Figure 2 ( A ) MCF7 and MDA - MB - 231 cells transfected with MYC - B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization . Representative images from three independent experiments are shown , Scale bars 5 µm . ( B ) Quantification of cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell was plotted ( n = 35 for MCF - 7 cells ; n = 25 for MDA - MB - 231 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C ) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies . ( D ) Cells expressing either vector or SFB - B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively . ( E ) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively . ( F ) Localization of PPM1G in cells expressing either empty vector ( control ) , full length B56δ ( FL ) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies . Representative images from two independent experiments are shown , Scale bars 10 µm . ( G ) Cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell expressing either vector , B56δ full length ( FL ) or mutant versions was quantified and plotted ( n = 10 ) . Error bars represents SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies.(D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.(E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.(F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 3 ( A ) Endogenous association of PPM1G with α - Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α - Catenin antibody . ( B ) SFB - PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads . Interaction with α - catenin was determined by western blotting . ( C ) In vitro phosphorylated MBP - tagged α - Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant ( D496A ) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represents mean absorbance from three independent experiments . Error bars indicate SD , * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( D ) In vitro phosphate release assay was performed using wild type α - Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted , n = 3 , Error bars indicate SD ; * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( E ) MDA - MB - 231 breast cancer cells were transfected with vector , wild type PPM1G or catalytically inactive PPM1G D496A ( D / A ) mutant . Levels of phosphorylated α - catenin were detected by using phospho - specific antibodies . ( F ) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies . ( G ) Cells transfected with vector , SFB - PR130 and SFB - B56δ and levels of pS641 α - catenin was detected using western blotting . ( H ) 293T Cells transfected with either empty vector , full length B56δ or indicated mutants were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody . ( I ) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody . Cells were transduced with shRNAs as indicated and the effect of ( J ) B56δ expression on α - Catenin phosphorylation was assessed . , K ) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α - Catenin phosphorylation was assessed . ( L ) SFB - PPM1G and SFB - PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells . Lysates were pulled down using streptavidin beads and interaction with α - catenin was detected by western blotting using α - catenin antibody . ( M ) Control shRNA and B56δ shRNA cells were transfected with vector , MYC - PPM1G and MYC - PPM1G ΔNLS in indicated combination and pS641 α - catenin levels were determined using western blotting .

O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody.(B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(D) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(E) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies.(F) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies.(G) Cells transfected with vector, SFB-PR130 and SFB-B56δ and levels of pS641 α-catenin was detected using western blotting.(H) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.(I) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.Cells were transduced with shRNAs as indicated and the effect of (J) B56δ expression on α-Catenin phosphorylation was assessed., K) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α-Catenin phosphorylation was assessed.(L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody.(M) Control shRNA and B56δ shRNA cells were transfected with vector, MYC-PPM1G and MYC-PPM1G ΔNLS in indicated combination and pS641 α-catenin levels were determined using western blotting.


Figure 4 ( A ) Cells transduced with the indicated shRNAs were fixed and localization of α - catenin and β - catenin was determined by imaging with a confocal microscope after staining with antibodies against α - catenin and β - catenin . Representative images from three independent experiments are shown . Scale bars , 10 μm . ( B ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4A was plotted ( n = 40 cells ) . Error bars indicate SD . * * * p < 0 . 001 , ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C ) Control , PPM1G and B56δ shRNA transduced cells were transfected with SFB - α - catenin . Cell lysates were pulled down using streptavidin beads and interaction with β - catenin was determined by western blotting . ( D ) F - Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope . Scale bars , 20 μm . E ) Localization of α - catenin and β - catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB - PPM1G and SFB - PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies . Representative images are shown , Scale bars , 10 μm . ( F ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4F was plotted ( n = 30 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting.(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 5 ( A , B ) MDA - MB - 231 cells were transfected with vector control , PPM1G wild type , catalytically inactive PPM1G D496A mutant or α - Catenin and transwell migration assay was performed . Phase contrast microscopic images ( A ) were taken after staining the migrated cells with crystal violet and ( B ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C , D ) MCF - 7 cells were transfected with SFB - vector , SFB - B56δ or indicated mutants and transwell migration assay was performed . Phase contrast microscopic images ( C ) were taken after staining the migrated cells with crystal violet and ( D ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( E , F ) SFB - B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells . Representative images from transwell migration assay ( E ) are shown and ( F ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( G , H ) SFB - PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells . Representative images from transwell migration assay ( G ) are shown and ( H ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( I , J ) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC - PPM1G and MYC - PPM1G ΔNLS . Representative images from transwell migration assay ( I ) performed with these cells are shown and ( J ) quantified data for migrated cells from three independent experiments per field was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( K , L ) SFB - tagged α - catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown . Representative images from transwell migration assay ( K ) performed with these cells are shown and ( L ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(I, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 1Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hSynI - mWwox [ total n = 18 , spontaneously dead n = 6 , mice taken out for electrophysiology / electron microscopy / analysis , are shown in yellow , n = 12 ] compared to mice injected with AAV9 - hSynI - GFP ( n = 6 ) or the non - injected ( n = 8 ( P < 0 . 0001 , Log - rank Mantel - Cox test ) . Immunofluorescence images showing the expression of WWOX ( red ) in brain tissue ( cortex ) at P17 from WT and Wwox null mice that were injected with AAV9 - hSynI - mWwox ( 2x1010 ) . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . White arrows show the transgene expression in NeuN positive cells . Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain ( Cortex , Hippocampus and Cerebellum ) from KO mice injected with AAV - mWwox at P17 . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - mWwox injected mice ( n = 3 ) brains .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test).Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells. Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains.


Figure 2Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hWWOX [ total n = 16 , alive n = 6 , spontaneously dead n = 6 , 4 mice ( shown in yellow ) were taken out for analysis ) compared to the non - injected ( n = 8 ) ] ( P < 0 . 0001 , Log - rank Mantel - Cox test ) . D - F . Immunofluorescence images showing the expression of WWOX ( magenta ) in cortex ( D ) , Hippocampus ( E ) and Cerebellum ( F ) at P19 from WT and Wwox null mice that were injected with AAV - hWWOX ( 2x1010 ) or Wwox null alone . Neurons are labelled with anti - NeuN antibody ( green ) . The square white box shows the magnified area . Graph represents the percentage of neurons showing the transgene ( NeuN and WWOX positive ) expression at different parts ( cortex , hippocampus and cerebellum ) of the KO brain at P19 after AAV injection . NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV - hWWOX injected mice ( n = 3 ) brains .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test).D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.   Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.


Figure 3Representative traces of cell - attached recordings performed in WT ( blue ) , KO ( red ) and KO injected with either with AAV9 - hSynI - mWwox ( KO + A - mW shown in purple ) or AAV9 - hSynI - hWWOX ( KO + A - hW shown in green ) pups at P18 - 21 days . Traces represent spontaneous neocortical activity ( action potentials shown ) . The panels represent 12 s recording with an inset presenting a zoom - in of an 0 . 5 s interval . The graph shows the recordings ( P18 - 21 ) averages over 30 neurons from WT ( n = 3 ) , 30 neurons from KO + A - mWwox ( n = 3 ) , 30 neurons from KO + A - hWWOX ( n = 3 ) , and 45 neurons from KO ( n = 3 ) . Immunofluorescence staining with anti - GFAP ( shown in green ) and anti - Iba1 ( shown in magenta ) antibodies showing the expression of astrocytes and microglia at P19 from WT , KO and KO + A - hWWOX . The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes . F , G . Graphs represents the quantification of GFAP ( F ) and Iba1 ( G ) positive cells from the images shown in E . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group ( n = 3 per each genotype ) . H . Immunocytochemical images showing astrocytes ( anti - GFAP ) and microglia ( anti - Iba1 ) in CA3 region of hippocampus from WT ( n = 3 ) and rescued ( KO + AAV - hWWOX ) ( n = 3 ) mice at 9 months . Magnified area is shown with square box . I , J . Graphs represents the quantification of GFAP ( I ) and Iba1 ( J ) positive cells from the images shown in H . The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections ( paraffin embedded ) from CA3 region per group ( n = 3 per each genotype ) .

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Figure 3Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.   The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).   Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes. F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype). H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box. I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).


Figure 4Images of whole brain sagittal sections that were immunolabelled with anti - MBP at P17 from indicated mice ( n = 3 for each group ) . MBP staining ( shown in gray ) in cortex ( green dotted line represents the border of the cortex ) , hippocampus and cerebellum is presented ( magnified from the top panel ) . Sagittal section of the brain ( P17 ) tissues immunostained for CC1 ( green ) and PDGFRα ( magenta ) . Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV - mWwox compared to Wwox null injected with AAV - GFP virus . Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum ( area 0 . 5 mm2 ) of WT ( n = 3 ) , KO ( n = 3 ) and KO + A - mWwox ( n = 3 ) counted from three similar brain sagittal sections per mouse in each genotype .

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Figure 4Images of whole brain sagittal sections that were immunolabelled with anti-MBP at P17 from indicated mice (n=3 for each group). MBP staining (shown in gray) in cortex (green dotted line represents the border of the cortex), hippocampus and cerebellum is presented (magnified from the top panel).Sagittal section of the brain (P17) tissues immunostained for CC1 (green) and PDGFRα (magenta). Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV-mWwox compared to Wwox null injected with AAV-GFP virus.   Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum (area 0.5 mm2) of WT (n=3), KO (n=3) and KO+A-mWwox (n=3) counted from three similar brain sagittal sections per mouse in each genotype.


Figure 5Electron micrograph ( EM ) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9 - hSynI - mWwox - injected mice compared to Wwox - null mice ( KO ) at P17 . Graph showing average number of myelinated axons in corpus callosum from WT ( n = 3 ) , KO ( n = 3 ) or KO mice injected with AAV9 - hSynI - mWwox ( n = 3 ) per field of view ( FOV ) . g - ratio analysis showing reduced myelin thickness of axons in corpus callosum KO ( n = 3 ) compared to the KO + AAV - Wwox ( n = 3 ) . Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum ( total no of axons n = 350 per each group ) . EM images showing increased number of myelinated axons in optic nerve in AAV9 - hSynI - mWwox injected mice compared to KO mice at P17 . Graph showing the average number of myelinated axons ( counted per FOV ) in optic nerve from WT ( n = 3 ) , KO ( n = 3 ) and KO + AAV - mWwox ( n = 3 ) . g - ratio ' s of myelinated axons from optic nerves showing improved myelin thickness ( less g - ratio ) after WWOX restoration ( KO + AAV - mWwox ) compared to KO alone ( n = 3 per genotype , total no of axons n = 200 per each group ) .

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Figure 5Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.   Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).   g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).   EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.   Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).   g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).


Figure 6Representative images of open field test showing tracking pattern ( open arena ) of WT ( females , n = 7 , males n = 6 ) and AAV - mWwox - injected KO mice ( females n = 7 , males n = 5 ) at 8 - 10 weeks . Dot plot graphs represent the velocity ( B ) , total distance travelled ( C )

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Figure 6Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C)


Figure 1A . CAF and WI - 38 cells pretreated with EVs from MDA - MB - 231 or MCF10A cells , or PBS for 48 h were unstarved ( U ) or starved ( S ) for 2 h in EBSS prior to addition of a mixture of leucine , arginine , and lysine ( LRK ) or a cocktail of essential AAs ( EAA ) . Cells were analyzed by Western blot analysis in a time course as indicated . B . Western blot analysis of puromycin ( Puro ) - labeled fibroblasts ( CAF , normal human mammary fibroblasts , WI - 38 , and NIH3T3 ) after 2 - h starvation and EAA add - back in the presence or absence of cycloheximide ( CHX ) . C . Western blot analysis of Puro - labeled fibroblasts cultured in growth medium .

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Figure 1A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated.B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium.


Figure 2B . TORCAR FRET reporter assay in WI - 38 or NIH3T3 cells after 2 - h starvation and EAA add - back at time 0 ( n = 3 biological replicates for each group ) . Data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA ) .

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Figure 2B. TORCAR FRET reporter assay in WI-38 or NIH3T3 cells after 2-h starvation and EAA add-back at time 0 (n=3 biological replicates for each group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA).


Figure 3A , B . Decreases of leucine , arginine , and lysine ( A ) as well as production of ammonium ( B ) in the conditioned medium ( CM ) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS ( n = 3 biological replicates for each group ) . C . Intracellular levels of leucine , arginine , and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells ( n = 3 biological replicates for each group ) .

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Figure 3A,B. Decreases of leucine, arginine, and lysine (A) as well as production of ammonium (B) in the conditioned medium (CM) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS (n=3 biological replicates for each group).C. Intracellular levels of leucine, arginine, and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells (n=3 biological replicates for each group).


Figure 4A . Western blot analysis of WI - 38 and NIH3T3 cells treated with EVs or PBS for 48 h . B . Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h . C . Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX . D . Western blot analysis of NIH3T3 cells that had been transfected with a pRK5 - RAGC - HA - GST expression plasmid or control vector and treated with EVs or PBS for 48 h , and then labeled with Puro . E . Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h .

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Figure 4A. Western blot analysis of WI-38 and NIH3T3 cells treated with EVs or PBS for 48 h.B. Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h.C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.E. Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h.


Figure 5A . Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti - miRNAs or control for 24 h and then treated with EVs or PBS for 48 h . C . Dual luciferase assay of the psiCHECK reporters containing the 3 ' UTR of RRAGC that was wild - type , with all three miR - 105 sites mutated , or with the miR - 204 site mutated . NIH3T3 cells were co - transfected with the reporter and indicated miRNA mimic ( n = 3 biological replicates for each group ) . D . Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h ( n = 3 biological replicates for each group ) .

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Figure 5A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h.C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group).D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).


Figure 6D . A heatmap showing the comparison of log2 fold change ( FC ) of the abundance of each identified 13C6 - labeled protein and its corresponding mRNA level determined by RNA - seq .

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Figure 6D. A heatmap showing the comparison of log2 fold change (FC) of the abundance of each identified 13C6-labeled protein and its corresponding mRNA level determined by RNA-seq.


Figure 7A , B . A breast cancer tissue array was analyzed for correlations among RagC , miR - 105 , and miR - 204 in tumor stroma by IHC / ISH - determined scores . Representative IHC and ISH images ( A ) and correlations determined by Kendall ' s tau correlation tests ( N = 24 independent tumors ; Kendall ' s tau and two - sided P value as indicated ) ( B ) are shown . Bar = 200 μm . C , D . Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR - 105 or empty vector were collected from NSG mice for RagC IHC ( n = 5 tumors for each group ) . The relative intensity of RagC stain in tumor stroma was quantified by ImageJ . Bar = 50 μm . In ( D ) , data are presented as mean ± SD . * P < 0 . 05 ( Student ' s t - test ) .

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Figure 7A,B. A breast cancer tissue array was analyzed for correlations among RagC, miR-105, and miR-204 in tumor stroma by IHC/ISH-determined scores. Representative IHC and ISH images (A) and correlations determined by Kendall's tau correlation tests (N=24 independent tumors; Kendall's tau and two-sided P value as indicated) (B) are shown. Bar=200 μm.C,D. Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR-105 or empty vector were collected from NSG mice for RagC IHC (n=5 tumors for each group). The relative intensity of RagC stain in tumor stroma was quantified by ImageJ. Bar=50 μm. In (D), data are presented as mean ± SD. *P<0.05 (Student's t-test).


Figure 1 ( A ) BMDMs from WT ( Pml + / + ) and Pml - / - mice were pre - treated with zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 30 min followed by co - treatment with AT - 406 ( 1 μM ) . Cell viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT and Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) and Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . ( D ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) , and GSK872 ( 10 μM ) , as indicated , for 8 h . Cell viability was determined by ATP assay . Data show mean ± SD of three biological replicates . ( E ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , IKK inhibitor BMS - 345541 ( 10 mM ) and Nec - 1 , as indicated , for 6 h and then cell death was assayed . Data show mean ± SD of five biological replicates , except the Nec - 1 - containing experiment ( n = 2 ) .

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Figure 1(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. (D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).


Figure 2 ( A , B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) ( A ) or TNF + zVAD ( Tz ) ( B ) , as in Fig . 1 , for the indicated times . Contents of RIPK1 , p - RIPK1 [ S166 ] , RIPK3 , p - RIPK3 , MLKL , and p - MLKL in cell lysates was analysed by immunoblots . Bottom panels , quantitation of p - RIPK1 [ S166 ] from three biological replicates using normalized intensity of p - RIPK1 [ S166 ] in WT cells at 4 h as 1 . Data show mean ± SD . ( D ) BMDMs were treated with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) for 18 h , and TNF generated was then analyzed by ELISA . Data show mean ± SD of four biological replicates . ( E ) BMDMs were treated with zVAD + AT - 406 ( 1 μM ) , with or without anti - TNF for 18 h , and cell viability quantitated . Data show mean ± SD of three biological replicates .

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Figure 2(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.


Figure 3 ( A ) Pml + / + and Pml - / - BMDMs were treated with zVAD for 30 min , followed by AT - 406 ( zA ) treatment at the indicated time - points , before preparing total cell lysates ( TCL ) . TCL were immunoprecipitated with anti - FADD and the amounts of p - RIPK1 [ S166 ] , RIPK1 and caspase - 8 in the precipitates and TCL were analysed . ( B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) as in ( A ) , and TCL were then harvested . TCL were immunoprecipitated with anti - caspase - 8 , and the contents of p - RIPK1 [ S166 ] and RIPK1 in the precipitates and TCL was analysed .

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Figure 3(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.


Figure 4 ( A , B ) WT ( Pml + / + ) and Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g body weight ) , and rectal body temperature ( A ) and survival ( B ) were determined at the indicated time - points . # , the temperature of only six mice was measured at the indicated time - points . Mean ± S . E . M is shown . * P < 0 . 05 as determined by two - way ANOVA ( A ) , or by Log - rank ( Mantel - Cox ) test ( B ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).


Figure 5 ( A ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated timeframes . The contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 and MK2 was analysed by immunoblotting . ( B ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times , and levels of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , and p38 at the indicated time points were then determined . ( C , D , E , F ) WT ( Pml + / + ) and Pml - / - mice were treated with DMSO or Nec - 1s ( 6 μg / g ) or MK2 inhibitor PF - 3644022 ( 75 μg / mouse ) by intraperitoneal injection 15 min before and 60 min after intravenous TNF ( 1 . 5 μg / g body weight ) injection . Body temperature ( C , E ) and survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined. (C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).


Figure 6 ( A , B ) WT and Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) ( A ) or with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) ( B ) , and then the contents of p - RIPK1 [ S321 ] , RIPK1 , p - p38 , p38 was determined at the indicated time - points . ( C ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with zVAD ( 20 μM ) , AT - 406 ( 1 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 18 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method . ( D ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method . ( F ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - mice were injected i . v . with TNF ( 1 . 5 mg / kg ) , and survival was monitored for 24 h . Data represent a combination of two independent experiments ( n = 10 ) . * P < 0 . 05 for Log - rank ( Mantel - Cox ) test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.


Figure 7 ( D ) WT and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times . p38 MAPK was immunoprecipitated , and its association with MK2 was then analysed . ( E ) Recombinant Flag - PML - I , GST - MK2 ( 60 ng ) and His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 and then PML , MK2 and p38 contents in the precipitate and reaction mixture ( input ) were detected by immunoblotting . ( F ) Recombinant Flag - PML - I , GST - MK2 ( 100 ng ) and His - p38 ( 200 ng ) were incubated , as indicated , at 30 ° C in in vitro kinase assay buffer ( 20 mM HEPES , 10 mM MgCl2 , 100 nM ATP , 1 mM DTT ) for 20 min . The levels of p - MK2 , MK2 , p38 , and PML ( FLAG ) in the reaction mixture were then determined .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.


Figure 1 ( B ) ARSA activity quantification ( nmol / h / mg ) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days . Data represent mean ± SD of 7 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against negative control ( E ) Analysis of different retinoids ' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug ( Adapalene 1 µM ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against DMSO control : * *

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days. Data represent mean ±SD of 7 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against negative control(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: **


Figure 2 ( D ) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) simultaneously treated with 10 and 20 µM tazarotene and bexarotene , respectively , up to 21 days . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent treatment times . # # # # p < 0 . 0001 . Difference against 0 days control : * p < 0 . 05 , * * * * p < 0 . 0001 . ( E ) Analysis and quantification of increased sulfatase activities different to ARSA , namely ARSB , GALNS , and STS in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( F ) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations ( FGE Gly247Arg , FGE Gly263Val , FGE Ala279Val , FGE Arg349Trp ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * * p < 0 . 0001 . ( G ) Quantification of ARSA activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 6 independent experiments ( biological replicates ) . Unpaired t - test . * * * * p < 0 . 0001 . ( H ) Quantification of SGSH activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . Unpaired t - test . * p < 0 . 05 , * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(D) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively, up to 21 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent treatment times. #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001.(E) Analysis and quantification of increased sulfatase activities different to ARSA, namely ARSB, GALNS, and STS in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001, **** p<0.0001.(F) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations (FGE Gly247Arg, FGE Gly263Val, FGE Ala279Val, FGE Arg349Trp) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001.(G) Quantification of ARSA activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 6 independent experiments (biological replicates). Unpaired t-test. **** p<0.0001. (H) Quantification of SGSH activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 3 independent experiments (biological replicates). Unpaired t-test. * p<0.05, *** p<0.001.


Figure 3 ( A ) Representative confocal images of control and MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green fluorescence ) and DAPI ( nuclei , blue ) . ( B ) Representative confocal images of control and MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green fluorescence ) and DAPI ( nuclei , blue ) . ( C ) Quantification of total intensity of LAMP1 - green fluorescence . N = 20 images and 13 z - series optical sections per condition with a step - size of 0 . 26 microns , displayed at maximum extension and automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD cells compared to DMSO treated control cells ) , # p < 0 . 05 ( MSD DMSO vs MSD treated ) . RFU : relative fluorescence units . ( D ) Quantification of LAMP1 - green fluorescence vesicle size ( µm ) . N = 20 images and 13 z - series optical sections per condition with a step - size of 0 . 26 microns , displayed at maximum extension and automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD cells compared to DMSO treated control cells ) , # # p < 0 . 01 ( MSD DMSO vs MSD treated ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (B) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (C) Quantification of total intensity of LAMP1- green fluorescence. N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), # p<0.05 (MSD DMSO vs MSD treated). RFU: relative fluorescence units. (D) Quantification of LAMP1- green fluorescence vesicle size (µm). N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), ## p<0.01 (MSD DMSO vs MSD treated).


Figure 4 ( A ) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene , 20 µM bexarotene , and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan - RAR receptor antagonist AGN 193109 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 3 - 11 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . Difference against 0 µM DMSO control ( B ) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene , 20 µM bexarotene , and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan - RXR receptor antagonist HX 531 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 4 - 14 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 . Difference against 0 µM DMSO control : * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RAR receptor antagonist AGN 193109 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 3-11 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01, ### p<0.001. Difference against 0 µM DMSO control (B) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RXR receptor antagonist HX 531 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 4-14 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01. Difference against 0 µM DMSO control: **** p<0.0001.


Figure 6 ( A ) ARSA protein amount quantification after treatment of MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) with tazarotene , bexarotene , and tazarotene / bexarotene in combination for 6 days referred to β - actin amounts and normalization of ARSA activity based on ARSA protein amount ( specific ARSA activity ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations . # p < 0 . 05 . Difference against 0 / 0 µM control : * p < 0 . 05 , * * * p < 0 . 001 . ( C ) Quantification of ARSA activities in CRISPR / Cas9 generated ARPE19 SUMF1 - / - cells and appropriate controls ( ARPE19 wild type , MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM for up to 21 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent concentrations . # # p < 0 . 01 . Difference against 0 days control : * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) ARSA protein amount quantification after treatment of MSD primary fibroblasts (variant FGE Gly247Arg homozygous) with tazarotene, bexarotene, and tazarotene/bexarotene in combination for 6 days referred to β- actin amounts and normalization of ARSA activity based on ARSA protein amount (specific ARSA activity). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0/0 µM control: * p<0.05, *** p<0.001.(C) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSD primary fibroblasts (variant FGE Gly247Arg homozygous)) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM for up to 21 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 days control: **** p<0.0001.


Figure 7 ( A ) Pulse - chase - experiment in HT1080 FGE Ser155Pro cells after pre - treatment with tazarotene / bexarotene and DMSO ( control ) for 3 days . Upper panel : representative autoradiogram of intracellular ( C ) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours . Lower panel : quantification of 35S isotope labelled intracellular FGE protein amounts . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Pulse-chase-experiment in HT1080 FGE Ser155Pro cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (C) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ** p<0.01, *** p<0.001.


Figure 1 ( B ) Pull - Down of GST - tagged α11 . 2 segments ( top immunoblot ; IB ) by immobilized MBP - β2AR C - terminus ( residues 326 - 413 of human β2AR ) . GST fusion proteins were detected by an anti - GST antibody and MBP fusion proteins by an anti - MBP antibody . Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP - β2AR . ( C ) Quantification of ( B ) ( * * * p < 0 . 001 , ANOVA ) . ( D ) Pull - Down of GST - CTC but not GST - CTD or GST - CTE ( middle IB ) by immobilized MBP - β2AR C - terminus ( left ) but not MBP alone ( right ) , all of which were present at comparable amounts ( bottom and top IBs , respectively ) . GST fusion proteins were detected by an anti - GST antibody and MBP fusion proteins by an anti - MBP antibody . ( E ) Quantification of ( D ) ( * * * p < 0 . 001 , ANOVA ) . ( F ) IP of β2AR in the presence of 10 μM GST ( Control ; Ctr ) or GST - tagged C - terminal fragments as indicated . CT4 and CTC specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle of same IB ) from β2AR ( bottom of same IB ) . Use of non - specific IgG ( left lane in right panels ) indicates specificity of IP . ( G , H ) . For quantification of coIPs , α11 . 2 and GluA1 immunosignals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) . ( I ) Representative IB showing amounts of the GST fusion proteins that were added in ( F ) , as detected by anti - GST antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Pull-Down of GST-tagged α11.2 segments (top immunoblot; IB) by immobilized MBP-β2AR C-terminus (residues 326-413 of human β2AR). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP-β2AR.(C) Quantification of (B) (***p<0.001, ANOVA).(D) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.(E) Quantification of (D) (***p<0.001, ANOVA).(F) IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST-tagged C-terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non-specific IgG (left lane in right panels) indicates specificity of IP.(G, H). For quantification of coIPs, α11.2 and GluA1 immunosignals were normalized to β2AR signals (***p<0.001, ANOVA).(I) Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti-GST antibodies.


Figure 2 ( A ) Titration of fluorescence polarization of FITC - peptides ( 100 nM ) spanning α11 . 2 aa 1906 - 1959 with purified MBP - β2AR C - terminus . Kd value was obtained by fitting a non - linear direct binding curve to Pep2 . ( B ) IP of β2AR in the absence ( Control ) or presence of 10 μM peptides , as indicated . Pep2 specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle , same IB ) from β2AR ( bottom , same IB ) . Use of non - specific IgG ( left lane ) indicates specificity of coIPs . ( C , D ) . For quantification , α11 . 2 and GluA1IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) . ( E ) IP of β2AR in the absence ( Control ) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated ( phPep2 ) , which did not displaced α11 . 2 . ( F ) α11 . 2 and GluA1 IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Titration of fluorescence polarization of FITC-peptides (100 nM) spanning α11.2 aa 1906-1959 with purified MBP-β2AR C-terminus. Kd value was obtained by fitting a non-linear direct binding curve to Pep2.(B) IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non-specific IgG (left lane) indicates specificity of coIPs.(C, D). For quantification, α11.2 and GluA1IB signals were normalized to β2AR signals (***p<0.001, ANOVA).(E) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).


Figure 3Forebrain slices from WT ( A - F ) and S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , and sequential IB for pS1928 , pS1700 , and α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of the blots , as indicated . All the α11 . 2 IPs in A - C and G - I were from the same samples ( which were split in half for parallel IP ) as the β2AR IPs in D - F and J - L , respectively ( for quantification of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , the time - dependent increase in S1928 and S1700 phosphorylation ( A - C ) paralleled the decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) and of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt the α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For quantification of α11 . 2 phosphorylation , pS1928 and pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) . Forebrain slices from WT ( A - F ) and S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , and sequential IB for pS1928 , pS1700 , and α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of the blots , as indicated . All the α11 . 2 IPs in A - C and G - I were from the same samples ( which were split in half for parallel IP ) as the β2AR IPs in D - F and J - L , respectively ( for quantification of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , the time - dependent increase in S1928 and S1700 phosphorylation ( A - C ) paralleled the decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) and of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt the α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For quantification of α11 . 2 phosphorylation , pS1928 and pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) . ( E , F , K , L ) For quantification of coIP , α11 . 2 and GluA1 signals were normalized to β2AR ( * * * p < 0 . 001 , One Way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).(E,F,K,L) For quantification of coIP, α11.2 and GluA1 signals were normalized to β2AR (***p<0.001, One Way ANOVA).


Figure 4Hippocampal cultures were transfected with FLAG - β2AR and α11 . 2 - HA at 6 days in vitro ( DIV ) , treated with vehicle ( water ) or 1 M isoproterenol ( ISO ) for 5 min at 18 DIV , fixed and surface labeled for HA and FLAG . ( A , B ) Representative immunofluorescence microscopy images obtained by wide - field microscopy at lower and higher resolutions ( scale bar in A is 5 m ) . ( C ) Quantification of distance between centers of HA and FLAG puncta ( * * p < 0 . 001 , Mann - Whitney Rank Sum Test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.(A,B) Representative immunofluorescence microscopy images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).


Figure 5Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) β2AR was IPed before IB for α11 . 2 ( top part of IB ) , GluA1 ( middle part of IB ) , and β2AR ( bottom part of IB ) , as indicated . ( B , C ) For quantification , α11 . 2 and GluA1 IB signals were normalized to β2AR ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( A , lanes 2 - 6 ) lasted at least 3 min but not 20 min ( compare lanes 6 and 7 ) . Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( D ) α11 . 2 and GluA1 were concurrently IPed from same samples as in ( A ) by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 ( top three panels ) and middle part for pS845 , pS831 , and total GluA1 ( bottom three panels ) . ( E - H ) For quantification , pS1928 and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( see A ) rendered α11 . 2 ( but not GluA1 ) refractory to re - phosphorylation of S1928 and S1700 upon a second ISO application of α11 . 2 ( D , compare lanes 5 and 6 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).


Figure 6Forebrain slices from S1928A KI mice were treated with vehicle ( lanes 1 , 5 ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) α11 . 2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 and middle part for pS845 , pS831 , and total GluA1 . In S1928A KI mice , S1700 re - phosphorylation after a 3 min wash out of ISO was not blunted ( lane 6 ) in contrast to WT mice but rather augmented ( compare to lane 5 ) . ( B - E ) For quantification , pS1928 ( virtually absent ) and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).