A - C . Immunofluorescence images ( A ) and quantifications ( B , C ) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with / without AZ - 3146 for 30 min to inhibit MPS1 . In all kinetochore intensity graphs , each dot represents a cell , horizontal lines indicate the median and vertical bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines . 30 cells from 3 experiments . Scale bars = 5µM . D . Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1 . 25 µM AZ - 3146 . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Immunofluorescence images of MAD1 and MAD1 - pT716 kinetochore levels in nocodazole - arrested RPE1 cells treated with or without AZ - 3146 . Scale bars = 5µM .

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A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.


B . Electron micrographs of rotary - shadowed MAD193 - 718 : MAD2 ( top row ) , MBP - MAD1 : MAD2 ( middle row ) , and MBP - MAD1 : MAD2 in complex with GST - CDK1 : Cyclin B1 ( bottom row ) . Scale bar = 50 nm .

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B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.


C . Quantifications ( top ) and corresponding immunofluorescence images ( underneath ) of relative kinetochore MAD1 and MAD1 - pT716 levels in nocodazole - arrested of MAD1 - WT - C14 and MAD1 - 3EK - C12 cells treated with different doses of AZ - 3146 for 30 min . MG132 was included at the time of AZ - 3146 addition to prevent mitotic exit . Each dot represents a cell , horizontal lines indicate the median and error bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . 30 cells from 3 experiments . Both kinetochore intensity graphs display data that is relative to the WT - C14 untreated controls , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.


D . Duration of mitotic arrest in MAD1 - WT C14 or MAD1 - 3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ - 3146 . Note , the 1 . 25 µM AZ - 3146 data is displayed in Figure 4D , but also included here to allow comparison with other drug doses . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Representative images of immunofluorescence of α - smooth muscle actin ( α - SMA ) , pan Cytokeratin , Pan - hMENA , hMENA11a and E - cadherin expression in CAFs and autologous cancer cells ( Ep - PDAC ) obtained from enzymatically digested primary PDAC tissue of patient # 36 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 20 μm .

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A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.


B . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal fibroblasts derived from transplant donor ( P - NF ) , pancreatic serous cystadenoma # 71 and cancer associated fibroblasts ( n = 10 ) obtained from primary PDAC tissues . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression . Quantification of P # 110 is relative to the sample shown in the WB on the left .

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B. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71 and cancer associated fibroblasts (n=10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENA∆v6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left.


C . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal lung fibroblasts ( L - NF ) , cancer associated fibroblasts obtained from NSCLC tissues ( n = 4 ) and normal dermal fibroblasts ( HNF ) . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression .

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C. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer associated fibroblasts obtained from NSCLC tissues (n=4) and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENA∆v6 expression.


A . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in the primary PDAC tissue of patient # 138 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . αSMA ‑ positive CAFs are also positive for Pan - hMENA ( arrow ) .

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A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).


B . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in NSCLC case # 484 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . As in A , α - SMA signal is evident in stromal cells which are also positive for Pan - hMENA ( arrow ) .

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B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).


C . Boxplots showing the mean mRNA expression of ENAH gene ( hMENA ) for 22 patients in the 10 groups of cell types ( Peng et al . , 2019 ) ( total number of cells : 38487 , mean of cells for patient : 1603 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Expressions from each cell - type group were compared to all other groups by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method . Stromal cell - type groups with significantly up - regulated ENAH expression respect to other stromal groups are : Fibroblast , * * * q = 0 . 0002 ; Stellate , * * * q = 1 . 5e - 07 . Non - stromal cell - type groups with significantly up - regulated ENAH expression with respect to other non - stromal groups are : Ductal 1 , * * q = 0 . 0013 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.


D . Boxplots showing the mRNA expression of ENAH gene ( hMENA ) in the 7 groups of cell types from ( Lambrechts et al . , 2018 ) ( total n = 52 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Cell expression from each group were compared to all other stromal / not - stromal cells by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method ( Fibroblast group vs other stromal groups , * * q = 0 . 0022 ; Other comparisons , q > 0 .

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D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05


A . Quantification of in vitro matrigel invasion assay ( bottom ) of P - CAF and L - CAF ( P - CAF # 36 , 138 and L - CAF # 189 , 484 ) transfected with control siRNA ( CNT ) or hMENA siRNA ( hMENA ( t ) ) indicating that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 reduces the invasive ability of CAFs with respect to siCNT CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot showing hMENA / hMENAΔv6 expression ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) of the CAFs employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


B . Quantification of in vitro matrigel invasion assay ( bottom ) of P - NF and L - NF and P - CAF # 110 and L - CAF # 400 transfected with control or hMENA ∆ v6 expressing vectors , demonstrating that the overexpression of hMENA ∆ v6 isoform induced the invasiveness of P - NFs and L - NFs and / or P - and L - CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot of hMENAΔv6 expression ( detected by the specific anti - hMENAΔv6 antibody ) in fibroblasts employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


A . Quantification of in vitro matrigel invasion assay of PANC - 1 cells cultured for 48 h with conditioned media ( CM ) of NFs ( P - NFs - CM ) , CAF low # 44 and # 110 and CAFs high # 36 and 138 . Histograms show the number of invading cells measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates , performed at least in duplicate each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A. Quantification of in vitro matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. * P <0.05, **P<0.01, ***P<0.001.


B . Quantification of in vitro matrigel invasion assay of PANC - 1 cultured for 48 h with CM derived from control P - CAFs # 36 ( siCNT - P - CAF - CM # 36 ) and hMENA / hMENA ∆ v6 silenced P - CAFs ( sihMENA ( t ) - P - CAF - CM # 36 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects PANC - 1 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

B. Quantification of in vitro matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENA∆v6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


C . Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media ( culture medium ) or conditioned media ( CM ) of L - CAF low # 400 and CAFs high # 189 , as described above . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

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C. Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


D . Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L - CAFs # 484 ( siCNT - L - CAF - CM # 484 ) and hMENA / hMENA ∆ v6 silenced L - CAFs ( sihMENA ( t ) - L - CAF - CM # 484 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects H1975 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of six replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENA∆v6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


E . Representative immunoblot ( top ) and densitometry quantification ( bottom ) of hMENA ∆ v6 expression level in P - NFs grown in RPMI control medium ( - ) or PANC - 1 - CM for 24 h ( n = 3 ) . Data are represented as fold increase with respect to control medium ± SD ( set as 1 ) . Data were analyzed using two ‐ sided Student ' s t ‐ test . * P < 0 . 05 .

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E. Representative immunoblot (top) and densitometry quantification (bottom) of hMENA∆v6 expression level in P-NFs grown in RPMI control medium (-) or PANC-1-CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two‐sided Student's t‐test. *P < 0.05.


F . Representative images ( top ) and quantification ( bottom ) of collagen gel - contraction ability of CAFs ( monoculture ) or CAFs co - cultured with siCNT ( co - siCNT ) or sihMENA ( t ) PANC - 1 cells ( co - sihMENA ( t ) ) . Dashed white circles illustrate the margins of the gel area . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 005 , followed by Bonferroni ' s multiple comparison test ANOVA : * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01.


G . Representative immunoblot ( top ) and quantification ( bottom ) of hMENA ∆ v6 and α - SMA expression in P - CAFs # 110 treated with DMEM medium ( - ) or with conditioned medium ( CM ) derived from siCNT ( siCNT PANC - 1 ) or sihMENA ( t ) PANC - 1 cells ( ( sihMENA ( t ) PANC - 1 ) ( n = 3 ) . Data are represented as fold increase with respect to DMEM ( set as 1 ± SD ) . Data presented were analyzed using two ‐ sided Student ' s t ‐ test : * P < 0 . 05 .

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G. Representative immunoblot (top) and quantification (bottom) of hMENA∆v6 and α-SMA expression in P-CAFs #110 treated with DMEM medium (-) or with conditioned medium (CM) derived from siCNT (siCNT PANC-1) or sihMENA(t) PANC-1 cells ((sihMENA(t) PANC-1) (n = 3). Data are represented as fold increase with respect to DMEM (set as 1± SD). Data presented were analyzed using two‐sided Student's t‐test: *P<0.05.


A . Heat map of proteins identified by LC - MS / MS analysis as differentially secreted in CM of normal fibroblasts ( P - NF # 1 and P - NF # 2 ) , CAFs with low level of hMENA ∆ v6 expression ( P - CAFs low # 44 and # 49 ) , CAFs with high level of hMENA ∆ v6 expression ( P - CAFs high # 67 and # 36 ) . The boxed hMENA ∆ v6 associated signature represents proteins exclusively present in the CM derived from CAFs high . Color code is shown in the upper left corner . Co - variance = 0 . 2 , q value = 0 . 1 ( 10 % false discovery rate ) , adjusted P value = 0 . 0074542 . N = 36 . The values of mass spec identified for each protein were plotted in log2 scale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Heat map of proteins identified by LC-MS/MS analysis as differentially secreted in CM of normal fibroblasts (P-NF#1 and P-NF#2), CAFs with low level of hMENA∆v6 expression (P-CAFs low#44 and #49), CAFs with high level of hMENA∆v6 expression (P-CAFs high #67 and #36). The boxed hMENA∆v6 associated signature represents proteins exclusively present in the CM derived from CAFs high. Color code is shown in the upper left corner. Co-variance = 0.2, q value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log2 scale.


B . Quantification of GAS6 secretion levels , as detected by ELISA , in the CM of P - NF ( # 1 ) , P - CAFlow ( # 44 and # 49 ) and P - CAF high ( # 67 and # 36 ) . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

B. Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P-NF (#1), P-CAFlow (#44 and #49) and P-CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


C . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in NFs ( P - NF # 1 ) , P - CAF low and P - CAF high , as described above . Data are presented as the mean ± SD of three replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

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C. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in NFs (P-NF#1), P-CAF low and P-CAF high, as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one-way ANOVA P=0.001, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


D . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in PANC - 1 and P - CAF ( n = 3 ) , showing a low GAS6 expression in PANC - 1 cells . Data are presented as the mean ± SD . P values were calculated by two ‐ sided Student ' s t ‐ test . * * * P < 0 . 001 .

D. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in PANC-1 and P-CAF (n=3), showing a low GAS6 expression in PANC-1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t‐test. ***P<0.001.


E . Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts ( white ) ( n = 15 ) versus primary NSCLC fibroblasts ( light blue ) ( n = 15 ) ( GSE22862 data set ) . In each boxplot the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) are shown . Statistical significance was calculated by Mann - Whitney U - test ( two - sided ) ( P = 0 . 0208 ) .

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E. Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) (n=15) versus primary NSCLC fibroblasts (light blue) (n=15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line) are shown. Statistical significance was calculated by Mann-Whitney U-test (two-sided) (P=0.0208).


F , G . Real time qRT - PCR analysis of P # 138 CAF ( F ) and L # 189 CAF ( G ) transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) as representative cases . The siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells ( set as 100 ) . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test * * P < 0 . 01 , * * * P < 0 . 001 .

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F, G. Real time qRT-PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENA∆v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test **P<0.01, ***P<0.001.


H . Quantification of matrigel invasion assay of PANC - 1 cultured for 48 h with DMEM ( culture medium ) , or conditioned medium ( CM ) derived from control siRNA P # 106 CAFs ( siCNT P - CAF - CM ) , GAS6 siRNA ( siGAS6 P - CAF - CM ) , hMENA ( t ) siRNA ( sihMENA ( t ) P - CAF - CM ) , hMENA ( t ) siRNA plus rGAS6 ( sihMENA ( t ) P - CAF - CM + rGAS6 ) . Number of invaded PANC - 1 cells after 48 h of treatment was measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 004 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Quantification of matrigel invasion assay of PANC-1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P-CAF-CM), GAS6 siRNA (siGAS6 P-CAF-CM), hMENA(t) siRNA (sihMENA(t) P-CAF-CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P-CAF-CM +rGAS6). Number of invaded PANC-1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.004, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


A . Immunoblot of AXL expression in PANC - 1 and A549 cancer cells upon transfection of control siRNA ( CNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a reduction of AXL protein expression .

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A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.


B . Real time qRT - PCR analysis of the relative AXL mRNA expression in PANC - 1 and A549 cancer cells transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a significant reduction of mRNA AXL expression . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * * P < 0 . 01 , * * * P < 0 . 001 .

B. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001.


C . Real time qRT - PCR analysis of the relative levels of mature AXL mRNA or AXL pre - mRNA in PANC - 1 ( left ) and A549 ( right ) cell lines , transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA , sihMENA ( t ) . Data represent percent of AXL mRNA or pre - mRNA levels in hMENA ( t ) silenced cells relative to siCNT control cells ( set at 100 ) . Data are presented as the mean ± SD of two biological replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

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C. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001.


D . Immunoblot analysis with the indicated antibodies of PANC - 1 cells transfected with control siRNA ( CNT ) or hMENA ( t ) siRNA , showing that the knock ‐ down of total hMENA isoforms , ( hMENA ( t ) ) inhibits GAS6 - mediated pAXL and pAKT expression . Cells were serum starved overnight and subsequently stimulated with DMSO ( 0 . 02 % ) in control culture medium ( - ) or rGAS6 ( 200 ng / mL ) , for 30 and 60 min . The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported .

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D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.


E . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) toward rGAS6 as chemo - attractant ( siCNT + rGAS6 and sihMENA ( t ) + rGAS6 ) , showing that the knock ‐ down of hMENA ( t ) reduced cancer cell invasion toward GAS6 . The number of invading cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * P < 0 . 01 .

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E. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01.


F . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) , untreated ( - ) or treated with conditioned media derived from CAFs ( P - CAF # 36 - CM ) for 48 h . The number of invaded cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

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F. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.


A . Overall survival ( OS ) curves in Pancreatic Adenocarcinoma patients ( PDAC ) ( n = 172 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in PDAC .

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A. Overall survival (OS) curves in Pancreatic Adenocarcinoma patients (PDAC) (n=172) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in PDAC.


B . Overall survival ( OS ) curves in Lung Squamous cancer patients ( LUSC ) ( n = 501 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in LUSC .

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B. Overall survival (OS) curves in Lung Squamous cancer patients (LUSC) (n=501) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in LUSC.


Components of the RLCco - precipitate with ER markers in density gradient fractionation . The post - nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient . ER markers Calnexin , Ribophorin I and Climp63 , co - sedimented with ribosomes ( RPS6 , ribosomal protein subunit 6 ) , mitochondria ( Tim23 ) and RLC proteins at fractions 3 - 5 . The bottom graph shows the density of each fraction .

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Components of the RLCco-precipitate with ER markers in density gradient fractionation. The post-nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers Calnexin, Ribophorin I and Climp63, co-sedimented with ribosomes (RPS6, ribosomal protein subunit 6), mitochondria (Tim23) and RLC proteins at fractions 3-5. The bottom graph shows the density of each fraction.


B ) TRBP , PACT and Ago2 partially co - localize with the ER marker mCherry - CLIMP63 . Hippocampal neurons ( 10 DIV ) expressing mCherry - CLIMP63 were immunostained with TRBP , PACT or Ago2 antibodies . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 10μm ) .

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B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm).


C ) GFP - Dicer partially co - localizes with mCherry - CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons . Neurons were imaged at 10 DIV using total internal reflection fluorescence ( TIRF ) microscopy using a penetration depth of 150nm . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 2μm or 10μm ) .

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C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm).


D ) The majority of Dicer , TRBP and PACT are associated with neuronal membranes . Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons . The relative quantity of depicted proteins was analyzed in three independent experiments ( p < 0 . 005 , t - test type 3 , n = 3 , error bars s . d . ) . Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions , respectively .

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D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively.


E ) TRBP is cytoplasmic in primary astrocytes . Sequential detergent extraction was performed in primary astrocyte cultures . Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions , respectively .

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E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively.


F ) The majority of Dicer , TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells , isolated using sequential detergent extraction .

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F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.


A ) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes . Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation . Protein intensity values were normalized to calnexin in each of three independent experiments ( p = 0 . 008 , t - test type 3 , n = 3 , error bars ; s . d . ) .

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A) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes. Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation. Protein intensity values were normalized to calnexin in each of three independent experiments (p=0.008, t-test type 3, n=3, error bars; s.d.).


B ) Endogenous TRBP partially co - localizes with calnexin at the neuronal cell soma and primary dendrites . Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation . Boxed insets are magnifications of the primary dendrite ( 15μm in length ) . Arrows point to overlapping puncta ( scale bars ; 20μm ( GFP ) or 10μm ) .

B) Endogenous TRBP partially co-localizes with calnexin at the neuronal cell soma and primary dendrites. Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation. Boxed insets are magnifications of the primary dendrite (15μm in length). Arrows point to overlapping puncta (scale bars; 20μm (GFP) or 10μm).


C ) BDNF leads to a decrease in co - localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons . Manders co - localization co - efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites ( 15μm ) using GFP as a morphological mask . Average co - localization values were normalized to controls in each experiment ( p < 0 . 008 , t - test type 3 , n = 4 , error bars ; s . d . ) .

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C) BDNF leads to a decrease in co-localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons. Manders co-localization co-efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites (15μm) using GFP as a morphological mask. Average co-localization values were normalized to controls in each experiment (p<0.008, t-test type 3, n=4, error bars; s.d.).


D ) BDNF decreases mCherry - CLIMP63 co - localization with TRBP , but not with PACT or Ago2 . Hippocampal neurons were transfected with GFP and mCherry - CLIMP63 at 7DIV and immunostained three days later , following BDNF or control stimulation . Manders co - localization was measured at the cell soma using GFP as a mask as indicated ( p = 0 . 01 , t - test type 3 , n = 4 , error bars ; s . d . ) .

D) BDNF decreases mCherry-CLIMP63 co-localization with TRBP, but not with PACT or Ago2. Hippocampal neurons were transfected with GFP and mCherry-CLIMP63 at 7DIV and immunostained three days later, following BDNF or control stimulation. Manders co-localization was measured at the cell soma using GFP as a mask as indicated (p=0.01, t-test type 3, n=4, error bars; s.d.).


E ) BDNF leads to an increase in the levels of TRBP ( p = 0 . 03 ) and PACT ( p = 0 . 004 ) in the cytoplasmic fraction . Cortical neurons ( 7 DIV ) were control - or BDNF - stimulated and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each experiment . The position of the star corresponds to the TRBP band used for analysis ( ~ 43kDa ) ( t - test type 3 , n = 4 , error bars ; s . d . ) .

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E) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).


F ) The BDNF - induced increase in cytoplasmic TRBP is dependent on intracellular calcium . Cortical neurons ( 7 DIV ) were treated with vehicle control , BDNF and BAPTA - AM as indicated , and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each independent experiment ( p = 0 . 03 , n = 3 , error bars ; s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).


BDNF stimulation causes a decrease in the TRBP - Dicer interaction . Young cortical neurons were treated with BDNF or vehicle control and co - immunoprecipitations ( co - IPs ) were performed using 2μg control IgG or polyclonal anti - TRBP antibodies . Dicer binding in co - IP was normalized to inputs in each experiment ( p = 0 . 001 , t - test type 2 , n = 3 , error bars represent s . d . ) .

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BDNF stimulation causes a decrease in the TRBP-Dicer interaction. Young cortical neurons were treated with BDNF or vehicle control and co-immunoprecipitations (co-IPs) were performed using 2μg control IgG or polyclonal anti-TRBP antibodies. Dicer binding in co-IP was normalized to inputs in each experiment (p=0.001, t-test type 2, n=3, error bars represent s.d.).


B ) Short BDNF stimulation changes the levels of only a few microRNAs , such as miR - 16 - 5p and miR - 551b - 5p . Volcano plot of small RNA sequencing data from BDNF - or control - treated cortical neurons ( 6DIV ) was based on differential expression analysis using the edgeR package . Individual points represent the average fold change obtained in three independent experiments for each miRNA , and plotted against obtained p - values . MiRNAs above the red dashed line are significant ( p < 0 . 05 , t - test type 2 , n = 3 ) .

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B) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).


C ) BDNF stimulation leads to a transient decrease in mature miR - 16 - 5p levels . 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR . Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation ( n = 3 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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C) BDNF stimulation leads to a transient decrease in mature miR-16-5p levels. 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR. Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation (n=3, p=0.02, t-test type 3, error bars; s.d.).


D ) BDNF stimulation leads to a transient increase in the levels of pre - miR16 after 10 minutes of BDNF stimulation . Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation ( n = 4 , t - test type 3 , error bars ; s . d . ) .

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D) BDNF stimulation leads to a transient increase in the levels of pre-miR16 after 10 minutes of BDNF stimulation. Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation (n=4, t-test type 3, error bars; s.d.).


E ) BDNF causes a decrease in pre - miR16 binding to TRBP . Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti - TRBP antibody . Ct values measured in rtPCR were normalized to respective inputs ( n = 3 , p = 0 . 002 , t - test type 3 , error bars represent s . d . ) .

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E) BDNF causes a decrease in pre-miR16 binding to TRBP. Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti-TRBP antibody. Ct values measured in rtPCR were normalized to respective inputs (n=3, p=0.002, t-test type 3, error bars represent s.d.).


A ) BDNF changes the canonical isomiR proportion of a subset of miRNAs . IsomiR levels were compared to the total amount of the corresponding miRNA . Shown are the miRNAs that had significant changes in the canonical isomiR ( annotated as ' 0 ' , p < 0 . 05 , t - test type 2 , n = 3 ) . Results are shown in order of decreasing significance . Shown in red are miRNAs of the miR16 family , miR - 16 - 5p and miR - 15b - 5p . Black squares correspond non - existing isomiRs . Numbers on the top of the table represent the trimming ( negative ) or addition ( positive ) of nucleotides to the 3 ' - or 5 ' - end of the canonical 5p or 3p isomiRs , respectively . Only templated nucleotide additions and trimmings , which might reflect Dicer - mediated pre - miRNA cleavage , were considered for analysis .

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A) BDNF changes the canonical isomiR proportion of a subset of miRNAs. IsomiR levels were compared to the total amount of the corresponding miRNA. Shown are the miRNAs that had significant changes in the canonical isomiR (annotated as '0', p<0.05, t-test type 2, n=3). Results are shown in order of decreasing significance. Shown in red are miRNAs of the miR16 family, miR-16-5p and miR-15b-5p. Black squares correspond non-existing isomiRs. Numbers on the top of the table represent the trimming (negative) or addition (positive) of nucleotides to the 3'- or 5'-end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated pre-miRNA cleavage, were considered for analysis.


B ) BDNF stimulation leads to a decrease in the canonical ( 22 nucleotides ) isomiRs of the miR16 family , miR - 16 - 5p ( p = 0 . 005 ) and miR - 15b - 5p ( p = 0 . 02 ) . In both cases , this is accompanied by an increase in the levels of shorter isomiRs ; the 19 - nucleotide isoform of miR - 16 - 5p ( p = 0 . 04 ) and the 21 - nucleotide isoform of miR - 15b - 5p ( p = 0 . 007 ) . The relative isomiRs proportion was calculated as described in A ( t - test type 2 , n = 3 , error bars ; s . d . ) .

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B) BDNF stimulation leads to a decrease in the canonical (22 nucleotides) isomiRs of the miR16 family, miR-16-5p (p=0.005) and miR-15b-5p (p=0.02). In both cases, this is accompanied by an increase in the levels of shorter isomiRs; the 19-nucleotide isoform of miR-16-5p (p=0.04) and the 21-nucleotide isoform of miR-15b-5p (p=0.007). The relative isomiRs proportion was calculated as described in A (t-test type 2, n=3, error bars; s.d.).


B ) Endogenous miR - 16 - 5p activity in developing hippocampal neurons . Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ( ' LNA Ctrl ' ) or the anti - sense miR - 16 - 5p inhibitor ( ' LNA - miR16 ' ) . Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ( ' GFP - only ' ) was compared to the total number of transfected neurons . LNA - miR16 reduces the amount of repression of the miR16 sensor compared to LNA - Ctrl ( n = 4 , p = 0 . 02 , t - test type 2 , error bars represent s . d . ) .

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B) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.).


C ) Short - term BDNF stimulation inhibits endogenous miR - 16 - 5p activity . Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B . Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment ( n = 4 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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C) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


D & E ) Inhibition of miR - 16 - 5p increases dendritic complexity and occludes BDNF - induced dendritogenesis . D ) Hippocampal neurons were transfected with the control sensor ( A ) , and stimulated with control or BDNF 24 hours before fixation . Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites ( scale bars are 10μm ) . E ) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis . Values were normalized to LNA - Ctrl and control treatment ( n = 3 , p < 0 . 04 , t - test type 3 , error bars represent s . d . ) .

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D&E) Inhibition of miR-16-5p increases dendritic complexity and occludes BDNF-induced dendritogenesis. D) Hippocampal neurons were transfected with the control sensor (A), and stimulated with control or BDNF 24 hours before fixation. Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites (scale bars are 10μm). E) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis. Values were normalized to LNA-Ctrl and control treatment (n=3, p<0.04, t-test type 3, error bars represent s.d.).


F & G ) Overexpression of miR - 16 - 5p blocks BDNF - induced dendritogenesis . F ) Hippocampal neurons were transfected with control sensor and either control miRNA mimic ( miR - Ctrl ) or a miR - 16 - 5p mimic ( miR16 ) and treated two days later with BDNF or control vehicle . DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope ( scale bars ; 10μm ) . G ) Total number of intersections was quantified in Sholl analysis and values were normalized to miR - Ctrl , control treatment in each experiment ( n = 4 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

F&G) Overexpression of miR-16-5p blocks BDNF-induced dendritogenesis. F) Hippocampal neurons were transfected with control sensor and either control miRNA mimic (miR-Ctrl) or a miR-16-5p mimic (miR16) and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). G) Total number of intersections was quantified in Sholl analysis and values were normalized to miR-Ctrl, control treatment in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


H & I ) Overexpression of the Dicer - binding deficient deletion mutant of TRBP ( GFP - TRBP Δ ) occludes BDNF - induced dendritogenesis . H ) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP - TRBP Δ , and treated two days later with BDNF or control vehicle . DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope ( scale bars ; 10μm ) . I ) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment ( n = 5 , p < 0 . 02 , t - test type 3 , error bars ; s . d . ) .

H&I) Overexpression of the Dicer-binding deficient deletion mutant of TRBP (GFP-TRBP Δ) occludes BDNF-induced dendritogenesis. H) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP-TRBP Δ, and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). I) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment (n=5, p<0.02, t-test type 3, error bars; s.d.).


B ) MiR - 16 - 5p targets the 3 ' UTR of the BDNFmRNA . Cortical neurons were transfected with 100ng pmiRGlo dual - luciferase reporter consisting of the 3 ' UTR of BDNF ( 3 ' UTR WT ) or a miR - 16 - 5p - binding site mutant ( 3 ' UTR Mut ) and either miR - Ctrl or miR - 16 . The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co - transfection ( n = 3 , p = 0 . 02 , t - test type 2 ) .

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B) MiR-16-5p targets the 3'UTR of the BDNFmRNA. Cortical neurons were transfected with 100ng pmiRGlo dual-luciferase reporter consisting of the 3'UTR of BDNF (3'UTR WT) or a miR-16-5p-binding site mutant (3'UTR Mut) and either miR-Ctrl or miR-16. The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co-transfection (n=3, p=0.02, t-test type 2).


C ) BDNFstimulation leads to an increase in the translation of 3 ' UTR WT ( p = 0 . 02 , t - test type 3 ) but not 3 ' UTR Mut luciferase reporter , which is rescued by miR - 16 - 5p overexpression ( p = 0 . 002 ; t - test type 2 ) . Cortical neurons were transfected as in ( B ) , stimulated with BDNF or vehicle control for 20 minutes , and lysed two hours after stimulation ( n = 4 ) .

C) BDNFstimulation leads to an increase in the translation of 3'UTR WT (p=0.02, t-test type 3) but not 3'UTR Mut luciferase reporter, which is rescued by miR-16-5p overexpression (p=0.002; t-test type 2). Cortical neurons were transfected as in (B), stimulated with BDNF or vehicle control for 20 minutes, and lysed two hours after stimulation (n=4).


D ) Pre - miR16 is present in the neuronal cell soma and processes of hippocampal neurons . Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately . Pre - miR137 and pre - miR341 were used to assay somatic and neurite enrichment , respectively . Raw Ct values were normalized to GAPDH mRNA in each experiment ( n = 3 ) .

D) Pre-miR16 is present in the neuronal cell soma and processes of hippocampal neurons. Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately. Pre-miR137 and pre-miR341 were used to assay somatic and neurite enrichment, respectively. Raw Ct values were normalized to GAPDH mRNA in each experiment (n=3).


More Dicer binds to membrane - targeted GFP - TRBP ( mGFP - TRBP ) compared to GFP - TRBP . HEK293T cells were transfected with GFP , GFP - TRBP or mGFP - TRBP and cell lysates were processed for co - IP using anti - GFP antibody . Dicer binding was normalized to respective input lanes in each condition ( n = 4 , p = 0 . 02 ; t - test type 2 , error bars are s . d . ) .

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More Dicer binds to membrane-targeted GFP-TRBP (mGFP-TRBP) compared to GFP-TRBP. HEK293T cells were transfected with GFP, GFP-TRBP or mGFP-TRBP and cell lysates were processed for co-IP using anti-GFP antibody. Dicer binding was normalized to respective input lanes in each condition (n=4, p=0.02; t-test type 2, error bars are s.d.).


B ) mGFP - TRBP but not GFP - TRBP blocks the BDNF - induced decrease in miR - 16 - 5p activity in dual - fluorescence sensor assay . Hippocampal neurons were transfected with 200ng miR16 - or control - sensor ( see Fig 5A ) plus 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - TRBP . Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti - GFP antibody . BDNF led to a decrease in miR - 16 - 5p activity in GFP - transfected neurons ( p = 0 . 03 , n = 4 , t - test type 3 ) , which was significantly different from mGFP - TRBP - expressing neurons ( p = 0 . 04 , n = 4 , t - test type 2 , error bars are s . d . ) .

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B) mGFP-TRBP but not GFP-TRBP blocks the BDNF-induced decrease in miR-16-5p activity in dual-fluorescence sensor assay. Hippocampal neurons were transfected with 200ng miR16- or control-sensor (see Fig 5A) plus 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-TRBP. Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti-GFP antibody. BDNF led to a decrease in miR-16-5p activity in GFP-transfected neurons (p=0.03, n=4, t-test type 3), which was significantly different from mGFP-TRBP-expressing neurons (p=0.04, n=4, t-test type 2, error bars are s.d.).


C & D ) BDNF - induced dendritogenesis is blocked by mGFP - TRBP overexpression . C ) Hippocampal neurons ( 4DIV ) were transfected for three days with 200ng dsRed and 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - BDNF . Neurons were treated with BDNF or vehicle control 24 hours before fixation . Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites ( scale bars are 10μm ) . D ) The total number of intersections was calculated in Sholl analysis , and values were normalized to control treatment in each of three independent experiments . BDNF led to a significant increase in dendritic complexity ( p = 0 . 04 ; t - test type 3 ) , which was significantly different in cells transfected with mGFP - TRBP , but not GFP - TRBP ( n = 3 , p = 0 . 03 ; t - test type 2 , error bars ; s . d . ) .

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C&D) BDNF-induced dendritogenesis is blocked by mGFP-TRBP overexpression. C) Hippocampal neurons (4DIV) were transfected for three days with 200ng dsRed and 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-BDNF. Neurons were treated with BDNF or vehicle control 24 hours before fixation. Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites (scale bars are 10μm). D) The total number of intersections was calculated in Sholl analysis, and values were normalized to control treatment in each of three independent experiments. BDNF led to a significant increase in dendritic complexity (p=0.04; t-test type 3), which was significantly different in cells transfected with mGFP-TRBP, but not GFP-TRBP (n=3, p=0.03; t-test type 2, error bars; s.d.).


B , Boxplots showing the cell size ( y - axis ) over time ( x - axis ) of all the samples as they progressed in the cell cycle . The boxplot graphs were generated with R language functions . Each box is drawn from the first to the third quartile , with a horizontal line drawn in the middle to denote the median . The whiskers show the interquartile range ( IQR ) , and they were drawn at 1 . 5xIQR . The replicates were all biological ones .

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B, Boxplots showing the cell size (y-axis) over time (x-axis) of all the samples as they progressed in the cell cycle. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.


Each plot shows the relative mass isotopomer distribution ( MID ) values for metabolite species detected from cell extracts in each of the five independent experiments shown in Figure 1 . Each value was divided by the average value of the entire time series ( x - axis ) for that species from the same experiment , and Log2 - transformed ( y - axis ) . Loess curves and the std errors at a 0 . 95 level are shown . The Log2 ( relative abundance ) values of pyruvate ( M1 ) and leucine ( M6 ) showed the most significant

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Each plot shows the relative mass isotopomer distribution (MID) values for metabolite species detected from cell extracts in each of the five independent experiments shown in Figure 1. Each value was divided by the average value of the entire time series (x-axis) for that species from the same experiment, and Log2-transformed (y-axis). Loess curves and the std errors at a 0.95 level are shown. The Log2(relative abundance) values of pyruvate(M1) and leucine(M6) showed the most significant changes


B , The indicated strains ( all in the prototrophic CEN . PK background ; were spotted at 10 - fold serial dilutions on solid Synthetic Minimal Medium ( SMM ) agar plates . Exogenous amino acids were added at 1mM final concentration , as indicated in each case . The plates were incubated at 30 ° C and photographed after 3 - days .

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B, The indicated strains (all in the prototrophic CEN.PK background; were spotted at 10-fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Exogenous amino acids were added at 1mM final concentration, as indicated in each case. The plates were incubated at 30 °C and photographed after 3-days.


C , DNA content profiles of BAT1 and bat1Δ cells from asynchronous cultures , in SMM medium . Where indicated , exogenous amino acids were added at 1mM final concentration . On the x - axis of the histograms is fluorescence per cell , while on the y - axis is the cell number . Peaks corresponding to cells in G1 with unreplicated ( 1N ) and cells in G2 and M phases with fully replicated ( 2N ) DNA are indicated . The percentage of cells with G1 DNA content ( % G1 ) from 3 independent measurements is shown in each case ( mean and sd ) .

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C, DNA content profiles of BAT1 and bat1Δ cells from asynchronous cultures, in SMM medium. Where indicated, exogenous amino acids were added at 1mM final concentration. On the x-axis of the histograms is fluorescence per cell, while on the y-axis is the cell number. Peaks corresponding to cells in G1 with unreplicated (1N) and cells in G2 and M phases with fully replicated (2N) DNA are indicated. The percentage of cells with G1 DNA content (%G1) from 3 independent measurements is shown in each case (mean and sd).


A , The indicated strains were spotted at 10 - fold serial dilutions on solid Synthetic Minimal Medium ( SMM ) agar plates . Rapamycin was added at 30 ng / mL , as shown in each case . The plates were incubated at 30 ° C and photographed after 5 and 7 days , as indicated .

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A, The indicated strains were spotted at 10-fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Rapamycin was added at 30 ng/mL, as shown in each case. The plates were incubated at 30 °C and photographed after 5 and 7 days, as indicated.


B , Immunoblots of total cell extracts from asynchronous BAT1 and bat1Δ cells , from four independent experiments . The signal from Pgk1 ( α - Pgk1 ) is shown on the blot at the bottom , and from phosphorylated Rps6 ( α - Rps6 - P ) is on the blot above , indicated for two exposures ( 2 min , top ; 20 sec , middle ) .

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B, Immunoblots of total cell extracts from asynchronous BAT1 and bat1Δ cells, from four independent experiments. The signal from Pgk1 (α-Pgk1) is shown on the blot at the bottom, and from phosphorylated Rps6 (α-Rps6-P) is on the blot above, indicated for two exposures (2 min, top; 20 sec, middle).


D , Exogenous addition of valine leads to sustained activation of TORC1 and phosphorylation of Rps6 in cells lacking Bat1 . Wild type ( BAT1 + ) and bat1 ∆ ( two independent isolates , # 4 , and # 5 ) strains were grown overnight in minimal ( SMM ) medium , diluted to 1E + 06 cells / mL in fresh SMM media containing the indicated amino acid ( at 1mM ) , and harvested when they reached 5E + 06 cells / mL . The levels of phosphorylated Rps6 ( α - Rps6 - P ) and Pgk1 ( α - Pgk1 ) are shown in immunoblots from total cell extracts in each case . The relative levels of Rps6 - P / Pgk1 are shown in each case at the bottom . The boxplot graphs were generated with R language functions . Each box is drawn from the first to the third quartile , with a horizontal line drawn in the middle to denote the median . The whiskers show the interquartile range ( IQR ) , and they were drawn at 1 . 5xIQR . The replicates were all biological ones .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Exogenous addition of valine leads to sustained activation of TORC1 and phosphorylation of Rps6 in cells lacking Bat1. Wild type (BAT1+) and bat1∆ (two independent isolates, #4, and #5) strains were grown overnight in minimal (SMM) medium, diluted to 1E+06 cells/mL in fresh SMM media containing the indicated amino acid (at 1mM), and harvested when they reached 5E+06 cells/mL. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in immunoblots from total cell extracts in each case. The relative levels of Rps6-P/Pgk1 are shown in each case at the bottom. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.


A , Immunoblots of total cell extracts from synchronous , elutriated wild - type ( CEN . PK ) cells in minimal ( SMM ) medium with exogenous Leu or Val added at 1mM immediately after elutriation . At the top , the percent of budded cells ( % B ) , cell size ( in fL ) , and time ( in min ) are indicated . The levels of phosphorylated Rps6 ( α - Rps6 - P ) and Pgk1 ( α - Pgk1 ) are shown in each case .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Immunoblots of total cell extracts from synchronous, elutriated wild-type (CEN.PK) cells in minimal (SMM) medium with exogenous Leu or Val added at 1mM immediately after elutriation. At the top, the percent of budded cells (%B), cell size (in fL), and time (in min) are indicated. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in each case.


B , Quantification of the levels of phosphorylated Rps6 and Pgk1 from independent experiments done as in A . The relative levels of each protein across the cell cycle is shown on the y - axis , as a function of cell size ( x - axis ) . Loess curves and the std errors at a 0 . 95 level are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Quantification of the levels of phosphorylated Rps6 and Pgk1 from independent experiments done as in A. The relative levels of each protein across the cell cycle is shown on the y-axis, as a function of cell size (x-axis). Loess curves and the std errors at a 0.95 level are shown.


B Western blot analysis of liver lysates ( 100 µg ) shows that intein - mediated protein trans - splicing efficiently reconstitutes full - length eGFP . Single AAV : n = 5 ; AAV - intein : n = 5 . Arrows indicate both full - length eGFP and excised inteins . C Quantification of eGFP protein bands . Values are reported as mean ± SEM . Each dot represents the eGFP protein band quantification from animals injected with either single AAV n = 5 or AAV intein n = 5 .

B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.


B Western blot analysis of lysates of HEK293 cells 72 hours post - transfection with the various F8 variants . Neg , non - transfected cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.


C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± SEM . Each dot within the same group corresponds to a biological replicate : Neg n = 5 ; Wild - type n = 8 ; N6 n = 8 ; SQ n = 7 ; V3 n = 6 . Significant differences between groups were assessed using The Kruskal - Wallis test followed by the posthoc analysis : Nemenyi ' s All - Pairs Rank Comparison Test . The Kruskal - Wallis test p value = 2 . 88e - 05 . * * indicates the significant difference between the N6 and the Wild - type groups : p value ≤ 0 . 01 . * indicates the significant difference between the V3 and the Wild - type groups : p value ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05.


A Western blot ( WB ) of protein lysates of HEK293 cells 72 hours post - transfection ( n = 3 ; biological replicates ) with either Npu inteins or Heterologous ( N - intein DnaB + C - intein DnaE ) split - inteins . I + II , N6 split - intein proteins ; I + II ( Het . ) , heterologous split - intein proteins ; I , 5 ' N6 coding sequence ( CDS ) - N - DnaE protein ; I ( N - int B ) , 5 ' N6 CDS - N - DnaB . II , C - DnaE - 3 ' N6 CDS protein . Excised inteins ( ∼ 12kDa ) are present only in the down part of the blot when I + II ( Npu inteins ) are provided . Arrows indicate the full - length N6 protein , single halves and excised inteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.


B WB of medium of the transfected cells showing the secreted proteins ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein as well as single halves and excised inteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.


C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± SEM . Each dot within each group represent a different biological replicate ( n ) : I + II n = 3 ; I + II ( Het . ) n = 3 ; I ( N - int E ) n = 3 ; I ( N - int B ) n = 3 ; II ( C - int E ) n = 3 ; Neg n = 4 . Significant differences between groups were assessed using Kruskal - Wallis rank sum test Kruskal − Wallis , p value = 0 . 013 . * indicates the significant difference between the I + II and the I ( N - int E ) groups : p value ≤ 0 . 05 . * * * indicates the significant difference between the I + II and the Neg groups : p value ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.


A Western blot ( WB ) of protein lysates of HEK293 cells 72 hpt with the AAV - N6 split - intein plasmids and with the codon - optimised set . I + II , N6 split - intein proteins ; I , 5 ' N6 CDS - N - DnaE protein ; II , C - DnaE - 3 ' N6 CDS protein . ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein , excised inteins and both single halves . Codop : codon - optimised .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.


B WB of medium from the transfected cells showing increased secretion of the Codop - N6 full - length protein compared to the non - codon - optimised . ( n = 3 ; biological replicates ) . Arrows indicate the full - length N6 protein , excised inteins and both single halves .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves.


C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units / deciliter ( IU / dl ) ( n = 3 ; biological replicates ) . Data are presented as mean ± SEM . Significant differences between groups were assessed using Kruskal - Wallis test p value = 0 . 027 . * indicates the significant difference between the I + II ( N6 split - intein ) and the II ( C - DnaE - 3 ' N6 CDS ) groups : p value ≤ 0 . 05 . * * indicates the significant difference between the Codop I + II and the I ( 5 ' N6 CDS - N - DnaE ) codop groups : p value ≤ 0 . 01 . * * indicates the significant difference between the Codop I + II and the II ( C - DnaE - 3 ' N6 CDS ) codop groups : p value ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01.


A Alkaline gel Southern blot analysis of AAV DNA . AAV DNA was hybridized to a probe specific for the HLP promoter . Neg , AAV DNA treated with Dnase I ; CodopV3 , AAV - CodopV3 ; I , AAV - 5 ' N6 - N - intein ; II , AAV - C - intein - 3 ' N6 .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.


B Chromogenic assay performed on plasma samples to detect F8 activity in AAV - treated mice compared to controls groups . Data are presented as mean ± SEM . The same animals were analysed at the various time - points . Each dot within the same group and within different groups of treatment corresponds to a single animal . The baseline includes all animals ( n = 20 ) before the treatment : CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . The statistical difference between groups has been assessed at 4weeks post - injection ( wpi ) with the One - way Anova p value = 0 . 056 ; at 8 wpi with the Kruskal - Wallis test p value = 0 . 68 ; at 12wpi with the Kruskal - Wallis test p value = 0 . 45 ; at 16wpi with the Kruskal - Wallis test p value = 0 . 58 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.


C Activated partial thromboplastin time ( aPTT ) assay performed on plasma samples both at the baseline and at the last time - point of the analysis ( 16 wpi ) . Data are presented as mean ± SEM . Each dot within each group of treatment corresponds to different animals : The baseline includes all animals before the treatment n = 21 ; CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . Significant differences between groups were assessed using the Kruskal - Wallis test . * * indicates the significant difference between the baseline and the N6 intein groups : p value ≤ 0 . 01 . * indicates the significant difference between the baseline and the CodopN6 intein groups : p value ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05.


D Tail - clip assay reported as bleeding time in seconds . Significant differences between groups were assessed using One - way Anova test p value is = 0 . 11 . Data are presented as mean ± SEM . Each dot corresponds to a different animal . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=


E Haemoglobin content measured as Optical Density ( OD ) at 416 nm after the tail - clip assay . Significant differences between groups were assessed using One - way Anova test . Data are reported as mean ± SEM . Each dot corresponds to a single animal . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n = 6 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.