the transfection methods. Cells were detached by trypsin and washed twice with 1XPBS. Cell pellets were lysed with RIPA buffer supplemented with a protease inhibitor cocktail and RNAse inhibitor (Invitrogen #10777019) 160 units/mL. Cell lysates were sonicated 3 × 20 seconds on ice and incubated for 30 minutes at 4 ̊C and pre-cleared with 25 ul of protein A magnetic beads (Invitrogen, #10-003D). Magnetic beads were pre-bound to the antibodies by incubating 5 μg of anti-SRSF-1 (Invitrogen, #PA5-30220) or Rabbit IgG (Cell Signaling Technologies, #2729) with 50 ul of beads for 30 minutes at room temperature. Input samples were collected, and pre-bound beads were incubated with pre-cleared supernatant for 3 hours at 4C. Beads were washed 6 times in RIPA buffer and transferred to 150 ul of RIPA buffer containing of 1% SDS and 1 mg/mL of proteinase K. Beads were eluted by incubating at 50 ̊ C for 30 minutes. Eluted beads were separated by a magnetic and RNA was extracted using a Qiagen RNAeasy mini kit (Qiagen, #74134) according to manufactures instructions. 500 ng of Immunoprecipitated RNA as well as corresponding input RNA was subjected to cDNA synthesis using the same protocol listed in RT-qPCR section. Exon 4 specific primers were used to amplify cDNA using SYBR Green master mix. Resulting Ct values were analyzed to be expressed as % of transcript bound to the antibody by using the % input method (100 X 2^[Ct (input) - Ct (IP)].
Statistical analysis
Data obtained from western blot, RT-qPCR, and GC-MS were analyzed using students t-test and done in biological triplicate ±SD or S.E.M. Quantification from immunofluorescence data was analyzed by two-way ANOVA followed by Tukey’s HSD post hoc analysis. More details regarding p-values and analysis can be found in the figure legends.