Supplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell

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1 Supplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell Supplementary Figures S1-S3 Supplementary Methods

2 Supplementary Figure S1. Identification of CD44v + and CD44v subpopulations of 4T1 cells. (a) Schematic representation of CD44s and CD44v coding regions and the positions of primers for RT-PCR analysis. E5 and E16, exons 5 and 16. (b) Flow cytometric analysis of 4T1 cells and sorting of CD44v + and CD44v subpopulations. Cells were stained with antibodies to pan-cd44 and to CD44v. (c) Immunoblot analysis of CD44 isoforms in CD44v + or CD44v subpopulations of 4T1 cells with antibodies to pan-cd44. α-tubulin was analyzed as a loading control. (d) Total RNA isolated from CD44v + and CD44v subpopulations of 4T1 cells was subjected to RT-PCR analysis with specific primers targeted to E5 and E16 of CD44 cdna. Direct sequencing of the PCR products revealed that CD44v8-10 mrna was the predominant isoform in CD44v + 4T1 cells.

3 Supplementary Figure S2. Differences in ROS defense ability and GSH content between 4T1PT and 4T1LM cells and the impact of GSH depletion on lung colonization by CD44v + cells. (a) 4T1PT or 4T1LM cells were incubated with the indicated concentrations of H 2 O 2 for 20 min, stained with DCFH-DA, and subjected to flow cytometric analysis. Data are means ± s.d. from four independent experiments. *P < 0.05, **P < 0.01 (Student s t-test). (b) Intracellular GSH content of 4T1PT and 4T1LM cells. Data are means ± s.d. from five independent experiments. *P < 0.05 (Student s t-test). (c) Intracellular GSH content of CD44v + 4T1 cells incubated with or without 500 µm BSO for 36 h. Data are means ± s.d. from five independent experiments. **P < 0.01 (Student s t-test). (d) IntegriSense images of lung metastases formed five days after intravenous injection of mice with BSO-treated or nontreated CD44v + 4T1 cells ( ), as well as quantitative analysis of the total fluorescence intensity per lung metastatic lesion. Cells were incubated with or without 500 µm BSO for 48 h before injection. Quantitative data are means ± s.d. for four animals per group. **P < 0.01 (Student s t-test).

4 Supplementary Figure S3. Effects of CD44v8-10 expression in shesrp1 CD44v + cells. (a) Quantitative RT-PCR analysis of ESRP1 mrna in shc CD44v + cells, shesrp1 CD44v + cells, or shesrp1 CD44v + cells stably transfected with an expression vector for CD44v8-10 or with the empty vector (mock). Data were normalized by the amount of GAPDH mrna and are means ± s.d. from three independent experiments. **P < 0.01 (Student s t-test). (b) Mock-transfected or CD44v8-10 expressing shesrp1 CD44v + cells were stained with DCFH-DA and subjected to flow cytometric analysis (upper). Mean ± s.d. values for RFI from five independent experiments are also shown (lower). NS, not significant (Student s t-test).

5 Supplemetary Methods Sulfasalazine treatment. CD44v + 4T1 cells ( ) were injected into the tail vein of 8- to 10-week-old female wild-type Balb/c mice, which were then injected intraperitoneally with saline (control) or sulfasalazine (8 mg/body) twice daily for 2 weeks. Sulfasalazine (Sigma) was freshly prepared immediately before each injection. Mice were killed 3 weeks after cell injection and the lungs removed for quantification of lung metastasis. Quantitative and semiquantitative RT-PCR analysis. Total RNA was extracted from cultured cells with the use of an RNeasy Mini Kit (Qiagen, Hilden, Germany), and portions of the RNA (1 µg) were subjected to RT with a Transcriptor First Strand cdna Synthesis Kit (Roche, Mannheim, Germany). Semiquantitative PCR was performed with the use of an RNeasy Mini Kit, and the PCR products were fractionated by agarose gel electrophoresis and stained with ethidium bromide. Quantitative PCR analysis was performed with a Thermal Cycler Dice Real Time System (TaKaRa Bio, Tokyo, Japan). The amplification protocol comprised an initial incubation at 95 C for 2 min and 40 cycles of 95 C for 30 s and 60 C for 30 s, followed by dissociation-curve analysis to confirm specificity. Primers for detection of CD44v cdna were targeted to exons 5 and 16. Semiquantitative PCR analysis was performed with mouse primer sets (forward and reverse, respectively) for CD44v (5 -GGAGATCAGGATGACTCCTTCT-3 and 5 -AGTCCTTGGATGAGTCTCGATC-3 ), Mena (5 -TGCTGGCCAGGAGGAGAAGAAT-3 and 5 -ACTGGGCTGTGATAAGGGTGTGG-3 ), (FGFR2 IIIb (5 -CACTCGGGGATAAATAGCTCC-3 and 5 -AGATGACTGTCACCACCATGCA-3 ), FGFR2 IIIc (5 -CGGTGTTAACACCACGGAC-3 and 5 -AGATGACTGTCACCACCATGCA-3 ), p120 catenin (5 -GGGTTTGTTTCTTAAAGGACTGG-3 and 5 -TCCCATCATCTGTGGTCTCC-3 ), and GAPDH as an internal control (5 -GTGAAGGTCGGTGTGAACG-3 and 5 -CTCACCCCATTTGATGTTAGTG-3 ). Quantitative PCR analysis was performed with mouse primer sets (forward and reverse, respectively) for ESRP1 (5 -CAGAGGCACAAACATCACAT-3 and 5 -AGAAACTGGGCTACCTCATTGG-3 ) and for GAPDH (5 -GTGAAGGTCGGTGTGAACG-3 and 5 -GACCATGTAGTTGAGGTCAATG-3 ).

6 Histology. Tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 µm. Sections were depleted of paraffin and then rehydrated in a graded series of ethanol solutions before staining with hematoxylin-eosin. GSH assay. Intracellular levels of GSH were determined with the use of a GSH-Glo Glutathione Assay kit (Promega, Tokyo, Japan). Cells ( per well) were plated in 96-well plates for the assay, which is based on the conversion of a luciferin derivative to luciferin by glutathione S-transferase in the presence of GSH. The signal generated in a coupled reaction with firefly luciferase is proportional to the amount of GSH in the sample. The assay results were normalized with the use of a GSH standard solution provided with the kit. Stable RNAi-mediated depletion of ESRP1. Expression vectors encoding an shrna specific for mouse ESRP1 mrna or a scrambled shrna were obtained from Origene Technologies (Rockville, MD) and were introduced into CD44v + 4T1 cells by transfection with the use of Lipofectamine 2000 (Invitrogen). Cells stably depleted of ESRP1 mrna were obtained by selection with 2.5 µm puromycin for 2 weeks. Stable expression of human CD44v in ESRP1-depleted cells. ESRP1-depleted CD44v + (shesrp1 CD44v + ) cells were transfected with the prc/cmv expression plasmid (Invitrogen) encoding human CD44v8-10 with the use of the FuGENE HD reagent (Roche, Tokyo, Japan). Cells stably expressing CD44v8-10 were obtained by selection with 0.5 to 1 mm G418 for 2 weeks and were then isolated by FACS with antibodies to human CD44v9. Microarray analysis. Total RNA was extracted from cells with the use of the Trizol reagent (Invitrogen) and an RNeasy Mini Kit (Qiagen). Cy3-labeled crna probes were synthesized from the total RNA and subjected to hybridization with a Whole Mouse Genome Microarray (Affymetrix). Raw intensity data for each experiment were analyzed with GeneSpring GX Software (Tomy Digital Biology, Tokyo, Japan). Microarray data are available in the GEO database under the accession number GSE35803.