1. Volume 20, Issue 6, Pages 881-890 (December 2005)
Cell Motility Is Controlled by SF2/ASF through Alternative Splicing of the Ron Protooncogene 
Claudia Ghigna, Silvia Giordano, Haihong Shen, Federica Benvenuto, Fabio Castiglioni, Paolo Maria Comoglio, Michael R. Green, Silvano Riva, Giuseppe Biamonti 
Molecular Cell 
Volume 20, Issue 6, Pages (December 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Ron Splicing Is Altered in Tumors and Is Regulated by Two Elements in Exon 12
(A) Schematic diagram of Ron alternative splicing is shown.
(B) Total RNAs from the indicated normal human tissues were analyzed by RT-PCR with Ron primers 2507 and 2991, annealing to exon 10 and 12, respectively. The histogram shows the ΔRon/Ron ratio quantified with the NIH Image program.
(C) Ron splicing pattern in 16 breast cancer specimens and in two normal human breast samples is shown. The ΔRon/Ron ratio has been determined as in (B).
(D) Ron splicing pattern and quantification of the ΔRon/Ron ratio in KATOIII, T47D, and 293 cells.
(E) Scheme of the Ron cassettes cloned into pcDNA3.1+. Plasmids names on the left refer to the cDNA sequence (GenBank NM ). Thick lines are exons, thin lines are introns, and dashed lines indicate internal deletions.
(F) RT-PCR analysis of KATOIII cells transfected with the indicated minigenes is shown. Numbers below each lane correspond to numbers on the right of the Ron cassettes in (E); t-Ron and t-ΔRon indicate minigene transcripts generated by inclusion and skipping of exon 11. The histogram shows the t-ΔRon/t-Ron ratio measured in three independent experiments. Error bars represent the mean ± standard deviation (SD).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The Strength of the ESE Parallels the Relative Abundance of the ΔRon Transcripts
(A) Scheme of Hβ minigenes and position of primers used in RT-PCR. Abbreviations are as follows: INH and E, inhibitor sequence and enhancer from the mouse IgM gene, respectively; ESS and ESE, silencer and enhancer from the Ron gene, respectively.
(B) KATOIII or T47D cells were transfected with the indicated plasmids. 24 hr after transfection, RNAs were analyzed by RT-PCR with primer set A-B to detect both spliced and unspliced transcripts (Total) and with primer set A–C specific for spliced molecules (Spliced).
(C) The intensity of the bands Total and Spliced in (B) were quantified with the NIH Image program; the ratio between Spliced and Total was plotted in the histogram by using the Hβ vector as a reference value (average of three independent experiments). Error bars represent the mean ±SD.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 SF2/ASF Controls the Strength of the ESE Element
(A) Scheme of Hβ minigenes and position of primers used in RT-PCR are shown. ESE and M indicate wild-type (nt 2925–2991) and mutated enhancer. The sequence of the wild-type and the mutated SF2/ASF binding site is indicated.
(B) RT-PCR analysis of KATOIII cells transfected with the indicated plasmids.
(C) Western blot analysis of SF2/ASF, SRp20, and α-tubulin protein levels in the indicated cell lines.
(D) Hβ minigenes were cotransfected into T47D cells either with the plasmids encoding the indicated splicing factors or the empty vector. Expression of T7-tagged SR protein was verified by Western blot by using the anti-T7 mAb. RNAs were analyzed in RT-PCR with primer set A–D (see [A]) to simultaneously detect spliced (S) and unspliced (U) transcripts. The histogram shows the percentage of spliced transcripts (average of three independent experiments). Error bars represent the mean ±SD.
(E) KATOIII cells were transfected with p and with a derivative mutated in the ESE sequence (p2507–2991-Mut), and the p minigene was cotransfected into T47D cells with the T7-tagged SF2/ASF or the empty vector. Total RNAs were analyzed by RT-PCR as in Figure 1F.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 SF2/ASF Directly Binds the Ron Enhancer
(A) The His-tagged SR proteins were UV crosslinked to an RNA probe containing a portion of the Ron enhancer (nt 2953–2991), either wild-type or mutated in the putative SF2/ASF binding site.
(B) UV crosslinking reactions with nuclear extracts were immunoprecipitated with antibodies specific for the indicated SR proteins. RNA probe is entire Ron enhancer (nt 2925–2991), either wild-type or mutated.
(C) The indicated His-tagged SR proteins were analyzed by using a band-shift assay with the same probes used in (B).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 SF2/ASF Triggers the Production of the ΔRon Isoform
(A) T47D cells were transfected with T7-SF2/ASF or T7-FF-DD cloned into pIRES-hrGFP-2a. GFP-positive cells were selected by FACS, and splicing of the endogenous Ron transcripts was analyzed by RT-PCR as in Figure 1B. The splicing pattern in nontransfected T47D and KATOIII cells is also shown.
(B) 293 cells were transfected with pCGT7-SF2 or pCGT7-SF2(FF-DD) with an efficiency of about 60%, and RNAs were analyzed with RT-PCR as in (A).
(C) 293 cells were stably transfected with T7-SF2/ASF (cl.SF2) or with the empty vector (“vector”). Cells of clones cl.SF2 and vector were analyzed by RT-PCR (as in [A]) and by Western blot with the anti-SF2/ASF mAb96.
(D) Ron was immunoprecipitated from cl.SF2, vector, and KATOIII cells and probed with anti-Ron β chain antibody. Ron immunoprecipitated from cl.SF2 and vector cells was also probed with anti-P-tyr antibody. The position of the uncleaved 165 kDa ΔRon isoform and of the 150 kDa Ron β chain is indicated.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 SF2/ASF Promotes Cell Scattering
(A) Cl.SF2 and vector cells were stained with a mAb specific to β-catenin and with phalloidin for visualization of actin-containing structures; cells morphologies were examined under a phase-contrast microscope (magnification 60×).
(B) The same cells were stained with an antibody specific to E-cadherin. RNAs were also analyzed by RT-PCR with primers for E-cadherin and GAPDH genes.
(C) Transwell motility assay of cl.SF2 and vector cells and of 293 cells transiently transfected with pCGT7-SF2 (SF2/ASF), pCGT7-SF2(FF-DD), or with the empty vector (“Mock”). Cells that migrated to the lower side of the filter were quantified with the vector or mock value as reference (average of triplicate determinations). Error bars represent the mean ±SD.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
RNAi-Mediated Knockdown of SF2/ASF or Ron Reduces Cell Migration
(A) KATOIII cells transfected with siRNAs against SF2/ASF (oligos 1 and 2) or the Luciferase gene (GL2-Luc). Cells were analyzed by Western blotting with antibodies against SF2/ASF and α-tubulin and by RT-PCR to reveal the splicing pattern of the endogenous Ron mRNA. Cell migration has been determined as in Figure 6C.
(B) Cl.SF2 cells were transfected with a pool of four siRNAs specific for Ron or with GL2-Luc siRNA. Cells were analyzed by RT-PCR with Ron primers Ex4-up and Ex4-dw (both in exon 4) or with GAPDH primers and by Western blot with anti-Ron α chain and anti-α-tubulin antibodies. Cells were also analyzed by immunofluorescence with phalloidin to stain actin-containing structures and by RT-PCR to determine the expression level of E-cadherin. Cell morphologies were examined under phase-contrast microscope (magnification 60×). The histogram shows the effect of RNAi on cell migration (average of triplicate determinations).
(C) Cl.SF2 cells were transfected with two siRNAs against Ron (oligos 1 and 2), two siRNAs against ΔRon (oligos 1 and 2), or with GL2-Luc. The cells were analyzed by RT-PCR with primers Ex4-up and Ex4-dw to detect all Ron transcripts or with primers specific for ΔRon mRNA (2507 and ΔRon, a primer that overlaps the junction between exon 10 and 12). The effect on cell migration was determined as in Figure 6C.
Error bars represent the mean ±SD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions