Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment . The nuclear extracts of the frontal cortices and hippocampus from 8 - mon - old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1 , and were then conducted to HDAC1 activity assay . N = 6 mice per group . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . * p = 0 . 0203 ( WT vs Tg - Veh ) or 0 . 0408 ( Tg - Veh vs Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( %

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

Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8-mon-old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * p = 0.0203 (WT vs Tg-Veh) or 0.0408 (Tg-Veh vs Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%


Representative data of western blot for nuclear acetylated - histone 3 ( Lys 9 / 14 ) , total histone H3 , and quantification of protein levels . N = 6 mice per group . * p = 0 . 0105 or * * * p = 0 . 0008 by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Representative data of western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * p = 0.0105 or *** p = 0.0008 by multiple comparison. Data information: All data are presented as mean ± SEM (%)


Representative data of western blot for nuclear HDAC1 levels and semi - quantification of the expression levels . N = 6 mice per group . * * p = 0 . 0022 ( Tg ) / 0 . 0012 ( Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Representative data of western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** p = 0.0022 (Tg)/ 0.0012 (Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%)


Left panel : representative data of western blot for cell cycle - and DNA damage - related proteins . Right histogram : semi - quantification of protein levels . N = 6 per group . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by multiple t - test with FDR correction , Q = 1 % . * * P ≤ 0 . 01 ; # # # P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 . Data information : All data are presented as mean ± SEM ( % )

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Left panel: representative data of western blot for cell cycle- and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by multiple t-test with FDR correction, Q = 1%. **P ≤ 0.01; ###P ≤ 0.001 and ****/####P ≤ 0.0001. Data information: All data are presented as mean ± SEM (%)


IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8 - mon - old of WT + Vehicle , Tg + Vehicle , Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0421 or * * * * p < 0 . 0001 by multiple comparison . Data information : number of cells in each view is presented .

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IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented.


Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 8 - mon - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0414 or * * * p = 0 . 0006 by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 8-mon-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm.* p = 0.0414 or *** p = 0.0006 by multiple comparison. Data information: All data are presented as mean ± SEM (%)


IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 12 - month - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * * p = 0 . 002 and * * * * p < 0 . 0001 by multiple comparison . Data information : where number of cells in each view is presented .

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IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 12-month-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. ** p = 0.002 and **** p < 0.0001 by multiple comparison. Data information: where number of cells in each view is presented.


Representative IF staining of TDP - 43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD - TDP . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

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Representative IF staining of TDP-43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD-TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells with co - mislocalized HDAC1 and TDP - 43 from each view of microscope . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of cells with co-mislocalized HDAC1 and TDP-43 from each view of microscope. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Linear regression analysis of cells with HDAC1 mislocalization and TDP - 43 proteinopathies . Total cell counts : 3000 per samples . P = 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Linear regression analysis of cells with HDAC1 mislocalization and TDP-43 proteinopathies. Total cell counts: 3000 per samples. P = 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD - TDP from each view of microscope . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

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Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD-TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells with γH2AX and Ki67 immunoreactivity . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Linear regression analysis of cells with DNA damage and aberrant cell cycle activity . Cell counts : 40 - 113 ( normal ) and 418 - 887 ( FTLD ) per samples . p < 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Cell counts: 40 -113 (normal) and 418-887 (FTLD) per samples. p < 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Dot - blot of HDAC1 and TDP - 43 in urea - soluble fractions from the frontal cortex of normal individuals and patients with FTLD - TDP .

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Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP.


Quantification of HDAC1 and TDP - 43 expression levels in urea - soluble fractions . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


( A ) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction . Phospho - to total proteomic changes were quantified from three independent biological replicates of Dox treated vs . untreated cells , via TMT ( Dataset EV8 ) . 872 phosphorylations were found to be significantly increased , while 82 phosphorylations were significantly decreased ( FDR < 0 . 05 ) . Significantly changing phosphorylations on RBPs whose RNA - binding activity was significantly increased ( red ) or decreased ( blue ) as per Fig 1C are highlighted on the plot .

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(A) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction. Phospho- to total proteomic changes were quantified from three independent biological replicates of Dox treated vs. untreated cells, via TMT (Dataset EV8). 872 phosphorylations were found to be significantly increased, while 82 phosphorylations were significantly decreased (FDR < 0.05). Significantly changing phosphorylations on RBPs whose RNA-binding activity was significantly increased (red) or decreased (blue) as per Fig 1C are highlighted on the plot.


( E ) KrasG12D induction enhances phosphorylation of CK2α ( T360 / S362 ) , in an Erk1 / 2 - dependent manner . IKras PDAC cells were grown in the absence of Dox for 48 hrs , before its addition to the indicated cells , with or without Trametinib ( 10 nM ) , for a further 24 hrs . Cells were then lysed and analyzed by immunoblotting ( IB ) with the indicated antibodies . The red triangle marks the main phospho band which overlaps with the total CK2α .

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(E) KrasG12D induction enhances phosphorylation of CK2α (T360/S362), in an Erk1/2-dependent manner. IKras PDAC cells were grown in the absence of Dox for 48 hrs, before its addition to the indicated cells, with or without Trametinib (10 nM), for a further 24 hrs. Cells were then lysed and analyzed by immunoblotting (IB) with the indicated antibodies. The red triangle marks the main phospho band which overlaps with the total CK2α.


( G ) Analysis of the RNA - binding activity of WT , Phospho - defective ( S4A ) , and phospho - mimicking ( S4D ) mutants of Ncl by OOPS . IKras PDAC cells were transiently transfected with myc - tagged WT , S4A , and S4D Ncl constructs . Cells were subsequently grown in the absence of Dox for 48 hrs , followed by Dox addition for 24 hrs to induce KrasG12D expression . Cells were then subjected to OOPS to isolate the interface ( RNA - bound proteins ) , or whole cell lysis ( total lysate ) , followed by immunoblotting with anti - Ncl antibody . For comparison , Gapdh , which also binds RNA but does not show a change in its RNA - binding activity ( Dataset EV1 ) , was also blotted for . A fraction of each interface or total lysate sample was also subjected to RNA extraction , which was resolved and quantified by capillary electrophoresis ( CE ) as loading control .

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(G) Analysis of the RNA-binding activity of WT, Phospho-defective (S4A), and phospho-mimicking (S4D) mutants of Ncl by OOPS. IKras PDAC cells were transiently transfected with myc-tagged WT, S4A, and S4D Ncl constructs. Cells were subsequently grown in the absence of Dox for 48 hrs, followed by Dox addition for 24 hrs to induce KrasG12D expression. Cells were then subjected to OOPS to isolate the interface (RNA-bound proteins), or whole cell lysis (total lysate), followed by immunoblotting with anti-Ncl antibody. For comparison, Gapdh, which also binds RNA but does not show a change in its RNA-binding activity (Dataset EV1), was also blotted for. A fraction of each interface or total lysate sample was also subjected to RNA extraction, which was resolved and quantified by capillary electrophoresis (CE) as loading control.


( B ) Immunofluorescence analysis of WT , S4A , and S4D Myc - Ncl subcellular localization . IKras PDAC cells ectopically expressing either WT , S4A , or S4D Myc - Ncl were fixed and immunostained with anti - Myc - tag antibody , along with anti - Fibrillarin ( Fbl ) antibody as a Nucleolar marker , and Hoechst , followed by confocal microscopy analysis .

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(B) Immunofluorescence analysis of WT, S4A, and S4D Myc-Ncl subcellular localization. IKras PDAC cells ectopically expressing either WT, S4A, or S4D Myc-Ncl were fixed and immunostained with anti-Myc-tag antibody, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker, and Hoechst, followed by confocal microscopy analysis.


( C ) Distribution of WT , S4A , and S4D Myc - Ncl crosslink sites across the annotated 47S pre - rRNA genomic region . Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments .

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(C) Distribution of WT, S4A, and S4D Myc-Ncl crosslink sites across the annotated 47S pre-rRNA genomic region. Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments.


( D ) Quantification of the percentage of crosslinks in each region of the 47S pre - rRNA for WT , S4A , and S4D Myc - Ncl . Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments . ( E ) Quantification of the proportional density of crosslinks in each region of the 47S pre - rRNA for WT , S4A , and S4D Myc - Ncl . Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region .

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(D) Quantification of the percentage of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments. (E) Quantification of the proportional density of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region.


( A ) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , grown for 48hrs in presence or absence of Dox , prior to pulse labeling with FUrd . Cells were then fixed and immunostained with anti - FUrd antibody ( green ) to visualize nascent RNA , along with anti - Fibrillarin ( Fbl ) antibody as a Nucleolar marker ( red ) , and Hoechst ( blue ) as the Nuclear stain , followed by confocal microscopy analysis . ( B ) Quantification of Nucleolar FUrd levels in images from ( A ) . FUrd fluorescence densities in single nucleoli were quantified from 182 - 220 individual cells per condition , combined from 2 independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) .

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(A) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, grown for 48hrs in presence or absence of Dox, prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody (green) to visualize nascent RNA, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 182-220 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).


( C ) RT - qPCR analysis of 5 ' ETS - containing pre - rRNA transcript levels in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , and grown for 48hrs in presence or absence of Dox , before RT - qPCR analysis with a specific probe against the mouse 5 ' ETS region . A probe against mouse Actb mRNA was used as loading control for normalization . A total of 4 biological replicate experiments were quantified ( * * * : P < 0 . 001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) .

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(C) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 4 biological replicate experiments were quantified (***: P < 0.001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).


( E ) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D , by puromycinylation . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , and grown for 48hrs in presence or absence of Dox , before pulse labelling with Puromycin ( 10 μg / ml ) for 15 min to label nascent proteins . Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies . ( F ) Quantification of puromycin incorporation from ( E ) , as an indicator of the overall protein synthesis rate . Total Erk1 / 2 levels were used as loading control . A total of 4 independent biological replicate experiments were quantified . Error bars depict SD . ( * * : P < 0 . 01 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . (

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(E) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D, by puromycinylation. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before pulse labelling with Puromycin (10 μg/ml) for 15 min to label nascent proteins. Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies. (F) Quantification of puromycin incorporation from (E), as an indicator of the overall protein synthesis rate. Total Erk1/2 levels were used as loading control. A total of 4 independent biological replicate experiments were quantified. Error bars depict SD. (**: P < 0.01; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). (


( G ) Nascent RNA imaging of WT , S4A , and S4D Myc - Ncl expressing iKras PDAC cells , in presence or absence of KrasG12D . Vectors encoding Myc - tagged WT , S4A , and S4D Ncl were transiently transfected into iKras PDAC cells , before reseeding and growing the cells for 48hrs in presence or absence of Dox . Cells were then subjected to pulse labeling with FUrd , fixation , and immunostaining with anti - FUrd antibody ( green ) , anti - Myc - tag antibody ( red ) , and Hoechst ( blue ) , followed by confocal microscopy analysis . Scale bar = 10 µm . ( H ) Quantification of FUrd levels in Myc - positive nucleoli from ( E ) . FUrd fluorescence densities in single nucleoli were quantified from 160 - 281 individual cells per condition , combined from three independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) . (

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(G) Nascent RNA imaging of WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox. Cells were then subjected to pulse labeling with FUrd, fixation, and immunostaining with anti-FUrd antibody (green), anti-Myc-tag antibody (red), and Hoechst (blue), followed by confocal microscopy analysis. Scale bar = 10 µm. (H) Quantification of FUrd levels in Myc-positive nucleoli from (E). FUrd fluorescence densities in single nucleoli were quantified from 160-281 individual cells per condition, combined from three independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). (


( I ) RT - qPCR analysis of 5 ' ETS - containing pre - rRNA transcript levels in WT , S4A , and S4D Myc - Ncl expressing iKras PDAC cells , in presence or absence of KrasG12D . Vectors encoding Myc - tagged WT , S4A , and S4D Ncl were transiently transfected into iKras PDAC cells , before reseeding and growing the cells for 48hrs in presence or absence of Dox , followed by RT - qPCR analysis with a specific probe against the mouse 5 ' ETS region . A probe against mouse Actb mRNA was used as loading control for normalization . A total of 5 to 8 biological replicate experiments per condition were quantified ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) .

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(I) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox, followed by RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 5 to 8 biological replicate experiments per condition were quantified (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).


( A ) Colony formation of control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , followed by clonogenic assay for 7 days in presence or absence of Dox . Colonies were visualized by Crystal Violet staining . ( B ) Quantification of Crystal Violet staining levels from ( A ) . A total of 6 biological replicate experiments were quantified . Error bars depict SD . ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from two way ANOVA with Šídák ' s multiple comparisons test ) . (

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(A) Colony formation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, followed by clonogenic assay for 7 days in presence or absence of Dox. Colonies were visualized by Crystal Violet staining. (B) Quantification of Crystal Violet staining levels from (A). A total of 6 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (


( C ) 3D proliferation of control and Ncl depleted iKras PDAC cells , in presence or absence of KrasG12D . Cells were transfected with a non - targeting control siRNA , or two independent siRNAs against Ncl , before being reseeded onto 3D Collagen - I gels , with or without Dox , and allowed to grow for 48 hrs . Cells were subsequently imaged live by phase contrast microscopy . Scale bar = 200 µm . ( D ) Analysis of the relative percentage of viable cells in 3D collagen - I cultures from ( C ) . Cells were subjected to luminescence - based viability assay by CellTiter - Glo to quantify the percentage of viable cells . A total of 3 biological replicate experiments were quantified . Error bars depict SD . ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from two way ANOVA with Šídák ' s multiple comparisons test ) . (

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(C) 3D proliferation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, before being reseeded onto 3D Collagen-I gels, with or without Dox, and allowed to grow for 48 hrs. Cells were subsequently imaged live by phase contrast microscopy. Scale bar = 200 µm. (D) Analysis of the relative percentage of viable cells in 3D collagen-I cultures from (C). Cells were subjected to luminescence-based viability assay by CellTiter-Glo to quantify the percentage of viable cells. A total of 3 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (


( A ) Dose response analysis of CX - 5461 impact on nascent rRNA expression . IKras PDAC cells were grown in the absence of Dox for 48hrs . Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression , followed by pre - treatment with the indicated concentrations of CX - 5461 for 30 mins prior to pulse labeling with FUrd . Cells were then fixed and immunostained with anti - FUrd antibody to visualize nascent RNA ( green ) , anti - Ncl antibody to reveal the Nucleolus ( red ) , and Hoechst ( blue ) as the Nuclear stain , followed by confocal microscopy analysis . ( B ) Quantification of Nucleolar FUrd levels in images from ( A ) . FUrd fluorescence densities in single nucleoli were quantified from 251 - 479 individual cells per condition , combined from 2 independent biological replicate experiments ( * * * * : P < 0 . 0001 ; n . s . : not significant - calculated from one - way ANOVA with Šídák ' s multiple comparisons test ) .

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 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

(A) Dose response analysis of CX-5461 impact on nascent rRNA expression. IKras PDAC cells were grown in the absence of Dox for 48hrs. Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression, followed by pre-treatment with the indicated concentrations of CX-5461 for 30 mins prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody to visualize nascent RNA (green), anti-Ncl antibody to reveal the Nucleolus (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 251-479 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).


( G ) MRI imaging of orthotopic iKras tumors in untreated or CX - 5461 treated mice . IKras PDAC cells were engrafted into the pancreas of Dox - fed nude mice and allowed to form tumors for 4 days . Animals were then divided into two groups , with the first group treated by daily oral administration of CX - 5461 ( 50mg / kg ) for a further 10 days , whilst the second group was left untreated for the same period . T2 scans were taken on the indicated days , post - engraftment . Green areas mark the tumors .

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(G) MRI imaging of orthotopic iKras tumors in untreated or CX-5461 treated mice. IKras PDAC cells were engrafted into the pancreas of Dox-fed nude mice and allowed to form tumors for 4 days. Animals were then divided into two groups, with the first group treated by daily oral administration of CX-5461 (50mg/kg) for a further 10 days, whilst the second group was left untreated for the same period. T2 scans were taken on the indicated days, post-engraftment. Green areas mark the tumors.


( K ) H & E analysis of the extracted pancreas tissues from ( I ) . Representative pancreatic tissue images from the untreated and CX - 5461 treated mice , showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated , but a largely normal pancreas histology with a small malignant ( M ) component ( marked by the dashed line ) in the treated mice .

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(K) H&E analysis of the extracted pancreas tissues from (I). Representative pancreatic tissue images from the untreated and CX-5461 treated mice, showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated, but a largely normal pancreas histology with a small malignant (M) component (marked by the dashed line) in the treated mice.


B . The truncated receptor encoded by RNA1 is endogenously expressed . HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C ( RNA1 ) . SDS - lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 ( Figure EV1 ) . Mousehypothalamus was exposed 10 times longer . In brain , a band around 75kDa of unknown origin is detected by the antiserum ( indicated by a star ) .

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B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).


D . RT - PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition . Below is a quantification of three independent experiments . A statistical evaluation showed significant differences between oligo # 5 ( p = 0 . 00008 ) and oligo # 5 - 3 ( p = 0 . 0019 ) and control .

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D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.


A . Titration of oligo # 5 . An increasing amount of oligo # 5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT - PCR . - RT : negative control without reverse transcriptase . B . Quantification of three independent experiments . The half - maximal effect was seen at 13 . 1 nM concentration .

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A. Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT-PCR. -RT: negative control without reverse transcriptase.B. Quantification of three independent experiments. The half-maximal effect was seen at 13.1 nM concentration.


C . Influence of oligo # 5 shortening on alternative splicing of the serotonin 2C receptor . Oligo # 5 : 5 ' AGU AUU GAG CAU AGC CGC 3 ' ( 18mer ) ; Oligo # 5 - 3 : 5 ' GAG CAU AGC CGC 3 ' ( 12mer ) ; Oligo # 5 - 10 : 5 ' G CAU AGC CGC 3 ' ( 10mer ) were used in transfection assays analyzed by RT - PCR . D . Statistical analysis of the effect of oligonucleotide shortening . The effect of all oligos testes is significant , oligo # 5 : p = 0 . 0015 ; oligo # 5 - 3 : p = 0 . 031 ; oligo # 5 - 10 : p = 0 . 048 , n = 6 .

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C. Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5' AGU AUU GAG CAU AGC CGC 3' (18mer); Oligo#5-3: 5' GAG CAU AGC CGC 3' (12mer); Oligo#5-10: 5' G CAU AGC CGC 3' (10mer) were used in transfection assays analyzed by RT-PCR.D. Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: p= 0.0015; oligo#5-3: p=0.031; oligo#5-10: p= 0.048, n=6.


E . Influence of cy3 label on the 5 ' and 3 ' end of oligo # 5 . Using a phosphate linker , Cy3 was added to the 5 ' or 3 ' phosphate , respectively and the oligonucleotides assayed by RT - PCR . Oligo # 5 with cy3 at the 5 ' end showed 86 % inclusion , with Cy3 at the 3 ' end 17 % exon inclusion . F . Statistical analysis of the effect of oligonucleotide modification . The effect is significant , p = 0 . 009 , n = 4 .

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E. Influence of cy3 label on the 5' and 3' end of oligo#5. Using a phosphate linker, Cy3 was added to the 5' or 3' phosphate, respectively and the oligonucleotides assayed by RT-PCR. Oligo#5 with cy3 at the 5' end showed 86% inclusion, with Cy3 at the 3' end 17% exon inclusion.F. Statistical analysis of the effect of oligonucleotide modification. The effect is significant, p=0.009, n=4.


D . In vitro splicing assay : Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract ( NE ) under splicing conditions for two hours at 30 ºC . The structures of the in vitro splicing products are shown on the right . LI and LII are two lariats formed . Numbers indicate the expected product lengths . E . Quantification of selected bands from four independent experiments . The percentage of each form in the total products is shown . The differences of splice products ( Va + b - MINX ) is significant , p = 0 . 0055 ; n = 3 .

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D. In vitro splicing assay: Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract (NE) under splicing conditions for two hours at 30 ºC. The structures of the in vitro splicing products are shown on the right. LI and LII are two lariats formed. Numbers indicate the expected product lengths.E. Quantification of selected bands from four independent experiments. The percentage of each form in the total products is shown. The differences of splice products (Va+b-MINX) is significant, p=0.0055; n=3.


B . Effect of oligo # 5 on pre - mRNA splicing of the reporter gene , stably transfected into HeLa cells .

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B. Effect of oligo#5 on pre-mRNA splicing of the reporter gene, stably transfected into HeLa cells.


C . Effect of oligo # 5 on the truncated 5HT2C protein . Stable cell lines expressing the construct shown in panel ( A ) were treated with oligo # 3 and oligo # 5 . After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant . The fractions were analyzed by Western Blot using the anti - RNA1 antiserum ( Figure EV1 ) . The protein samples were on the same membrane , but the supernatant fractions were exposed about ten times longer . D . Effect of oligo # 5 on the full - length 5HT2C protein . The protein was prepared as in panel ( C ) and analyzed using a polyclonal anti - GFP antiserum . E . Quantification of panels C and D from three independent experiments . The ratio of protein signal after oligo # 3 and oligo # 5 treatment is shown for RNA1 ( p = 0 . 002 ) and RNA2 ( p = 0 . 0003 ) , n = 3 , respectively .

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C. Effect of oligo#5 on the truncated 5HT2C protein. Stable cell lines expressing the construct shown in panel (A) were treated with oligo#3 and oligo#5. After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant. The fractions were analyzed by Western Blot using the anti-RNA1 antiserum (Figure EV1). The protein samples were on the same membrane, but the supernatant fractions were exposed about ten times longer.D. Effect of oligo#5 on the full-length 5HT2C protein. The protein was prepared as in panel (C) and analyzed using a polyclonal anti-GFP antiserum.E. Quantification of panels C and D from three independent experiments. The ratio of protein signal after oligo#3 and oligo#5 treatment is shown for RNA1 (p=0.002) and RNA2 (p=0.0003), n=3, respectively.


F , G . Effect of oligo # 5 on localization of the EGFP - tagged serotonin 2C receptor . Confocal microscopy images of three representative pictures are shown . H . Quantitification of GFP signal found on the membrane after oligo # 5 addition . The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image . 200 cells from three individual experiments were analyzed , p < 0 . 008 , n = 200 .

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F, G. Effect of oligo#5 on localization of the EGFP-tagged serotonin 2C receptor. Confocal microscopy images of three representative pictures are shown.H. Quantitification of GFP signal found on the membrane after oligo#5 addition. The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image. 200 cells from three individual experiments were analyzed, p<0.008, n=200.


A . Accumulation of Cy3 - labeled oligo # 5 in the area around the 3rd ventricle one hour post injection , shown is the ventral part of a coronal section - 1 . 34 mm from Bregma . The injection coordinates were AP : - 1 . 82 mm , DV : - 4 . 5 mm , ML : 0 . B . Uptake of oligo # 5 in the arcuate nucleus region ( arc ) , enlarged from panel A .

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A. Accumulation of Cy3-labeled oligo#5 in the area around the 3rd ventricle one hour post injection, shown is the ventral part of a coronal section -1.34 mm from Bregma. The injection coordinates were AP: -1.82 mm, DV: -4.5 mm, ML: 0.B. Uptake of oligo#5 in the arcuate nucleus region (arc), enlarged from panel A.


C . Oligo # 5 enters neurons . Enlargement of hypothalamus region taking up oligo # 5 ( red ) , stained for the neuronal marker NeuN ( green ) . Most of the oligo # 5 signal is in neurons , but some is also in NeuN negative cells ( arrow ) , likely glia . D . Oligo # 5 enters some glia cells . Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo # 5 injection . The oligo ( red ) can be found in some GFAP - positive glia cells ( arrow ) , but numerous GFAP - positive cells ( green ) show no oligo # 5 uptake .

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C. Oligo#5 enters neurons. Enlargement of hypothalamus region taking up oligo#5 (red), stained for the neuronal marker NeuN (green). Most of the oligo#5 signal is in neurons, but some is also in NeuN negative cells (arrow), likely glia.D. Oligo#5 enters some glia cells. Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo#5 injection. The oligo (red) can be found in some GFAP-positive glia cells (arrow), but numerous GFAP-positive cells (green) show no oligo#5 uptake.


E . Effect of oligo # 5 on RNA1 / RNA2 ratios in the injected area . The area marked by Cy3 staining in the dorsal hypothalamus , consisting mainly of the arcuate nucleus was microdissected , RNA isolated and analyzed by qPCR . NS : non - significant changes ( 3 hrs : p = 0 . 01 ; 6 hrs : p = 0 . 000001 ; 9 hrs : p = 0 . 02 ; 12 hrs : p = 0 . 06 ; n = 3 )

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E. Effect of oligo#5 on RNA1/RNA2 ratios in the injected area. The area marked by Cy3 staining in the dorsal hypothalamus, consisting mainly of the arcuate nucleus was microdissected, RNA isolated and analyzed by qPCR. NS: non-significant changes (3 hrs: p=0.01; 6 hrs: p=0.000001; 9 hrs: p=0.02; 12 hrs: p=0.06; n=3)


F . Oligo # 5 does not change the RNA2 - editing pattern . The hypothalamic area showing oligo # 5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing . The percent of non - edited reads in total reads is shown ( at least 20 , 000 reads per lane ) .

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F. Oligo#5 does not change the RNA2-editing pattern. The hypothalamic area showing oligo#5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing. The percent of non-edited reads in total reads is shown (at least 20,000 reads per lane).


G . Effect of oligo # 5 on POMC expression . The RNA from panel D was analyzed using qPCR with primers against POMC , normalized to GAPDH ( 3 hrs : p = 0 . 0009 ; 6 hrs : p = 0 . 02 ; 9 hrs : p = 0 . 1 ; 12 hrs : p = 0 . 3 , n = 3 ) .

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G. Effect of oligo#5 on POMC expression. The RNA from panel D was analyzed using qPCR with primers against POMC, normalized to GAPDH (3 hrs: p=0.0009; 6 hrs: p=0.02; 9 hrs: p=0.1; 12 hrs: p=0.3, n=3).


H . Effect of oligo # 5 on food uptake in wild - type mice . C57BL / 6 wild - type mice received a guide canulae and did not have access to food for 16 hours . After injection of 2 µg oligonucleotides , they could freely access food . The food consumption was measured by weighing the food used . A total of 52 animals were used , p = 0 . 001 for the difference between oligo # 5 and control oligonucleotides . Each of the six individual experiments showed statistically significant differences between oligo # 5 and control ( p < 0 . 01 - 0 . 001 ) . Control oligo : an oligonucleotide against human SMN2 .

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H. Effect of oligo#5 on food uptake in wild-type mice. C57BL/6 wild-type mice received a guide canulae and did not have access to food for 16 hours. After injection of 2 µg oligonucleotides, they could freely access food. The food consumption was measured by weighing the food used. A total of 52 animals were used, p=0.001 for the difference between oligo#5 and control oligonucleotides. Each of the six individual experiments showed statistically significant differences between oligo#5 and control (p<0.01 - 0.001). Control oligo: an oligonucleotide against human SMN2.


I . Effect of oligo # 5 on food uptake in ob / ob mice . Ob / obmice received oligo # 5 through ICV injection , similar to panel F . However , the mice could freely access food at all times ( p = 0 . 04 , n = 3 ) .

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I. Effect of oligo#5 on food uptake in ob/ob mice. Ob/obmice received oligo#5 through ICV injection, similar to panel F. However, the mice could freely access food at all times (p=0.04, n=3).


A . 50 µg of cy3 - labelled - oligo # 5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected . ChP : choroid plexus , RSGc : retrosplenial granular cx , c , 3V : 3rd ventricle ; D3V : dorsal 3rd ventricle .

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A. 50 µg of cy3-labelled-oligo#5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected. ChP: choroid plexus, RSGc: retrosplenial granular cx,c, 3V: 3rd ventricle; D3V: dorsal 3rd ventricle.


B . Change of RNA2 / RNA1 ratios after carotid artery injection . The area around the arcuate nucleus was microdissected at the times indicated post injection . The ration of RNA1 / RNA2 was determined using real - time PCR . Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs : p = 0 . 3 ; 4 hrs : p = 0 . 003 , 8 hrs : p = 0 . 0006 , n = 5 )

B. Change of RNA2/RNA1 ratios after carotid artery injection.The area around the arcuate nucleus was microdissected at the times indicated post injection. The ration of RNA1/RNA2 was determined using real-time PCR. Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs: p=0.3; 4 hrs: p=0.003, 8 hrs: p=0.0006, n=5)


C . Effect of oligo # 5 delivered through a carotid artery catheter on food uptake in wild - type mice . C57BL / 6 wild - type mice with a pre - implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated .

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C. Effect of oligo#5 delivered through a carotid artery catheter on food uptake in wild-type mice. C57BL/6 wild-type mice with a pre-implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated.


D . Long term effect of oligo # 5 action . Food intake was measured for 70 hrs after one injection of 100 µg oligo # 5 through a carotid catheter at time zero .

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D. Long term effect of oligo#5 action. Food intake was measured for 70 hrs after one injection of 100 µg oligo#5 through a carotid catheter at time zero.


( B ) SEC - MALLS traces of different TRIM25

(B) SEC-MALLS traces of different TRIM25 and


( C ) TRIM32 constructs . The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve . The constructs were analysed over different concentration ranges and the data are reported for : T25R , 4 mg / mL ; T25RB1 , 3 . 2 mg / mL ; T25RB1B2 , 3 . 6 mg / mL ; T25RBCC , 0 . 5 mg / mL ; T32R , 3 mg / mL ; T32RB , 4 mg / mL ; T32RBCC , 4 mg / mL .

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(C) TRIM32 constructs. The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges and the data are reported for: T25R, 4 mg/mL; T25RB1, 3.2 mg/mL; T25RB1B2, 3.6 mg/mL; T25RBCC, 0.5 mg/mL; T32R, 3 mg/mL; T32RB, 4 mg/mL; T32RBCC, 4 mg/mL.


( A , C ) UBE2D3 ~ Ub discharge assays with different TRIM25 and TRIM32 constructs , respectively . Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min . The asterisk indicates the band for the TRIM construct .

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(A, C) UBE2D3~Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct.


( B , D ) Quantification of the discharge assays using UBE2D1 ~ UbAtto and TRIM25 or TRIM32 , respectively . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental triplicates ( ± s . d . ) .

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(B, D) Quantification of the discharge assays using UBE2D1~UbAtto and TRIM25 or TRIM32, respectively. The loss of UBE2D1~UbAtto is plotted as the average of experimental triplicates (± s.d.).


( E , G ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 and TRIM25 and TRIM32 , respectively . Reactions were incubated for 30 minutes and samples taken at indicated times . The asterisk indicates the band for the TRIM construct .

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(E, G) K63 poly-ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 minutes and samples taken at indicated times. The asterisk indicates the band for the TRIM construct.


( F , H ) Assays were quantified ( see Materials and Methods ) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32 , respectively . Assays were carried out in triplicate .

(F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate.


( A ) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2 + ions as grey spheres .

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(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.


( B ) Overlap of the RING dimers of TRIM32 ( cyan , 5FEY . pdb ) , TRIM25 ( yellow , 5FER . pdb ) and TRIM5 ( orange , 4TKP . pdb ) . The structures were overlapped on the circled RING domain . This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs .

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(B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.


( C ) Structure of the TRIM25 RING / E2 ~ Ub complex with the RING domains in the same colour scheme as in ( A ) , UBE2D1 in grey and the ubiquitin molecules in salmon and red .

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(C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.


( A ) Close - up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices ( left ) . Close - up of the interface between each RING monomer and the proximal ubiquitin ( right ) .

(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right).


( B ) UBE2D1 ~ UbAtto discharge assays with TRIM25 wild - type RING , the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin . Time point zero for the T25 - R Linker and T25 - R Linker V72R samples was taken before the addition of E3 as discharge is very fast . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental duplicates ( ± s . d . ) .

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(B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.).


( C ) UBE2D3 ~ Ub discharge assays with the same mutants as in ( B ) , stained with InstantBlue . ( D ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 . The asterisk indicates the band for the TRIM construct .

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(C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.


( A ) Role of E10 in the RING of TRIM25 ( blue ) in stabilizing the closed E2 ~ Ub conformation by contacting K11 from the proximal ubiquitin ( salmon ) and N71 of the opposite RING ( cyan ) which in turn contacts K33 of the same ubiquitin .

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(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.


( B ) E2 ~ Ub discharge and K63 poly - ubiquitination assays to test the role of E9 and E10 in TRIM25 activity . Substitution of Glu9 with Arg has no significant effect on activity , whereas the E10R mutation almost completely abolishes catalytic activity .

(B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.


( C ) Discharge and K63 poly - ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R . Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved .

(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.


( D and E ) Quantification of UBE2D1 ~ UbAtto discharge assays . The loss of E2 ~ Ub is plotted as the average of experimental duplicates ( ± s . d . ) .

(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).


( G ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( H ) Quantification of ∆ FosB + nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation , with different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the right column . Scale bar , 25μM . ( J ) Quantification of ∆ FosB + nuclei in cultured MSNs after different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM. (J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( K ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons ( Gad67Cre ; Alk4fl / fl ) and control mice ( Alk4fl / fl ) after exposure to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( L ) Quantification of ∆ FosB + nuclei in cultured MSNs from mutant and control mice after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( A ) Western blot analysis of Protein Kinase A phospho - substrates ( PKA Ⓟ - subs ) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with forskolin ( Fsk ) in the absence or presence of the PKA inhibitor PKI14 - 22 ( PKI ) . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . Lower blot shows the total amount of PCBP1 in the cell lysates . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(A) Western blot analysis of Protein Kinase A phospho-substrates (PKA Ⓟ-subs) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin (Fsk) in the absence or presence of the PKA inhibitor PKI14-22 (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot shows the total amount of PCBP1 in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( B ) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) , PKA inhibitor ( PKI ) and the ALK4 inhibitor SB431542 ( SB ) as indicated . Blots of nuclear and cytosolic fractions were reprobed with anti - PCNA and anti - αTubulin ( αTub ) antibodies , respectively . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle ( first column ) normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(B) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk), PKA inhibitor (PKI) and the ALK4 inhibitor SB431542 (SB) as indicated. Blots of nuclear and cytosolic fractions were reprobed with anti-PCNA and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( C ) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) or their combination as indicated . The Act + Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . The lower blot shows unaltered Smad3 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of Smad3 / PCBP1 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to PCBP1 levels . ( D ) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel ( C ) . The lower blot shows unaltered PCBP1 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of PCBP1 / Smad3 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to Smad3 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(C) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control. Reprobing with anti-PCBP1 antibodies is shown in the middle blot. The lower blot shows unaltered Smad3 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1 levels. (D) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel (C). The lower blot shows unaltered PCBP1 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation (coIP) over vehicle (first column) normalized to Smad3 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( E ) Representative immunofluorescence staining of Smad3 ( green ) and PCBP1 ( red ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM .

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(E) Representative immunofluorescence staining of Smad3 (green) and PCBP1 (red) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.


( F , G ) Quantification of PCBP1 + ( F ) or Smad3 + ( G ) nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying PCBP1 ( F ) or Smad3 ( G ) in the nucleus . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(F, G) Quantification of PCBP1+ (F) or Smad3+ (G) nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying PCBP1 (F) or Smad3 (G) in the nucleus. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( H ) Quantification of PCBP1 / Smad3 co - localization in nuclei of cultured MSNs using conditions identical to panel ( F ) . Results are expressed as average ± SEM ( biological replicates ) of % of MAP2 + cells displaying co - localization of PCBP1 and Smad3 in the nucleus . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(H) Quantification of PCBP1/Smad3 co-localization in nuclei of cultured MSNs using conditions identical to panel (F). Results are expressed as average ± SEM (biological replicates) of % of MAP2+ cells displaying co-localization of PCBP1 and Smad3 in the nucleus. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) RT - qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated ( same treatment in both treatment days ) . Results are expressed as average ± SEM of fold change over vehicle . No significant differences were found by one - way ANOVA followed with Tukey ' s multiple comparisons test ( p ≥ 0 . 05 ; N = 4 ) . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(I) RT-qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated (same treatment in both treatment days). Results are expressed as average ± SEM of fold change over vehicle. No significant differences were found by one-way ANOVA followed with Tukey's multiple comparisons test (p≥0.05; N=4). Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( J ) Representative images of PLA ( red ) between Smad3 and PCBP1 ( S3 / PCBP1 ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) , PKA inhibitor ( PKI ) or SB431542 ( SB ) as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( K ) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of PLA dots per cell . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(J) Representative images of PLA (red) between Smad3 and PCBP1 (S3/PCBP1) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF), PKA inhibitor (PKI) or SB431542 (SB) as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of PLA dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( E , F ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in HeLa cells after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in cultured MSNs exposed to consecutive stimulation as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( H , I ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative immunofluorescence staining of ∆ FosB ( red ) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with green fluorescent protein ( GFP , expressed by the lentiviruses ) and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( E ) Quantification of ∆ FosB + nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments . Results are expressed as average ± SEM of % of GFP + cells displaying ∆ FosB in the nucleus . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; two - way ANOVA followed with Tukey ' s multiple comparisons test in E ) . N denotes biological replicates .

O O O O O O O O O O O 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 O B-CONTROLLED_VAR O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.


( A - F ) RT - qPCR analysis of FosB ( A , D ) , c - Fos ( B , E ) and ∆ FosB ( C , F ) mRNAs in NAc of Gad67Cre ; Alk4fl / fl ( A - C ) mutant mice 1 hour ( A - C ) or 24 hour ( D - F ) after the second saline ( Sal ) or cocaine ( Coc ) injection of TIPS . Results are expressed as average ± SEM of fold increase over the saline group ( Sal / Sal ) of Gad67Cre mice . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative immunofluorescence staining of ∆ FosB in Nac of control ( Gad67Cre ) and mutant ( Gad67Cre ; Alk4fl / fl ) mice , 24 hours after the second injection of the TIPS protocol . Scale bars 20 μm . ( H ) Quantification of ∆ FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre ; Alk4fl / fl mice . Results are expressed as average ± SEM of the number of ∆ FosB + cells per mm2 . At least 10 images from both left and right hemispheres were analyzed per mouse and condition . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm. (H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Western blot analysis of FosB and ∆ FosB in in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Blots were probed with an antibody that recognizes both FosB ( upper band , 48kDa ) and ∆ FosB ( lower band , 38 kDa ) . Lower blot shows reprobing with anti - β - actin . ( J , K ) Quantification of FosB ( J ) and ∆ FosB ( K ) levels in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Results are expressed as average ± SEM of fold increase of FosB and ∆ FosB over the saline group ( Sal / Sal ) of Gad67Cre mice normalized to β - actin levels . N = 4s . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin. (J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


A . KD efficiency of three different Syt11 shRNAs in DRG neurons . Cultured DRG neurons were infected with control or shRNA - expressing lentiviruses at DIV 1 , and Western blotting for Syt11 , Syt1 , Syt4 , synaptobrevin 2 ( Syb2 ) , SNAP25 , complexin 1 / 2 ( Cpx1 , Cpx2 ) , clathrin , adaptor protein 2 ( AP - 2 ) , and β - actin were performed at DIV 6 - 7 . N = 4 independent experiments .

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A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.


B , C . Representative Cm traces induced by 200 - ms pulse depolarization ( arrows ) ib DRG neurons . DRG neurons were transfected with plasmids expressing shSyt11 - 2 ( Syt11 KD , KD ) or scrambled shRNA ( Sc ) and Cm recording was performed 5 days after transfection . Endo - 5s represents the Cm decay 5 s after stimulation . Insets show Ca2 + currents recorded in the same neurons .

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B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.


D . Representative Cm overshoot recorded from an Syt11 KD neuron . About 45 % of the KD neurons showed excessive membrane retrieval after a 200 - ms depolarizing pulse .

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D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.


E . Averaged Cm traces recorded from Control ( Ctrl ) , Sc , KD ( from all three shRNAs , n = 39 for shSyt11 - 2 , n = 7 for shSyt11 - 1 , n = 14 for shSyt11 - 3 ) , and rescued ( with the shSyt11 - 2 - resistant form of Syt11 ) DRG neurons .

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E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.


F - H . Statistics of Cm jumps , Endo - 5s , and Ca2 + current recorded from DRGneurons as in ( B - E ) . Data were collected from 4 ( Sc , Rescue ) , 8 ( Ctrl ) , and 16 ( KD ) independent experiments .

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F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.


I . Normalized Cm changes induced by two pulses of 200 - ms depolarization ( arrows ) at a 1 - s interval . Data were normalized to the capacitance jump induced by the first depolarization .

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I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.


J . Paired - pulse ratios of ΔCm with different interstimulus intervals as in ( I ) . DRG neurons were stimulated with two 200 - ms pulses at different intervals as indicated , and the paired - pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one .

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J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.


A , B . FM 1 - 43 uptake into DRG neurons stimulated by 100 mM K + for 30 s . The right - shifted cumulative frequency in ( B ) indicates the increased uptake level in KD neurons . Scale bars , 5 μm .

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A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.


C . Quantification of KCl - evoked FM 1 - 43 uptake with different stimulation times .

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C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.


D . Representative micrographs showing the increased constitutive FM 1 - 43 uptake ( for 5 min ) in KD neurons . Scale bars , 5 μm .

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D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.


E . Quantification of constitutive FM 1 - 43 uptake with different loading times .

E. Quantification of constitutive FM 1-43 uptake with different loading times.


F . Basal [ Ca2 + ] i in DRG neurons measured with Fura - 2 .

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F. Basal [Ca2+]i in DRG neurons measured with Fura-2.