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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 . ) .

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

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 ) .

O O O B-MEASURED_VAR 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 B-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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 . ) .

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

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 ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_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 O O O

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 .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 ) .

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

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 ) .

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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 ) .

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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 . ) .

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

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 . ) .

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

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

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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 =

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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 .

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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.


F Western blot analysis of liver lysates ( 100 µg ) from either CodopN6 intein - treated mice ( n = 6 ) or untreated haemophilic mice ( n = 2 ) . A lysate from HEK293 cells transfected with the N6 full - length plasmid was used as positive control ( 50 µg ) . Arrows point at N6 full - length protein ( N6 ) , the 5 ' half of N6 ( 5 ' N6 ) and 3 ' half of N6 ( 3 ' N6 ) .

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F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).


A The amount of anti - F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units / milliliter ( AU / ml ) . Each numbered bar represents a single mouse . CodopV3 , AAV - CodopV3 injected group n = 8 ; N6 intein , AAV - N6 intein injected group n = 5 ; CodopN6 intein , AAV - CodopN6 intein injected group n = 5 . Significant differences were assessed as follow : Paired T - test has been used for CodopV3 n = 5 : p value ≤ 0 . 001 ; Wilcoxon test for CodopV3 n = 3 : p value ≤ 0 . 05 ; Paired T - test has been used for N6 intein n = 5 : p value ≤ 0 . 05 ; Paired T - test has been used for CodopN6 intein n = 5 p value ≤ 0 . 01 .

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A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.


B Bethesda assay performed on plasma samples mice injected with AAV - CodopV3 ; on the plasma of animals injected with AAV - N6 intein n = 3 and the plasma of animals injected with AAV - CodopN6 intein n = 2 ; plasma of a wild - type animal was used as a control . The dotted line indicates the threshold above which anti F8 - antibodies are considered inhibitors .

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B Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.


C Pro - inflammatory cytokines levels followed over - time and reported as percentage of the baseline levels ( time point before the injection ) . Data are represented as mean ± SEM . Five animals belonging to the CodopV3 group were analysed . Significant differences were assessed using the Kruskal − Wallis test ; for IP - 10 : * indicates the p value ≤ 0 . 05 between 0 and 4 wpi ; * indicates the p value ≤ 0 . 05 between 0 and 8 wpi ; * indicates the p value ≤ 0 . 05 between 0 and 16 wpi . ( n = 20 ) ; for eotaxin : * indicates the p value ≤ 0 . 05 .

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C Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05.