Table 1.
Demographics and clinical characteristics of the patients.
Figure 1.
Expression of SRSF1, SRSF2 and phosphorylated SRSF2 proteins in NSCLCs.
A, Representative immunostaining from frozen section of normal lung parenchyma and lung cancer tissue with anti SRSF1 (a, b, c), anti SRSF2 (d,e,f) and anti phospho-SRSF2 (g, h, i) antibodies [(a, d, g) normal lung; (b, e, h) ADC; (c, f, i) SCC; immunoperoxidase and haematoxylin counterstaining]. B, Representative western blots demonstrating overexpression of SRSF1 and SRSF2 proteins in lung tumors compared with their matched normal lung tissues. (NL, normal lung; ADC, adenocarcinoma; SCC, squamous cell carcinoma).
Table 2.
Immunohistochemical analysis of SRSF1 and SRSF2 proteins expression in non-small cell lung cancer according to histological subtype.
Figure 2.
SRSF1 scores according to the clinico-pathological parameters in NSCLC subtypes.
Distribution of SRSF1 scores according to the tumor size (A), the nodal status (B), the presence of metastases at distance (C) and the pTNM stage (D), in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). Statistical analysis was done using Mann-Whitney’s U test.
Figure 3.
Relationship between SRSF2 overexpression and its phosphorylated status in NSCLC subtypes.
Distribution of phospho-SRSF2 scores in tumors displaying either normal SRSF2 expression (class 0) or SRSF2 overexpression (class +), in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). Statistical analysis was done using Mann-Whitney’s U test.
Figure 4.
Expression of SRSF2 and its phosphorylated form according to the clinico-pathological parameters in NSCLC subtypes.
Distribution of SRSF2 and phospho-SRSF2 scores according to the tumor size (A, C) and the stage (B, D), in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). Statistical analysis was done using Mann-Whitney’s U test.
Table 3.
Immunohistochemical analysis of SRPK1 and SRPK2 proteins expression in non-small cell lung cancer according to histological subtype.
Figure 5.
Expression of SRPK1 and SRPK2 proteins in NSCLCs.
A, Representative immunostaining from frozen section of normal lung parenchyma and lung cancer tissue with anti SRPK1 (a, b, c) and anti SRPK2 (d, e, f) antibodies [(a, d) normal lung; (b, e) ADC; (c, f) SCC; immunoperoxidase and haematoxylin counterstaining]. B, Left panels: Representative western blots illustrating overexpression of SRPK1 and SRPK2 in lung tumors compared with their matched normal lung tissues. (NL, normal lung; ADC, adenocarcinoma; SCC, squamous cell carcinoma). Right panels: Densitometric analysis of western blot experiment was performed using Image J software. Each signal was quantified and the SRPK1/2:tubulin ratio was calculated in each case. The value 1 was arbitrarily assigned to the ratio obtained in normal tissues and a relative ratio was calculated for each tumor sample according to its normal tissue pair.
Figure 6.
Relationship between SRPK2 protein expression and phospho-SRSF2 status in NSCLC subtypes.
A, Distribution of phospho-SRSF2 scores in tumors displaying normal (class 0), moderate (class +) or overexpression (class ++) of SRPK2, in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). B, Distribution of SRPK2 scores according to the tumor stage, in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). Statistical analysis was done using Mann-Whitney’s U test.
Figure 7.
SRSF1 overexpression confers a more aggressive phenotype.
Western blot analyses (A, C, E) and soft agar assays (B) were performed in H358-Ctl and H358-SRSF1 cells that were cultured at the same passage. (A) Expression of SRSF1 and activation of AKT/ERK signaling pathways were analyzed by western blotting. Tubulin was used as a loading control. (B) Upper panels: representative images of colonies in soft agar. Magnification, ×500. Lower panel: relative amounts of colonies in eight representative fields. Three independent experiments were performed in quadriplicate. (C) Western blot analysis of epithelial and mesenchymal markers. Tubulin was used as a loading control. (D) H1299 and H2170 cells were transfected either with control pcDNA3.1 or with myc-tagged SRSF1 plasmid. Transfected cells were selected during 6 days with G418 (800 µg/ml) and western blot analyses were performed using the indicated antibodies. Tubulin was used as a loading control. (E) H358-Ctl and H358-SRSF1 cells were cultured for 72 hours in the presence or absence of 500 nM wortmaninn or 10 µM U0126 as indicated. Expression of epithelial (E-cadherin) and mesenchymal (fibronectin, N-cadherin, vimentin) markers was analyzed by western blotting. Tubulin was used as a loading control.
Figure 8.
SRSF1 overexpression increases resistance to carboplatin and paclitaxel.
(A) H358 cells were treated for 24 hours with increasing amounts of etoposide, carboplatin or paclitaxel as indicated. SRSF1 protein level was analyzed by western blotting. Tubulin was used as a loading control. (B) 96-hours cell viability assays were performed in H358-Ctl cells (grey symbols) or H358-SRSF1 clones (black symbols) treated or not with increasing amounts of carboplatin, paclitaxel or etoposide. Results are expressed as the percentage of survival cells compared to untreated cells. Mean value of three independent experiments ± standard deviation performed in triplicate are presented.