D Quantification of the RCA product after co - IP of the C - circles with Nic96 - RFP showing the effect of ADA2 deletion . Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2 ∆ signals . Relative signals were calculated by setting the mean of est2 ∆ signals from each independent experiment to 1 . Means and s . e . m . of the six independent co - IPs experiments each with 2 - 6 clones per genotype are shown . Outliers were removed using ROUT robust outlier test . The p - values from one - way ANOVA were adjusted using the Bonferroni ' s correction for multiple comparisons . est2 ∆ versus est2 ∆ NIC96 - RFP ( p = 0 , 000007 ) , est2 ∆ NIC96 - RFP versus est2 ∆ ada2 ∆ NIC96 - RFP ( p = 0 , 00012 ) . * * * P < 0 , 0001 .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

D Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of ADA2 deletion. Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2∆ signals. Relative signals were calculated by setting the mean of est2∆ signals from each independent experiment to 1. Means and s.e.m. of the six independent co-IPs experiments each with 2-6 clones per genotype are shown. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,000007), est2∆ NIC96-RFP versus est2∆ ada2∆ NIC96-RFP (p=0,00012).***P<0,0001.


E Quantification of the RCA product after co - IP of the C - circles with Nic96 - RFP showing the effect of SUS1 deletion . The graph shows means and s . e . m . of the three independent co - IPs with 2 - 5 clones per genotype . Outliers were removed using ROUT robust outlier test . The p - values from one - way ANOVA were were adjusted using the Bonferroni ' s correction for multiple comparisons . est2 ∆ versus est2 ∆ NIC96 - RFP ( p = 0 , 002 ) , est2 ∆ versus est2 ∆ sus1 ∆ NIC96 - RFP ( p = 0 , 0006 ) . * * P < 0 , 001 , * * * P < 0 , 0001 .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O 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 Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of SUS1 deletion. The graph shows means and s.e.m. of the three independent co-IPs with 2-5 clones per genotype. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,002), est2∆ versus est2∆ sus1∆ NIC96-RFP (p= 0,0006). **P<0,001, ***P<0,0001.


A Senescence curves of two est2 ∆ and two est2 ∆ ADA2 - Myc clones used to generate type II survivors for Co - IP - RCA . This experiment is representative out of the 4 independent experiments performed during this study .

O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O 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 Senescence curves of two est2∆ and two est2∆ ADA2-Myc clones used to generate type II survivors for Co-IP-RCA. This experiment is representative out of the 4 independent experiments performed during this study.


C Detection of the C - circles co - immunoprecipitated with Ada2 - Myc in type II survivors . The DNA co - immunoprecipitated with Ada2 - Myc was amplified by RCA , loaded on a dot blot and detected with a telomeric TG1 - 3 probe . The numbers above the blot refer to clones in ( A ) and correspond to type II survivors produced in independent cultures . The lower panel shows the efficiency of Ada2 - Myc IP analyzed by western blot with anti - Myc antibody .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O 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-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O

C Detection of the C-circles co-immunoprecipitated with Ada2-Myc in type II survivors. The DNA co-immunoprecipitated with Ada2-Myc was amplified by RCA, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to clones in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot with anti-Myc antibody.


A Mean senescence profiles of the est2 ∆ ( n = 7 ) and est2 ∆ sus1 ∆ ( n = 8 ) clones isolated from the same heterozygous diploid . The error bars are SDs . B Mean senescence profiles of the est2 ∆ ( n = 10 ) and est2 ∆ sac3 ∆ ( n = 10 ) clones isolated from one heterozygous diploid . The error bars are SDs .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

A Mean senescence profiles of the est2∆ (n=7) and est2∆ sus1∆ (n=8) clones isolated from the same heterozygous diploid. The error bars are SDs. B Mean senescence profiles of the est2∆ (n=10) and est2∆ sac3∆ (n=10) clones isolated from one heterozygous diploid. The error bars are SDs.


A Mean senescence profiles of the est2 ∆ ( n = 10 ) and est2 ∆ shs1 ∆ ( n = 16 ) clones . The error bars are SDs . B Mean senescence profiles of the est1 ∆ ( n = 6 ) and est1 ∆ bud6 ∆ ( n = 7 ) clones . Est1 , similarly to Est2 , is essential for telomere maintenance . est1 ∆ has been used in this experiment instead of est2 ∆ because EST2 and BUD6 genes are linked on chromosome XII . The error bars are SDs .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

A Mean senescence profiles of the est2∆ (n=10) and est2∆ shs1∆ (n=16) clones. The error bars are SDs. B Mean senescence profiles of the est1∆ (n=6) and est1∆ bud6∆ (n=7) clones. Est1, similarly to Est2, is essential for telomere maintenance. est1∆ has been used in this experiment instead of est2∆ because EST2 and BUD6 genes are linked on chromosome XII. The error bars are SDs.


Results of three independent microscopy - based screens in U2OS cells . Knockdown of Luciferase ( blue ) and the positive controls PPM1D and βTrCP ( red ) are indicated . A low / high Z - score indicates less / more phospho - histone H3 positive cells compared to the control , reflecting reduced / increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR . Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides . Grey represents a recovery value that is similar to the Luciferase control ( < 0 . 5 SD ) and the red represents an altered recovery value ( > 0 . 5 SD ) in two independent experiments .

O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Results of three independent microscopy-based screens in U2OS cells. Knockdown of Luciferase (blue) and the positive controls PPM1D and βTrCP (red) are indicated. A low/high Z-score indicates less/more phospho-histone H3 positive cells compared to the control, reflecting reduced/increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR. Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides. Grey represents a recovery value that is similar to the Luciferase control (<0.5 SD) and the red represents an altered recovery value (>0.5 SD) in two independent experiments.


U2OS cells ( D ) and RPE1 hTert immortalized primary epithelial cells ( E ) were transfected with siRNA oligos targeting the hits in a similar setup as described in A ) , but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments .

B-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O 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-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

U2OS cells (D) and RPE1 hTert immortalized primary epithelial cells (E) were transfected with siRNA oligos targeting the hits in a similar setup as described in A), but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.


RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA , in conditions used for the primary screen ( 2 Gy of IR ) . Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA, in conditions used for the primary screen (2 Gy of IR). Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.


U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen , but with 10 Gy of IR and in the presence of ATM and ATR inhibitors . Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments .

B-CELL_LINE O O O O 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-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen, but with 10 Gy of IR and in the presence of ATM and ATR inhibitors. Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.


U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0 , 2 and 24 h after 5 Gy IR . Samples were stained with antibodies for γH2AX ( D ) or 53BP1 ( E ) and analyzed through automated confocal microscopy and specifically developed image analysis software . From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown . Shown are the number of γH2AX ( D ) and 53BP1 ( E ) foci per cell . In the box plots , the median with the first and third quartile is indicated , and the whiskers are drawn down to the 10th percentile and up to the 90th .

B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0, 2 and 24 h after 5 Gy IR. Samples were stained with antibodies for γH2AX (D) or 53BP1 (E) and analyzed through automated confocal microscopy and specifically developed image analysis software. From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown. Shown are the number of γH2AX (D) and 53BP1 (E) foci per cell. In the box plots, the median with the first and third quartile is indicated, and the whiskers are drawn down to the 10th percentile and up to the 90th.


U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls .

B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O

U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls.


U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated

B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodies


U2OS WT , PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP - PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3

B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 staining


U2OS WT , PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP - PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated

B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodies


U2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence . In ( F ) is presented 53BP1 IRIF intensity . In ( G ) is shown the number of γH2AX foci per cell .

B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O

U2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell.


U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis . The comet tail moment was analyzed in at least 50 individual cells .

B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells.


U2OS cells stably expressing GFP - PHF6 and mCherry - NBS1 were laser - irradiated and cells were analyzed by time - lapse imaging .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O

U2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated and cells were analyzed by time-lapse imaging.


U2OS cells stably expressing GFP - PHF6 were laser - irradiated and cells were analyzed by time - lapse imaging . In ( B ) , the PARP inhibitor Olaparib was added 30 min before laser - induced damage . At least 60 cells were analyzed in three individual experiments and error bars represent the SD ( lower panel ) . Scale bar is 5 μM .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells stably expressing GFP-PHF6 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.


GC92 SV40 immortalized human fibroblasts containing the I - SceI NHEJ reporter were transfected with the indicated siRNA oligos and I - SceI . 48 h later the cells were fixed and analyzed by flow cytometry . Represented is the relative repair efficiency as compared to the Luciferase control .

B-CELL_LINE B-ORGANISM O B-ORGANISM B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O

GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control.


Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2 , 3 or 4 Gy IR . Shown is the relative survival as compared to the undamaged control . Error bars represent the SEM of three individual experiments .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3 or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.


GC92 SV40 immortalized human fibroblasts containing the I - SceI NHEJ reporter were transfected with the indicated siRNA oligos and I - SceI . 48 h later genomic DNA was extracted and repair of I - SceI cut - sites analyzed through junction analysis ( n = independently derived sequences , see materials and methods for details ) .

B-CELL_LINE B-ORGANISM O B-ORGANISM B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).


A . RGFP966 ( 966 ) - dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth ( 24h in culture ) compared to HDAC class II , HDAC1 / 2 and HDAC1 or 3 inhibitions via 69A , 963 and 233 respectively . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test ) .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).


B , C . Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth ( average neurite outgrowth after 24h in culture , Tuj1 positive neurons ) on PDL - laminin or Myelin substrates . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

O O O O O O O O B-GENEPROD O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


D , E . Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


F , G . AAV - mediated mutant HDAC3 ( Y298H , V5 ) infection of DRG neurons induces neurite outgrowth ( Tuj - 1 / V5 double positive neurons ) after 36h in culture , which is repressed by wildtype HDAC3 overexpression . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus V5 vs AAV - GFP ANOVA followed by Bonferroni test ) .

O O O O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O O O O O O O

F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).


A . IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased , primarily in the nucleus 24h after sciatic nerve axotomy ( SNA ) but not spinal dorsal column axotomy ( DCA ) . Scale bars , 100 μm .

O O B-EXP_ASSAY O B-TISSUE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O

A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm.


B - C . Bar graphs show mean fold change levels ( B ) or % of pHDAC3 + cells ( C ) ( pHDAC3 + threshold was set at the minimum intensity level of sham cells ) ± s . e . m . DAPI counter staining was used to label nuclei N = 9 - 10 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham or laminectomy ( lam ) ( ANOVA followed by Bonferroni test ) .

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B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).


D . Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA . E . Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham ( Student ´ s t - test ) .

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D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test).


F . HDAC3 activity ( HDAC3 IP and activity assay ) is strongly reduced after SNA but not DCA . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

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F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


PP4 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

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PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


PP2 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


C . SNA but not DCA significantly enhances calcium levels in DRG . Data is expressed as mean ± s . e . m . N = 4 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham or lam ( ANOVA followed by Bonferroni test ) .

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C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).


D - F . Immunoblotting of DRG homogenates shows that nerve injury - dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve ( Axo = Axotomy ) . Cytoplasmic pERK levels are used as control of axotomy - dependent signalling . ( E - F ) . Data is expressed as mean fold change of band intensity levels ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 , * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

O O O O B-EXP_ASSAY O B-TISSUE O O O B-DISEASE I-DISEASE O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-DISEASE B-TISSUE I-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


Significant Pearson correlations between calcium levels ( Fluo4AM ) and H3K9ac , r2 : 0 . 7802 ( G ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin ( calcium and H3K9ac , r2 : 0 . 0007 Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

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Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


Significant Pearson correlations between calcium levels ( Fluo4AM ) ; or pHDAC3 , r2 : 0 . 6366 ( H ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3 , r2 : 0 . 3025 ) . Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

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Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


J - L . Immunofluorescence ( J ) in DRG tissue sections 24h after injury in vivo shows that the PP2a / PP4 inhibitor Fostriecin ( F ) inhibits the injury - induced decrease in pHDAC3 ( K ) and the increase in H3K9ac ( L ) . Scale bar , 10 μm . ( K - L ) . Data is expressed as mean fluorescence intensity in arbitrary units ± s . e . m . N = 3 ( L ) or 4 ( K ) biological replicates ( * p < 0 . 05 ; * * p < 0 . 01 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

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J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


M - N . Pharmacological inhibition of PP2a / PP4 impairs conditioning - induced DRG neurite outgrowth ex vivo ( 12h in culture ) . M . DRG neurite outgrowth ( TUJ1 positive cells ) 24h after sham or sciatic nerve axotomy ( SNA ) prior to i . t . administration of vehicle ( V ) or Fostriecin ( F ) ( 240µM ) . Scale bar , 100µm . ( N ) Data is expressed as average neurite length per neuron ± s . e . m . N = 3 biological replicates per condition , technical triplicate . * * * p < 0 . 005 indicate significant difference versus sham / vehicle ( One way ANOVA followed by Bonferroni ) .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).


A - E . PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl ( A - C , arrows ) , both in the nucleus and the cytoplasm ( D ) . Scale bar , 20 μm . ( C , D ) Data is expressed as mean fold change vs scrambled ( scr ) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 ; * * * p < 0 . 005 ) indicate significant difference versus scrambled siRNA ( Student ' s t - test ) . ( E ) PCR against PP4c demonstrates silencing of PP4c mRNA . F - G . PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 ( F ) . ( G ) Data is expressed as mean fold change of immunoblot band intensity ± s . e . m . N = 3 biological replicates ( * p < 0 . 05 ; * * * * p < 0 . 001 ) indicate significant difference versus control siRNA ( Student ' s t - test ) .

O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test).


H - I . Genetic modification of activity - dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth . ( H ) DRG neurite outgrowth after transfection of control ( V5 control ) , WT HDAC3 - V5 , phospho - dead HDAC3 ( S424A ) - V5 or phospho - mimetic HDAC3 ( S424D ) - V5 and after 24h in culture . Scale bar , 50µm . ( I ) Data is expressed as mean ± s . e . m . N = 8 - 9 biological replicates , approximately 35 V5 positive cells each . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference ( One way ANOVA followed by Bonferroni ) .

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H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).


A . The graph shows the number of differentially expressed ( DE ) genes 24h following SNA or DCA compared to their respective controls .

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A. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls.


B . RNAseq read counts show differential occupancy of H3K9ac on specific gene regions , including an increased occupancy close to the transcriptional start site ( TSS ) after SNA only , when compared to corresponding controls .

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B. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls.


C . Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA ( RNAseq ) with an increased or decreased H3K9ac occupancy ( ChIPseq ) , respectively . D . Odds ratio analysis of the enrichment / depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in ( C ) ; the numbers in red represent the p value given by Fisher ' s exact test ( ns = not significant ) .

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C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant).


A , B . Co - immunofluorescence of anti - neuronal and anti - regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle . Shown is increased protein expression for regeneration - associated targets in NF200 positive DRG neurons ( arrows ) after 966 treatment ( 5 weeks following SCI ) . Scale bar , 50 μm . ( B ) Data is expressed as fold change of fluorescence intensity in NF200 + cells , 966 versus vehicle ± s . e . m . N = 4 - 7 biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference versus vehicle ( Student ' s t - test ) .

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A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).


A - B . AAV - HDAC3 mutant ( Y298H ; dominant negative deacetylase inactive ) promoted DRG regenerative growth after spinal cord injury vs AAV - V5 control ( injected 5 weeks prior to SCI in the sciatic nerve bilaterally ) as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , ( A - B ) 200 μm .

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A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.


C - F . Immunofluorescence for H3K9ac . AAV - HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons , but not in surrounding nuclei of satellite cells . Scale bar , ( C - F ) 50 μm .

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C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.


G - J . New synaptic formations ( VGlut1 + ) from regrowing axons are shown beyond the lesion site after injection of AAV - HDAC3mut . ( G ) Scale bar 200 μm , ( H - J ) High magnification insets depict co - localization of Dextran and vGlut1 staining . Scale bar 50 μm .

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G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.


K - L . Quantification of dextran positive axons shows that AAV - HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site . Data is expressed as percentage of dextran + axons at each distance vs dextran + axons at - 700μm from the lesion margin ( K ) or distance from the caudal margin of the lesion to the most rostral dextran + axon tip for each animal ( L ) . N = 10 animals per condition , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 indicate a significant difference ( ANOVA followed by Bonferroni test ) .

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K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).


A - B . Intrathecally administered RGFP966 ( 966 ) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , 200 μm .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O B-SUBCELLULAR B-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O 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-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.


C - D . Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site . Data is expressed as ratio of dextran + axons vs dextran + axons at - 700 μm from the lesion site ± s . e . m ( C ) or distance from the caudal margin of the lesion to the last dextran + axon tip ( D ) . N = 6 - 10 animals per condition . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O 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-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


E . Immunofluorescence for H3K9ac and Neurofilament 200 ( NF200 ) shows upregulated H3K9ac in vivo in DRG neurons after 966 . Scale bar , 50 µm .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-TISSUE B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O

E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.


F - G . Data is expressed as fold change of fluorescence intensity mean levels ( F ) or % of H3K9ac + cells ( G ) ( H3K9ac + threshold was set at the mean intensity level of vehicle treated animals , DAPI counter staining was used to label nuclei from NF200 + or NF200 - cells ) vs veh ± s . e . m . N = 11 - 12 biological replicates . ( * * * p < 0 . 005 ) indicate a significant difference vs respective Veh ( ANOVA followed by Bonferroni test ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).


( B ) Western blot analysis of Atxn1 levels in cerebellar granule neurons ( CGNs ) after knockdown of Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( C ) Western blot analysis of Atxn1 levels in the cerebella of wild - type ( n = 3 ) , Zbtb7a + / - ( n = 4 ) , Zbtb7b + / - ( n = 3 ) , Zbtb7a + / - ; Zbtb7b + / - ( n = 4 ) mice . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( D ) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( E ) Genetic interaction of wild - type human ATXN1 ( 30Q ) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes . Co - expression of ATXN1 ( 30Q ) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels . Scale bar : 100

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(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm


( C ) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild - type ( WT ) , zinc - finger domain mutant ( ZFmut ) or BTB domain mutants ( BTBmut1 and BTBmut2 ) ( n = 3 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) qRT - PCR results of Atxn1 mRNA levels in the cerebella of wild - type ( n = 3 ) , Zbtb7b + / - ( n = 4 ) , and Zbtb7b - / - mice ( n = 4 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) MA plot ( M : log ratio ; A : mean average ) of the differentially expressed genes ( DEG ) analysis from RNA - seq samples of the cells overexpressing either WT or ZFmut ZBTB7B . Four filters and the number of genes kept after applying each filter are described . Two genes that passed the 4th filter are labeled .

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(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.


( F ) Expression heatmap of the 16 genes that passed the 3rd filter . Color scale : row z - score .

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(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.


( G ) qRT - PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( H ) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation . ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line ( sgRSK3 ) and control cell lines ( WT and sgControl ) ( n = 3 ) . ZBTB7B + RSK3 : co - overexpression of both ZBTB7B and RSK3 . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) qRT - PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( J ) qRT - PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice ( n = 6 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

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(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( K ) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter . Start and End position numbers are described based on TSS . The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( L ) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

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(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( A ) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells . Bottom left panel : qRT - PCR results of RSK3 mRNA . Bottom right panel : densitometry of the western blot image in the top panel ( n = 3 except for shATXN1 where n = 1 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( B ) Western blot analysis of ATXN1 after overexpression of RSK3 , ZBTB7B , or eGFP in Daoy cells ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( C ) Western blot analysis of ATXN1 ( 82Q ) after overexpression of either wild - type or inactive RSK3 in Daoy cells expressing ATXN1 ( 82Q ) ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) In vitro kinase assay of RSK3 with ATXN1 . MSK1 was used as a positive control . Asterisk : auto - phosphorylated RSK3 .

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(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.


( E ) In vitro kinase assay of four different RSK members with ATXN1 .

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(E) In vitro kinase assay of four different RSK members with ATXN1.


( F ) In vitro kinase assay of RSK3 with N - terminal ( F1 ) , middle ( F2 ) , and C - terminal fragment ( F3 ) of ATXN1 .

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(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.


( G ) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3 .

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(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.


( H ) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) Western blot analysis of total and phospho - S776 ATXN1 after a co - treatment with MSK1 ( SB - 747651A ) and RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( A ) Images of Drosophila eyes expressing human ATXN1 ( 82Q ) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II . Scale bar : 100 μm ( top ) and 20 μm ( bottom ) .

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(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).


( B ) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1 ( 82Q ) in the central nervous system . Mean ± SEM , * P < 0 . 05 , Linear mixed - effect model ANOVA , n = 10 / genotype , four times of measurements .

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(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.


( C ) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice ( Atxn1154Q / 2Q ) cerebellum . Scale bar : 2 mm .

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(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.


( D ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl ( n = 3 ) , shRsk3 ( n = 3 ) , or shAtxn1 ( n = 2 ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the brainstem ( BS ) and cerebella ( CBM ) of 8 weeks old Atxn1154Q / 2Q ( n = 6 ) and Atxn1154Q / 2Q ; Rsk3 + / - mice ( n = 7 ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test .

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(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.


( F ) Rotarod motor performance test result of Atxn1154Q / 2Q ; Rsk3 + / - mice at 11 - 12 weeks old . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 35 , 31 , 34 , 38 for WT , Rsk3 + / - , Atxn1154Q / 2Q , Atxn1154Q / 2Q ; Rsk3 + / - genotype , respectively .

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(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.


( A ) Fluorescent western blot analysis of Atxn1 , Msk1 , and Rsk3 in seven different brain regions of wild - type mice at 9 weeks old ( n = 3 ) . Each protein levels were represented relative to the protein levels in brainstem . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . Data information : BS : brainstem , CBM : cerebellum , MB : midbrain , ST : striatum , HC : hippocampus , CTX : cortex , OB : olfactory bulbs .

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(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.


( B ) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild - type ( top ) and Rsk3 - / - mice ( bottom ) at 5 - 6 weeks old . The areas within the black squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM :

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(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( C ) Representative IF images of Msk1 in the whole brain sagittal sections of wild - type ( top ) and Msk1 - / - mice ( bottom ) at 6 months old . The areas within the white squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM :

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(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( D ) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice ( n = 4 ) . Relative Rsk3 and Msk1 levels were represented into log2 scale . The protein levels of the two kinases are significantly different between the two brain regions . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - way ANOVA . Data information : BS : brainstem , CBM :

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(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA. Data information: BS: brainstem, CBM: cerebellum


( E ) Western blot analysis of ATXN1 , RSK3 , pRSK3 , MSK1 , and pMSK1 in the postmortem cerebella , pons , and medulla oblongata of human controls and SCA1 patients ( n = 3 per genotype ) . Total protein was used for normalization , and protein levels are represented relative to cerebellar protein levels in control group . C : Cerebellum , P : Pons , M : medulla oblongata . Values from P and M are combined and presented in BS . Mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , two - way ANOVA , n = 3 ( CBM ) and 6 ( BS ) . Data information : BS : brainstem , CBM :

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(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS). Data information: BS: brainstem, CBM: cerebellum


( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Plethysmography data measuring breathing ability ( total ventilated gas volume per minute ) of 34 weeks old mice with different genotypes . Mean ± SD , * P < 0 . 05 , two - tailed t - test , n = 12 , 13 , 15 , 13 , 15 , 9 , 15 , 13 following the order of genotypes in the graph , respectively .

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(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.


( C ) Representative immunohistochemistry images of Iba1 + reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes . Yellow arrows indicates Iba1 + microglia , and larger pyramidal shaped cells are motoneurons . scale bar = 50 μm .

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(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.


( D ) Quantification of the number of Iba1 + cells shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( E ) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus ( white dashed line ) of 36 weeks old mice with different genotypes . The motoneurons are dark purple with pyramidal shape within the nucleus . C : central canal . Scale bar = 100 μm .

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(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.


( F ) Quantification of the number of motoneurons shown in ( E ) . Mean ± SD , * P < 0 . 05 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( G ) Kaplan - Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases . The data of Msk1 + / - , Rsk3 + / - , and Msk1 + / - ; Rsk3 + / - mice overlap those of wild - type mice . * P < 0 . 05 , Gehan - Breslow - Wilcoxon test , n = 15 , 15 , 12 , 13 , 22 , 11 , 20 , 19 following the order of genotypes in the legend on top of the figure , respectively .

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(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.


( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Rotarod motor performance test result of 11 - 12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 18 , 12 , 13 , 11 , 19 , 13 , 15 , 13 following the order of genotypes in the legend on top of the figure , respectively .

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(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.


( C ) Representative immunofluorescent ( IF ) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old , stained with anti - Calbindin 1 antibody . Scale bar = 100 μm .

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(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.


( D ) Quantification of the thickness of top and bottom molecular layers shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from three sections per mouse .

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(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.


( E ) Fold increase of Oct4 - GFP + colony number over control under forced expression of individual factors . n ≥ 5 , where each dot indicates an independent experiment . Box - plots show median , 1st and 3rd quartile values . One - sample Wilcoxon test p - values are as indicated , with p values < 0 . 05 shown in

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(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red


( B ) Heatmap of average gene expression normalised to β - actin over an EpiSC resetting time course in 2i + LIF . Each row is coloured according to the unique minimum and maximum for that gene . The values shown are average expression of four independent

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(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments


( C ) Gene expression for Stat3 , Klf2 , Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs . β - actin serves as an internal control . Mean + / - SEM , n = 4 independent experiments . * = p < 0 . 05 Student " s

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(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t


( E ) Representative confocal images ( top ) and quantification ( bottom ) of Oct4 - GFP reporter mean intensity . The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6 . Mean + / - SD of 9 technical replicates . One representative experiment of two is shown . Scale bars =

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(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm


( F ) Representative alkaline phosphatase ( AP ) staining images ( left ) and quantification ( right ) of AP + colonies after clonal replating , as described in

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(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel


( A ) Top : Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 ( + E / T ) . Bottom : Gene expression of EpiSCs harbouring empty vector ( grey ) or Esrrb / Tbx3 ( orange ) , capture at D0 ( F / A ) , D2 and D4 ( as described in Fig 3D ) . Dashed black line : expression levels in ESCs maintained in 2i + LIF . Data normalised to empty vector cultures in F / A . Gapdh serves as an internal control . Mean + / - SEM , n = 3 independent experiments . ( B ) As for panel A , comparison of control with dual expression of Esrrb and Klf4 ( + E / K , green in bottom plot ) . Mean + / - SEM , n = 3 independent experiments for bottom qRT - PCR

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(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels


( C ) Left : Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb - T2A - Klf4 construct and cultured in 2i + LIF with DOX for 2 and 4 days , with the indicated fraction of cells sorted for colony formation assay . Since the Venus reporter is under the control of a DOX responsive element , and the emission spectra of Venus and GFP fluorescence overlap , Oct4 - GFP reporter could not be fully distinguished from Venus expression . Right : Number of AP + colonies formed from 250 sorted cells from indicated fractions . Data points represent two technical replicates of one out of two independent

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(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments


( D ) Heatmap of single - cell expression measured by qRT - PCR of major ESC and EpiSC markers in un - induced EpiSCs ( black ) , established ESCs ( red ) , Day 2 High / Low ( dark and light blue ) , and Day 4 High cells ( green )

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(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green)


( E ) Scatterplots of the relative expression of pluripotency and transitional markers . Red bar corresponds to median gene expression , and each dot represents a single

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(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell


Left : 0 . 782 cABN predictions of the sequence of gene activation between gene pairs ( white , OFF ; blue , ON ) along the resetting trajectory , compared to single cell gene expression measured by RT - qPCR . Each table summarises the percentage of single cells at the indicated stage of resetting ( columns ) that have the indicated expression state ( rows ) . Right : Scatterplots showing single cell coordinates based on the expression of the gene

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Left: 0.782 cABN predictions of the sequence of gene activation between gene pairs (white, OFF; blue, ON) along the resetting trajectory, compared to single cell gene expression measured by RT-qPCR. Each table summarises the percentage of single cells at the indicated stage of resetting (columns) that have the indicated expression state (rows). Right: Scatterplots showing single cell coordinates based on the expression of the gene pair


( A ) 0 . 782 cABN predictions of essential or dispensable factors for EpiSC resetting , compared with the experiment results shown in panel ( B ) . ( B ) siRNA knockdown effects measured by Oct4 - GFP + colony formation at Day 6 of resetting . n = 4 independent

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(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments


( C ) Left , resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4 - GFP + colony formation at Day 6 of resetting . Data points represent two independent experiments . Right , representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i +

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(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF


( D ) Expression of naïve pluripotency , transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i + LIF . Expression is normalised to wild type EpiSCs in A / F , and β - actin was used as internal control . The values shown correspond to the average expression of three technical replicates from one representative experiment out of

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(D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two


( E ) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4 - GFP + colony formation at Day 6 of resetting in 2i + LIF . Data points represent two independent

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(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments


( F ) Comparison between the effect of single factor knockdowns on ESC maintenance ( n = 4 independent experiments ) and EpiSC resetting using experimental

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(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results