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50
51 A
B
C
52 Figure 1. Identification of LARP isoforms regulating lamellipodia formation.
(A) LARPs siRNAs efficiently decrease the expression of LARPs mRNA in SKOV3 cells.
LARPs mRNA expression levels were determined by quantitative real-time PCR. Data shown (mean ± s.e., n = 3) are the mRNA values normalized to β- actin. * P <0.05, ** P <
0.005, *** P <0.001 (B) SKOV3 cells (2 × 104) grown on glass-bottom dishes were transfected with 5 nM siRNAs for 48 h. Cells were fixed followed by staining with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Confocal microscopy representative images are shown and yellow arrows point to lamellipodia forming cell.
The data showed (mean ± s.e., n = 4) are the percentages of cells forming lamellipodia.
(C) Data shown (mean ± s.e., n = 4) are the percentages of cells forming lamellipodia,
*** P < 0.0001. Three LARPs isoforms regulate lamellipodia formation in SKOV3 cells (LARP3, LARP4 and LARP4b).
53 Figure 2. LARP4 knockdown promotes lamellipodia formation in SKOV3 cells
(A) Protein expression demonstrated by western blot showing LARP4 knockdown effect of siRNA. (B) Cells were fixed followed by staining with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Confocal microscopy representative images are shown and yellow arrows point to lamellipodia. (C) The data showed (mean ± s.e., n = 11) are the percentages of cells forming lamellipodia. * P < 0.05, *** P < 0.0001.
C B
A
54
Figure 3. LARP4 overexpression decrease lamellipodia formation in SKOV3 cells (A) Protein expression demonstrated by western blot showing LARP4 overexpression effect of LARP4 vector. (B) Cells were fixed followed by staining with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Confocal microscopy representative images are shown and yellow arrows point to lamellipodia. (C) The data showed (mean ± s.e., n
= 4) are the percentages of cells forming lamellipodia. * P < 0.05, *** P < 0.0001.
55 Figure 4. RhoA knockdown decreases lamellipodia formation in SKOV3 cells
Representative data for the percentage of lamellipodia forming cells counted by confocal microscopy after cell fixation and staining with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). The data shown (mean ± s.e., n = 11) are the percentages of cells forming lamellipodia. *** P < 0.0001.
56 Figure 5. LARP4 knockdown specifically promotes ovarian cancer cell migration, rather than invasion in SKOV3 cells
(A and C) SKOV3 (1 × 104) cells were transfected with 5 nM siRNAs for 48 h. Extracted proteins were submitted to immunoblot analysis using antibodies specific for LARP4 or β-actin. Equal amounts of protein were loaded in each lane and the representative images are shown with decreased LARP4 protein expression in LARP4 siRNA transfected cells. (B) Cell migration was assessed using the transwell assay. Data shown (mean ± s.e., n = 7-19 for SKOV3 cells) are the percentages of migrating cells, * P < 0.05, *** p <
0.0001. (D) Invasive activity assessed by matrigel-coated transwell assay as described in materials and methods. Data shown (mean ± s.e., n = 6 for SKOV3 cells) are the percentages of invading cells.
57 Figure 6. LARP4 knockdown promotes ovarian cancer cell migration and invasion in PC3 cells
(A and C) Extracted proteins were submitted to immunoblot analysis using antibodies specific for LARP4 and β-actin. Equal amounts of protein were loaded in each lane and the most representative picture is shown. (B) Cell migration was assessed using the transwell assay for 24 h. Data shown (mean ± s.e., n = 4) are the percentages of migrating cells. * P <0.05. (D) Cell invasion was assessed using the matrigel coated transwell assay for 24 h. Data shown (mean ± s.e., n = 4) are the percentages of migrating cells. * P <0.05.
58 Figure 7. LARP4 knockdown mediates its action through signaling pathway other than PI3K, MAPK, EMT and Tumor suppressor genes
Extracted proteins were submitted to immunoblot analysis using different antibodies. Equal amounts of protein were loaded in each lane and the most representative picture is shown.
59
60 Figure 8. LARP4 suppresses RhoA protein expression.
(A) Extracted proteins were submitted to immunoblot analysis using antibodies specific for LARP4, Rho GTPase family proteins or β-actin. Equal amounts of protein were loaded in each lane and the representative pictures are shown. (B, D and F) LARP4 protein level is significantly depleted in both LARP4 siRNA sequences (si1 and si2) (B) in RhoA protein expression
experiments. The data shown (mean ± s.e., n = 8) is the percentages of LARP4 protein expression.
*** P < 0.0001. (D) in Rac protein expression experiments. The data shown (mean ± s.e., n = 6) is the percentages of LARP4 protein expression. *** P < 0.0001. (F) in CDC42 protein expression experiments. The data shown (mean ± s.e., n = 6) is the percentages of LARP4 protein expression.
*** P < 0.0001. (D, E and F) Protein bands were quantified and the ratio values of individual proteins expression relative to β-actin are shown (mean ± s.e., n = 6 or 8). All statistical analyses were performed by unpaired, Student t-test. *** P < 0.0001.
61 Figure 9. Anti-metastatic potential of LARP4 in an ovarian cancer xenograft model.
(A) SKOV3 cells (1 × 104) transfected with 5 nM control si2 or LARP4 si2 for up to 96 h. Extracted proteins were submitted to immunoblot analysis using antibodies specific for LARP4 or β-actin.
The representative images are shown with persistence of LARP4 depletion up to 96 h. (B) SKOV3 cells (5 × 106 cells/mouse) transfected with 5 nM control si2 or LARP4 si2 for 24 h were inoculated into the peritoneal cavity of nude mice (n = 6 or 9). Four weeks later, mice were sacrificed and the number of metastatic nodules in the peritoneum was determined. The number of metastatic nodules were significantly higher in LARP4 depleted group. All statistical analyses were performed by one-tailed t-test. * P < 0.05.
62 A
B
63 C
D
64 E
F
65 Figure 10. Correlation of LARPs mRNA expressions with the overall survival probability in ovarian cancer patients
The graphs show the correlation between overall survival and mRNA expressions of LARPs in ovarian cancer patients. The red line represents patients with higher mRNA expression and the black line indicates those with lower mRNA expression. The numbers of patients used for the analyses are shown in the individual panels. Overall survival graphs of ovarian cancer based on LARPs expression were performed using publicly available database (KM plotter; www.kmplot.com) with the following options: auto select best cutoff, overall survival (OS), 120 month, the plot include addy-id and sample number. cut off values were as follow: LARP1 (19), SSB/LARP3 (1738), LARP4 (253), LARP4B (25), LARP6 (300), LARP7 (813). The LARP4-high patient group showed longer overall survival than the LARP4-low group (p < 0.031). Conversely, SSB/LARP3 mRNA expression was inversely correlated with overall survival (p < 0.0064). No significant correlation of overall survival with the mRNA expression of other LARPs was observed.