Figure in EIF4E. p-values are indicated as *<0.05,

Figure Legends:

Fig. 1: EIF4G1 protein expression is increased in prostate tissue sections of Prostate Cancer patients:

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

Analysis of EIF4G1 expression in human prostate tissue microarray:

a. Representative image of EIF4G1 IHC staining on prostate tissue microarray with different grade tumors (40X). b. Quantitation of EIF4G1 staining based on high, moderate and low expression. c. Staining intensity of EIF4G1 with normal prostate and tumor. d. Staining intensity of EIF4G1 in sample with normal prostate and different tumor grades. p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. 2: EIF4G1 is overexpressed in multiple clinical cohorts of PCa: Analysis of EIF4G1 expression in PCa samples from different databases: a. mRNA levels of EIF4G1 in Normal vs prostate tumor samples from TCGA dataset. b. mRNA levels of EIF4G1 in Paired Normal vs prostate tumor samples from TCGA dataset. c. Graded increase in EIF4G1 with increasing Gleason Score (GS) (TCGA). d. High expression of EIF4G1 decreases survival probability (TCGA data set, time in months). e. EIF4G1 is amplified and over expression in 16% patients in Metastatic Prostate Cancer data set. f. EIF4G1 is amplified and over expression in 59% patients in CRPC-Neuroendocrine Prostate Cancer data set, while no change is observed in EIF4E. p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. 3: EIF4G1 expression is increased in PCa cell lines and during tumor progression in tissue sections from TRAMP mice: EIF4G1 expression in cell lines and tumor tissue: a. EIF4G1 expression in PCa cell lines by Western Blot; (Adjacent graph is quantitation of blot). b. Immunohistochemistry for EIF4G1 expression on prostate tissue samples from wild type (WT) & 30 Week Old TRAMP mice (Images are 20X, inset 40X); (Adjacent graph is quantitation) Error bars represent ± SD. p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. 4: Knockdown of EIF4G1 impairs mRNA translation: Polysome profiles following knockdown of EIF4G1 in LNCaP and C4-2b cells: Cytoplasmic extracts from siControl and siEIF4G1 for LNCaP (a) & C4-2b (b) cells were centrifuged respectively through continuous sucrose gradients to obtain fractions of increasing density. Absorbance (A254) (y axis) versus increasing density (x axis) of fractions was monitored for presence of ribosomal subunits, ribosomes and polysome. Polysome (P)/Monosome (M) Ratio were calculated further. c. Western blotting showing knockdown of EIF4G1 in LNCaP and C4-2b. Adjacent graph is quantitation of blot. Fig. 5: Knockdown of EIF4G1 reduces the levels of cyclin D1 and p-Rb and delays cell cycle: Loss of Function studies showing the effects of knockdown of EIF4G1 on cell cycle: Knockdown of EIF4G1 decreases Rb/p-Rb, Cyclin D1 protein levels and delays cells at G0/G1 phase of cell cycle in LNCaP (a & b) and C4-2b (c & d). Adjacent is quantitation of cell cycle distribution in different phase. Fig. 6: EIF4G1 promotes cell growth and colony formation: Loss of Function studies following knockdown of EIF4G1 using siRNA: a & b. Cell proliferation was measured  by crystal assay at 48/72/96h with siControl and siEIF4G1, graph showed absorbance at 560 nm for  LNCaP (a) and C4-2b (b). c. Cell Viability assay for LNCaP and C4-2b (d) done by MTT assay at 24/48/72/96h with siControl and siEIF4G1 e. Colony formation for LNCaP and C4-2b (f) siControl and siEIF4G1, Lower panels: Colonies were counted using a ImageJ software. Error bars represent ± SD. g. Western blot showing knockdown of EIF4G1 in siControl and siEIF4G1 for LNCaP & C4-2b. p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. 7: Knockdown of EIF4G1 downregulates EMT markers and limits cell migration and in PCa Cells: Effect of EIF4G1 knockdown on EMT markers and cell migration in PCa cells: a. Representative image of immunoblotting for siControl and siEIF4G1 for LNCaP and C4-2b for N-Cadherin & Snail. b. Representative image of immunoblotting for VC and shEIF4G1 for LNCaP and C4-2b. Representative image and graph of Transwell® cell migration assays on LNCaP (c) & C4-2b (d) with Vector Control (VC) & ShEIF4G1. Graph is showing migratory cells per field. Error bars represent ± SD. p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. 8: Knockdown of EIF4G1 sensitize CRPC cells to bicalutamide and enzalutamide: a. Representative image of cell proliferation assay done by crystal violet with CRPC (C4-2b cell line) cells with siControl and siEIF4G1 treated with 10µM Enzalutamide (ENZ) and 10µM Bicalutamide (Bi) for 24h. b. Graph showed quantitation for above plate absorbance at 560 nm. Error bars represent ± SD. p-values are indicated as *<0.05, **<0.01, ***<0.001.   Fig. S1: Analysis of EIF4E expression in PCa samples from TCGA database: A. mRNA levels of EIF4G1 in Normal vs prostate tumor samples B. EIF4E with increasing Gleason Score (GS) with statistically insignificant p-value except with GS 6. Fig. S2: mRNA level of EIF4G1 & Cyclin D1 by knockdown of EIF4G1: Knockdown of EIF4G1 decreases mRNA level of EIF4G1 & Cyclin D1 in LNCaP (Left) and C4-2b (Right). p-values are indicated as *<0.05, **<0.01, ***<0.001. Fig. S3: EIF4G1 promotes cell proliferation: Representative image of cell proliferation assay done by crystal violet in LNCaP and C4-2b with siControl and siEIF4G1 at 48/72/96h. Fig. S4: RT-PCR data for EMT marker of siControl and siEIF4G1 in LNCaP & C4-2b cells: Real time PCR data showing Knockdown of EIF4G1 decreases EMT marker i.e. N-Cadherin, Vimentin, Zeb-1 & Snail in LNCaP (Top) and C4-2b (Bottom) with siControl and siEIF4G1. p-values are indicated as *<0.05, **<0.01, ***<0.001.