1. Volume 21, Issue 12, Pages 3406-3413 (December 2017)
SRSF1 Prevents DNA Damage and Promotes Tumorigenesis through Regulation of DBF4B Pre-mRNA Splicing 
Linlin Chen, Chunling Luo, Lei Shen, Yuguo Liu, Qianqian Wang, Chang Zhang, Ruochen Guo, Yanan Zhang, Zhiqin Xie, Ning Wei, Wenwu Wu, Jun Han, Ying Feng 
Cell Reports 
Volume 21, Issue 12, Pages (December 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 3406-3413DOI: (10.1016/j.celrep.2017.11.091)
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3. Figure 1 SRSF1 Is the Major Regulator for DBF4B Exon6 Splicing
(A) Schematic diagram of DBF4B variants including or lacking alternative exon6 (DBF4B-FL and -S).
(B) Diagrams for detection of DBF4B variants including or lacking alternative exon6 (E6+ or E6−). Primer pairs and product sizes for the two variants are shown.
(C) RNAs were extracted and RT-PCR was performed for analysis of DBF4B exon6 inclusion/skipping.
(D) RT-PCR analysis using RNA from 293T cells co-transfected with minigene and siRNA. PSI was shown at the bottom of the gel.
(E) RT-PCR analysis using RNA from 293T cells co-transfected with minigene and overexpression plasmids. PSI was shown at the bottom of the gel.
(F) CLIP, followed by RT-PCR analysis using primer pairs complementary to the indicated DBF4B exon-intron (E-I) boundary sequences.
(G) In vivo splicing analysis of DBF4B minigene and indicated minigene mutants in 293T cells. PSI was shown at the bottom.
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4. Figure 2
DBF4B-FL and SRSF1 Are Required for Proliferation and Survival of Human Colon Cancer Cells In Vitro and in Mice
(A) RKO or SW620 cells were treated with isoform-specific siRNA (si-DBF4B-FL#1, #2), or si-NC. KD efficiency was assessed by RT-PCR analysis.
(B and C) Clonogenic survival assay was performed with cells as described in (A). Three independent experiments were performed and representative cells stained with crystal violet were shown (B). The number of focal adhesions was quantified and results were shown as mean ± SD (C).
(D and E) RKO or SW620 cells were transiently transfected with SRSF1 siRNA (si-SRSF1#1 or #2), followed by clonogenic survival assay (D) and quantification (E).
(F) Stable RKO/shDBF4B-FL, RKO/shSRSF1 and RKO/shLuci cells were injected into nude mice. After 4 weeks after injection, tumors excised from the mice were shown.
(G) Tumors shown in (F) were formalin-fixed and sliced for Ki-67 and DAPI staining.
(H) RKO/shSRSF1 cells were infected with lentivirus expressing DBF4B-FL-HA, DBF4B-S-HA, or HA vectors, and selected for puromycin resistance. Three stable cell lines were then injected into nude mice. After 4 weeks after injection, tumors excised from the mice were shown.
(I) Tumor weight. Results are shown as mean ± SD of tumor weights (each with initial 10 injections).
(J) Tumors shown in (H) were formalin-fixed and sliced for Ki-67 and DAPI staining.
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5. Figure 3
Overexpression of DBF4B-FL Represses DSBs and Rescues Survival Defects of shSRSF1 Cells
(A) γH2AX level was examined by western blotting using whole cell lysates.
(B) Immunostaining with γH2AX (red) antibodies in RKO/shSRSF1, RKO/shDBF4B-FL, and RKO/shLuci cells. Merged images were shown.
(C) Immunostaining with γH2AX (red) antibodies using sections made from tumors as shown in Figure 2F. Merged images were shown.
(D) RKO/shSRSF1 and RKO/shLuci cells were infected with lentivirus expressing DBF4B-FL-HA or HA vectors and selected for puromycin resistance. γH2AX level was examined by western blotting.
(E) Immunostaining with γH2AX (red) antibodies in RKO cells described in (D).
(F) Clonogenic survival assay of RKO cells described in (D), along with stable RKO/shSRSF1+DBF4B-S-HA cell lines. Quantification of the number of colonies for the indicated cell lines was shown on the bottom. Three independent experiments were performed and error bars indicate SD of mean.
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6. Figure 4
Inclusion of DBF4B Exon6 Is Increased in CRC Samples, which Parallels Increased Expression of SRSF1
(A) Total RNAs from 60 paired human CRC and normal tissues were examined by RT-PCR using primer sets as described in Figure 1B. Representative results for detection of DBF4B exon6 splicing patterns are shown between CRC (T) and matched normal tissues (N) from the same patient. PSI was shown on the bottom.
(B) Quantification of data from RT-PCR results of 60 paired human CRC and normal tissues described in (A) for DBF4B-FL/S ratio.
(C) Western blotting analysis confirmed upregulated expression of SRSF1 protein in CRC samples.
(D) Positive correlation between DBF4BFL/S ratio and expression levels of SRSF1 was observed in CRC samples.
(E and F) Kaplan-Meier survival analysis showed patients with non-increased ratio of DBF4B FL/S (E) or low levels of SRSF1 (F) expression had significantly longer median survival than patients with increased ratio of DBF4B FL/S or high SRSF1 expression.
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