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Fig. 1
FRG1 expression levels in prostate tumor and cell lines: a. Representative images of tumor and uninvolved tissues of prostate, as seen in first (uninvolved) and second (tumor) column from left. b. Comparison of IRS between tumor and uninvolved tissue. Graph shows that the reduction of IRS in tumor tissue (N = 100) compared to uninvolved adjacent tissue (N = 100) was significant (chi square test, 2 tailed, df-5, p value < 0.0005). Median IRS score for FRG1 in tumor is 2.5 compared to adjacent uninvolved tissue, which is 3.5. c. Distribution of staining pattern for FRG1 in the prostate tumor (N = 100) and uninvolved tissue (N = 100). d. FRG1 expression levels in three different prostate cancer cells, western blot panel shows comparatively higher levels of FRG1 in PC3 and LNCaP than DU145 cells. e. Western blot to confirm ectopic expression of FRG1 in DU145 cells. f. Western blot showing reduced FRG1 levels after RNAi silencing in DU145. g. Western blot image validating ectopic expression of FRG1 levels in PC3 cells. h. Reduction in FRG1 levels in FRG1 silenced PC3 cells confirmed by western blot. **** represents p value < 0.0005, N represents number of patient samples
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Fig. 2
Effect of FRG1 expression on cell proliferation and scratch wound healing: a. Ectopic expression of FRG1 has no significant (N = 5, 2-tailed unpaired t-test, p value > 0.05) effect on cell proliferation in DU145, compared to empty vector control. b. Measurement of cell proliferation in DU145, knockdown of FRG1 showing significant increase in cell proliferation (N = 5, 2-tailed unpaired t-test, p value < 0.05), compared to scrambled vector control. c. Measurement of cell proliferation in PC3 with ectopic expression of FRG1, compared to empty vector control, showing significant reduction (N = 5, 2-tailed unpaired t-test, p value < 0.05). d. Quantitation of cell proliferation in PC3 with knockdown of FRG1, compared to scrambled vector control, showing no significant (N = 5, 2-tailed unpaired t-test, p value > 0.05) effect. e. Representative images of scratch wound healing assay of DU145 cells, with ectopic expression of FRG1 and respective vector control. f. Graph of scratch wound healing assay of DU145 cells with ectopic expression of FRG1, showing 52% reduced wound area, compared to empty vector control with 74% reduced wound area (N = 3, 2-tailed unpaired t-test, p value < 0.05). g. Representative images of scratch wound healing assay of DU145 with FRG1 knockdown and respective scrambled vector control. h. Graph of scratch wound healing assay of DU145 cells with FRG1 knockdown showing 91% reduced wound area, compared to scrambled vector control with 68% reduced wound area (N = 3, 2-tailed unpaired t-test, p value < 0.05). i. Representative images of scratch wound healing assay of PC3 cells with ectopic expression of FRG1 and respective vector control. j. Graph of scratch wound healing assay of PC3 cells with ectopic expression of FRG1, showing 57% reduced wound area, compared to empty vector control with 90% reduced wound are (N = 3, 2-tailed unpaired t-test, p value < 0.05). k. Representative images of scratch wound healing assay of PC3 with FRG1 knockdown and respective scrambled vector control. l. Graph of scratch wound healing assay of PC3 cells with FRG1 knockdown, showing 80% reduced wound area, compared to scrambled vector control with 70% reduced wound area (N = 3, 2-tailed unpaired t-test, p value < 0.005). # represents p value > 0.05, * represents p value ≤0.05, ** represents p value < 0.01, *** represents p value < 0.005, N represents experiment replicates
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Fig. 3
Effect of FRG1 expression on transwell migration and invasion: a. Representative images of transwell migration assay of DU145 cells with ectopic expression of FRG1 and respective vector control. b. Graphical representation of transwell migration assay of DU145 cells with ectopic expression of FRG1 (2183 ± 84), showing reduction in transwell migration, compared to empty vector control (2565 ± 94) (N = 3, 2-tailed unpaired t-test, p value < 0.01). c. Representative images of transwell migration assay of DU145 cells with FRG1 knockdown and respective scrambled vector control. d. Graph of transwell migration assay of DU145 cells with FRG1 knockdown (1532 ± 165), showing enhanced transwell migration, compared to scrambled vector control (987 ± 130) (N = 3, 2-tailed unpaired t-test, p value < 0.05). e. Representative images of transwell migration assay of PC3 cells with ectopic expression of FRG1 and respective vector control. f. Graphical representation of transwell migration assay of PC3 with ectopic expression of FRG1 (1869 ± 86), showing reduction (N = 3, 2-tailed unpaired t-test, p value < 0.005) in transwell migration, compared to empty vector control (2256 ± 81). g. Representative images of transwell migration assay of PC3 cells with FRG1 knockdown and respective scrambled vector control. h. Graphical representation of transwell migration assay of PC3 cells with FRG1 knockdown (1685 ± 120), showing enhanced (N = 3, 2-tailed unpaired t-test, p value < 0.01) transwell migration, compared to scrambled vector (1285 ± 71). i. Representative images of matrigel invasion assay of DU145 cells with ectopic expression of FRG1 and respective vector control. j. Graphical representation of matrigel invasion assay of DU145 cells with ectopic expression of FRG1 (231 ± 43) compared to empty vector control (357 ± 60) (N = 3, 2-tailed unpaired t-test, p value < 0.05). k. Representative images of matrigel invasion assay of DU145 with FRG1 knockdown and respective scrambled vector control. l. Representative graph of matrigel invasion assay of DU145 cells with FRG1 knockdown (412 ± 62) compared to scrambled vector control (234 ± 27) (N = 3, 2-tailed unpaired t-test, p value < 0.05). m. Representative images of matrigel invasion assay of PC3 cells with ectopic expression of FRG1 and respective vector control. n. Representative graph of matrigel invasion assay of PC3 cells with ectopic expression of FRG1 (208 ± 65) compared to empty vector control (365 ± 53) (N = 3, 2-tailed unpaired t-test, p value < 0.05). o. Representative images of matrigel invasion assay of PC3 with FRG1 knockdown and respective scrambled vector control. p. Representative graph of matrigel invasion assay of PC3 cells with FRG1 knockdown (318 ± 29) compared to scrambled vector control (207 ± 37) (N = 3, 2-tailed unpaired t-test, p value < 0.05). * represents p value ≤0.05, ** represents p value < 0.01, *** represents p value < 0.005, N represents experiment replicates
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Fig. 4
Effect of FRG1 expression on LNCaP cell properties. a. Western blot to confirm ectopic expression of FRG1 in LNCaP cells. Western blot to confirm depletion of FRG1 expression in LNCaP cells after RNAi silencing. b. Graphical representation of LNCaP cell proliferation assay showing that ectopic expression of FRG1 has no significant (N = 5, 2-tailed unpaired t-test, p value > 0.05) effect on cell proliferation, compared to empty vector control (pCMV6.XL5). c. Graph showing that knockdown of FRG1 (FRG1 KD) doesn’t change LNCaP cells proliferation significantly (N = 5, 2-tailed unpaired t-test, p value > 0.05), compared to control (pLKO.1sc). d. Representative images of transwell migration assay of LNCaP cells with ectopic expression of FRG1 (FRG1) and respective vector control (pCMV6.XL5). e. Graphical representation of transwell migration assay of LNCaP cells with ectopic expression of FRG1 (601 ± 54), showing no change in transwell migration, compared to empty vector control (622 ± 71) (N = 3, 2-tailed unpaired t-test, p value > 0.05). f. Representative images of transwell migration assay of LNCaP cells with FRG1 knockdown (FRG1 KD) and respective scrambled vector control (pLKO.1sc). g. Graph of transwell migration assay of LNCaP cells with FRG1 knockdown (645 ± 61), showing no change in transwell migration, compared to scrambled vector control (550 ± 55) (N = 3, 2-tailed unpaired t-test, p value > 0.05). h. Representative images of matrigel invasion assay of LNCaP cells with ectopic expression of FRG1 and respective vector control. i. Graphical representation of matrigel invasion assay of LNCaP cells with ectopic expression of FRG1 (483 ± 130) compared to empty vector control (484 ± 89) (N = 3, 2-tailed unpaired t-test, p value > 0.05). j. Representative images of matrigel invasion assay of LNCaP cells with FRG1 knockdown (FRG1 KD) and respective scrambled vector control (pLKO.1sc). k. Representative graph of matrigel invasion assay of LNCaP cells with FRG1 knockdown (666 ± 60) compared to scrambled vector control (589 ± 24) (N = 3, 2-tailed unpaired t-test, p value > 0.05). # represents p value > 0.05, N represents experiment replicates
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Fig. 5
Expression analysis of cytokines and MMPs: a. q-RT PCR expression analysis of genes listed in Additional file 1 in DU145 cells, transfected with FRG1 silencing vector compared to scrambled vector control, b. q-RT PCR expression data of genes listed in Additional file 1, in PC3 cells with knockdown for FRG1 versus scrambled vector control. In panel A and B, t-test (2 tailed, for unpaired samples) was used for comparison of fold change values between experimental and control group. Each experimental group had three replicates. Dotted line represents 1.5-fold cut off, ** represents p value < 0.01, **** represents p value < 0.0005
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Fig. 6
Reduced FRG1 expression enhances p38 MAPK phosphorylation: a. Panel of blots showing enhanced p38 MAPK phosphorylation with FRG1 knockdown in DU145 cells but no change in activation of ERK levels. b. Panel of blots showing enhanced p38 MAPK phosphorylation with FRG1 knockdown in PC3 cells but mild change in phospho ERK levels. si represents protein lysate from FRG1 knockdown cells and sc represents cells expressing scrambled vector control. c. Panel of blot shows neutralization of p38 activation, when FRG1 depletion is rescued by transfection with FRG1 expression vector, in DU145 cells d. Panel of blot shows neutralization of p38 activation, when FRG1 depletion is rescued by transfection with FRG1 expression vector, in PC3 cells. All the experiments were repeated three times
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Fig. 7
Anisomycin based p38 MAPK activation affects expression of various cytokines and MMPs: a. Treatment of the DU145 cells with 0.4 μg/ml of Anisomycin (p38 MAPK activator) led to enhanced phosphorylation of p38 MAPK compared to the mock. Activation state was observed at 2 h (Lane 1 and 2) and 6 h (Lane 3 and 4) time points. b. Treatment of the PC3 cells with 0.4 μg/ml of Anisomycin led to enhanced phosphorylation of p38 MAPK compared to the mock. Activation state was observed at 2 h (Lane 1 and 2) and 6 h (Lane 3 and 4). c. Expression of GM-CSF, MMP1 and PLGF levels were quantified in DU145 cells after 8 h of treatment with 0.4 μg/ml of Anisomycin. GM-CSF (Fold change = 18.97) and PLGF (Fold change = 2.73) were significantly up-regulated on Anisomycin treatment, no significant effect on MMP1 (Fold change = 0.67) levels were observed. d. Expression of GM-CSF, MMP1, PDGFA and CXCL1 levels were quantified in PC3 cells after 8 h of treatment with 0.4 μg/ml of Anisomycin. GM-CSF (Fold change = 82.24), MMP1 (Fold change = 2.07) and CXCL1 (Fold change = 11.98) were significantly up-regulated on Anisomycin treatment, no significant effect on PDGFA (Fold change = 1.36) levels were observed. In panel C and D, t-test (2 tailed, for unpaired samples) was used for comparison of fold change values between experimental and control group. Each experimental group had three replicates. # represents p value > 0.05, * represents p value < 0.05, ** represents p value < 0.01, *** represents p value < 0.001
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Fig. 8
p38 inhibition in FRG1 knockdown prostate cancer cells affects expression of various molecules: a. Representative Western blot panel showing reduction of phosphorylated p38 levels in the first lane after treatment with 0.5 μM SB203580 in DU145 FRG1KD cells (Si). Phosphorylated p38 levels were higher in second lane with 0.1 μM SB203580 and in third lane with untreated FRG1KD cells (Si). We could also observe lower p38 phosphorylation levels in DU145 pLKO.1sc (Sc) cells (lane 4) compared to FRG1KD (Si) (lane 3). Third row representing FRG1 panel shows knockdown of FRG1 in FRG1KD (Si) cells compared to pLKO.1 sc (Sc) cells. GAPDH levels were detected as loading control. b. Representative western blot panel showing reduction of phosphorylated p38 levels in the first lane after treatment with 0.5 μM SB203580 in PC3 FRG1KD cells (Si). Phosphorylation levels were higher in second lane with 0.1 μM SB203580 and in third lane with untreated FRG1KD cells (Si). We can also observe lower p38 phosphorylation levels in PC3 pLKO.1sc (Sc) cells compared to FRG1KD (Si). Third row representing FRG1 panel shows knockdown of FRG1 in FRG1KD (Si) cells compared to pLKO.1 sc (Sc) cells. GAPDH levels were detected as loading control. c. q-RT PCR based expression analysis of GM-CSF, MMP1 and PLGF in DU145 FRG1KD cells after 8 h of treatment with 0.5 μM SB203580 (p38 inhibitor). Fold change in FRG1KD and FRG1KD + SB (p38 inhibitor) groups was derived in comparison with DU145 pLKO.1sc. After 8 h of treatment significant reduction in expression was observed in GM-CSF (FRG1KD, FC = 1.94 vs. FRG1KD + SB, FC = 1.25) and PLGF (FRG1KD, FC = 2.59 vs. FRG1KD + SB, FC = 1.13) expression, no significant reduction was observed in case of MMP1 (FRG1KD, FC = 1.78 to FRG1KD + SB, FC = 1.49). d. q-RT PCR based expression analysis of GM-CSF, MMP1, PDGFA and CXCL1 in PC3 FRG1KD cells after 8 h of treatment with 0.5 μM SB203580 (p38 inhibitor). Fold change in FRG1KD and FRG1KD + SB groups was derived in comparison with PC3 pLKO.1sc. After 8 h of treatment significant reduction in GM-CSF (FRG1KD, FC = 4.26 vs. FRG1KD + SB, FC = 1.12), MMP1 (FRG1KD, FC = 4.69 vs. FRG1KD + SB, FC =1.17), PDGFA (FRG1KD, FC = 4.17 vs. FRG1KD + SB, FC = 1.21) and CXCL1 (FRG1KD, FC = 1.71 vs. FRG1KD + SB, FC = 1.05) expression was observed. In panel C and D, t-test (2 tailed, for unpaired samples) was used for comparison of fold change values between experimental and control group. Each experimental group had three replicates. # represents p value > 0.05, * represents p value < 0.05,, FC represents fold change
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Fig. 9
Representation model for possible molecular interaction during FRG1 knockdown in prostate cancer cells
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