Figure 3 ( B , C ) Percentage of GAPDH or 5 . 8 rRNA levels quantified by qPCR in reactions containing increasing doses of ( PR ) 20 ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . ( D ) HTM - mediated analysis of 5 - ethynyl - deoxyuridine ( EdU ) levels per nucleus found in U2OS cells exposed to 10 μM ( PR ) 20 for 4 h . EdU was added 30 ´ prior to fixation . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm . ( E ) HTM - mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM ( PR ) 20 for the indicated times . Note the accumulation of cells with persistent DNA damage ( γH2AX ) 10 h after IR in the presence of the DPR . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm .

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 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 3(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


Figure 4 ( D ) Immunofluorescence of Cy3 - labelled salmon protamine ( red ) and the nucleolar factor UBF1 ( green ) in U2OS cells treated with 30 μM Cy3 - protamine for 4 h . Scale bar ( white ) indicates 2 . 5 μm . ( E , F ) HTM - mediated quantification of the EU levels per nucleus ( E ) and O - propargyl - puromycin ( OPP ) levels per cell ( F ) in U2OS cells exposed to 30 μM protamine for 16 ( E ) or 24 ( F ) h . EU and OPP were added 30 ´ prior to fixation . Black lines indicate median values ( * * * * , p < 0 . 0001 ; t - 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 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 4(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).


Figure 6 ( D ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test . ( E ) Immunofluorescence of HA - ( PR ) 20 ( green ) and Cy5 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 4 μM of Cy5 - labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test . ( F ) Clonogenic survival assay of U2OS cells exposed to 7 . 5 μM with the indicated peptides or protamine ( PROT . ) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0 . 5 μM Heparin . ( G ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with heparin ( 0 . 5 μM ) . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - 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 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 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 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

Figure 6(D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.(E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


Figure 7 ( A ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without 2 μM of a non - coding 38nt oligonucleotide . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . Note that besides its impact on overall survival , the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide . ( B , C ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( B ) and NSC34DIFF ( C ) treated as in ( A ) ( n = 3 ) . Data represent mean values ± SEM . ( Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . ( D ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without heparin ( 0 . 5 μM ) . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . ( E , F ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( E ) and NSC34DIFF ( F ) treated as in ( D ) ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . S

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 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

Figure 7(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S


Figure 4 ( B ) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs , except in the heart and cerebellum . RT - PCR for GAPDH was used as control ( Ctrl . ) . ( D ) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks ( 3w ) ( 1 piglet , 1 retina , 1 duodenum ) and 1 year ( 1y ) ( 2 pigs , 1 retina each , 3 TRs ) . Arrow indicates the expected size of harmonin isoform a ( 72kDa ) . ( E ) Representative immunofluorescence staining of harmonin ( green ) of longitudinal cryosection through WT and USH1C pig retinas ( 2 pigs , 1y , 1 retina each , 3 TRs , scale bar 10µm ) demonstrated the absence of harmonin staining in the outer segment ( OS ) of photoreceptor cells and in the other layers below the outer limiting membrane ( OLM , arrow ) of the USH1C pig retina . ( F ) Representative Western blot analysis of retinal protein extracts , indicating that the abundance of the USH proteins SANS ( USH1G ) , whirlin ( USH2D ) or myosin7a ( USH1B ) is unaffected ( 1 piglet , 3w , 1 retina each , 2 TRs ) .

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 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 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 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

Figure 4(B) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT-PCR for GAPDH was used as control (Ctrl.).(D) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72kDa).(E) Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1y, 1 retina each, 3 TRs, scale bar 10µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina.(F) Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).


Figure 5 ( A , B ) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks ( 3w ) . ( A ) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM ( arrow ) to the ganglion cell layer ( GCL ) of the retina . The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis . IS , inner segment ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; scale bar : 20 µm . ( B ) Left panel : Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls . Anti - actin Western blot was used as loading control . Right panel : Quantification of Western blot bands in 4 gels by the LI - COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age - matched WT controls ( 2 piglets , 3w , 2 retinas each , 2 TRs , error bars represent standard deviation ( SD ) of the mean , two - tailed Student ' s t tests , * p < 0 . 05 ) . ( C , D ) Reduced synaptic width in USH1C pigs at an age of 1 year ( 1y ) . ( C ) Fluorescent microscopic analysis of the cone synapse phenotype . Left panel : Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre - synaptic marker PSD - 95 ( green ) and by fluorescent peanut agglutinin ( PNA , red ) for cone synaptic pedicles ( white arrows ) and counter - stained by DAPI for nuclear DNA ( blue ) . Scale bar 10µm . Middle panel : Higher magnification of a PNA - stained cone synaptic pedicle . Synapse width was determined as the maximum extension of consistent PNA signals . Scale bar 1µm . Right panel : Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA - stained sections indicated reduced synaptic width ( 2 pigs , 1y , 1 retina each , number of examined synapses indicated , error bars represent SD , two - tailed Student ' s t tests , * * * p < 0 . 001 ) . ( D ) Determining cone synaptic pedicles width by TEM . Left panel : Representative images of retinal cross sections . Scale bar 500nm . Right panel : Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs . ( 1 pig , 1y , 1 retina each , number of examined synapses indicated ,

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 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 5(A, B) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks (3w). (A) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM (arrow) to the ganglion cell layer (GCL) of the retina. The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; scale bar: 20 µm. (B) Left panel: Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls. Anti-actin Western blot was used as loading control. Right panel: Quantification of Western blot bands in 4 gels by the LI-COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age-matched WT controls (2 piglets, 3w, 2 retinas each, 2 TRs, error bars represent standard deviation (SD) of the mean, two-tailed Student's t tests, *p<0.05).(C, D) Reduced synaptic width in USH1C pigs at an age of 1 year (1y). (C) Fluorescent microscopic analysis of the cone synapse phenotype. Left panel: Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre-synaptic marker PSD-95 (green) and by fluorescent peanut agglutinin (PNA, red) for cone synaptic pedicles (white arrows) and counter-stained by DAPI for nuclear DNA (blue). Scale bar 10µm. Middle panel: Higher magnification of a PNA-stained cone synaptic pedicle. Synapse width was determined as the maximum extension of consistent PNA signals. Scale bar 1µm. Right panel: Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA-stained sections indicated reduced synaptic width (2 pigs, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, ***p<0.001). (D) Determining cone synaptic pedicles width by TEM. Left panel: Representative images of retinal cross sections. Scale bar 500nm. Right panel: Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs. (1 pig, 1y, 1 retina each, number of examined synapses indicated,


Figure 6 ( A - C ) Ciliary length measured by fluorescence microscopic analysis at 1 year of age ( 1y ) . ( A ) Representative immunostaining of the ciliary marker Gt335 ( green ) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas . Counterstaining by DAPI , scale bar 10µm . ( B ) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining . Scale bar 500nm . ( C ) Length of connecting cilia ( CC ) appear elongated in USH1C when applying a Fiji script to immunostained histological sections ( 2 pigs , 1y , 1 retina each , number of examined connecting cilia is indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) . ( D , E ) Measuring CC length in TEM sections . ( D ) Representative images of longitudinal cross sections and principle of determining CC length . Scale bar 500nm . ( E ) Elongation of CC length is confirmed in TEM sections of USH1C PRC . ( 1 pig , 1y , 1 retina each , number of examined connecting cilia indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) . Length of primary cilia in dermal fibroblasts . ( F ) Staining of ciliary structures in fibroblasts stained with ARL13B ( ciliary shaft , green ) and PCNT2 ( ciliary base , red ) , DAPI ( nucleus , blue ) . Scale bar upper panel 5 µm , lower panel 2 . 5 µm . ( H ) Similarly , ciliary length of fibroblasts from a human USH1CR31X / R80fs patient are elongated , compared to a healthy control ( 7 TR , number of cells examined indicated , KS - test , * * * * p < 0 . 0001 ) . ( I - M ) Outer segment ( OS ) PRC architecture by TEM of longitudinal retinal sections . ( I ) At 3w , WT show normal sub - cellular organization , including parallel stacking of photosensitive discs and calyceal processes ( black arrows , representative image from 2 pigs , 2 retinas , scale bar 600nm ) . ( J ) In 3w USH1C rods , membrane discs appear also in vertical orientation ( white arrows , representative image from 2 pigs , 2 retinas , scale bar 750nm ) . At 1y , interstitial gaps appeared in disc stacks of the OS in USH1C pigs ( K , arrow , scale bar 550nm ) and vesicles are found at the OS base ( L , asterisk , scale bar 500nm ) . ( M ) The disc architecture in 1y animals was substantially distorted ( arrows , representative image from 2 pigs , 2 retinas , scale bar 850nm ) .

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 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 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 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

Figure 6(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).(D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).(I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm).


Figure 7 ( A ) AAV transduction of retinal layers . AAV8 , AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo . Upper Panel : Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples . Dashed circles indicate bleb size . Lower panel : Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer ( OS , IS and ONL ) for all AAV . Increased exposure ( 4x ) documented transduction of MGC ( arrows ) by AAV9 and Anc80 but not by AAV8 ( representative images from 1 pig , 1 retina each , 2 TR , scale bars 25 µm ) . ( D ) Efficacy of AAV gene therapy was examined in vivo by sub - retinal injection of Anc80 virus capsid expressing harmonin _ a1 under a ubiquitous promoter into a USH1C pig ( see also Figure EV5 ) . Left panel : Sub - retinal injection reconstituted harmonin expression in the AAV - treated eye but not in the sham - treated eye ( 3 TR ) . Protein extract from a WT retina served as positive control , actin staining as loading control . The 72kD band correlates with harmonin _ a isoforms while higher MW indicates putative dimer formation . Middle panel : Immunofluorescence reveals absence of harmonin in the retina of the PBS - injected control eye and harmonin abundance ( green ) in the retina of the AAV - treated eye . ( Representative image from a single animal . Blue : DAPI , scale bar 25 µm ) . Right panel : Photopic gfERG reveal increased a - and b - wave response to single flash and flicker stimulation in the AAV - treated ( red ) , compared to the sham - treated ( black ) eyes of two USH1C pigs .

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 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

Figure 7(A) AAV transduction of retinal layers. AAV8, AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo. Upper Panel: Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples. Dashed circles indicate bleb size. Lower panel: Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer (OS, IS and ONL) for all AAV. Increased exposure (4x) documented transduction of MGC (arrows) by AAV9 and Anc80 but not by AAV8 (representative images from 1 pig, 1 retina each, 2 TR, scale bars 25 µm).(D) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.


Figure 2A - H ) slices through electron cryotomograms of H . gracilis cells ( left panels ) , central slices through subtomogram averages ( STA , middle panels ) and schematic representations ( right panels ) representing the various assembly stages observed . White arrow in ( A ) points to the MS - complex ; green arrows point to the novel 53 - nm - wide cytoplasmic ring . Scale bars in ( H ) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels . Empty panels represent stages where not enough particles were found to produce a STA . OM = outer membrane , IM = inner membrane .

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 2A-H) slices through electron cryotomograms of H. gracilis cells (left panels), central slices through subtomogram averages (STA, middle panels) and schematic representations (right panels) representing the various assembly stages observed. White arrow in (A) points to the MS-complex; green arrows point to the novel 53-nm-wide cytoplasmic ring. Scale bars in (H) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels. Empty panels represent stages where not enough particles were found to produce a STA. OM=outer membrane, IM=inner membrane.


Figure 3A - D ) slices through electron cryotomograms of H . pylori ( left panels ) indicating the presence of various flagellar intermediates . Middle panels show central slices through STAs . Right panels show schematic representations . Question marks in the schematic in ( C ) indicate that it is unclear whether the densities surrounding the upper part of the C - ring are the same as the novel cytoplasmic rings seen in the MS - complex . Empty panels indicate that there were not enough examples to produce a STA . E & F ) cryotomogram slices , STAs , and schematics of H . pylori fliP * cells showing the MS - complex ( E ) and the C - complex ( F ) . The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel . White arrows in ( A - F ) point to the particles . G & H ) central slices through the STAs of the MS - complexes of the motile H . pylori ( G ) and H . pylori FliP * mutant ( H ) with the diameters of their various constituent rings indicated .

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 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 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

Figure 3A-D) slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA.E & F) cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles.G & H) central slices through the STAs of the MS-complexes of the motile H. pylori (G)and H. pylori FliP* mutant (H) with the diameters of their various constituent rings indicated.


Figure 5A - G ) Slices through electron cryotomograms ( left panels ) , central slices of subtomogram averages ( middle panels ) , and schematic representations ( right panels ) of the MS - and C - complexes ( white arrows on the slices ) of different mutants in H . pylori fliP * . Light and dark green arrows in ( A and D ) point to the ~ 53 - nm and ~ 67 - nm cytoplasmic rings . Scale bars for cryotomogram slices are 50 nm , and 20 nm for STAs . Empty panels indicate that there were not enough examples to produce a STA .

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

Figure 5A-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.


Figure 1Treatment of HeLa cells with DFP ( 1mM for 24 h ) reduces the conjugation of SUMO - 2 / 3 ( n = 5 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change global SUMO - 1 - ylation ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to increased levels of SENP3 ( n = 8 , biological replicates ; * * p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change levels of SENP5 ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) . DFP treatment increases SENP3 stability . DFP ( 1mM for 24 h ) - treated HeLa cells were treated with cycloheximide ( CHX ; 50μg / ml ) for the indicated durations in a chase experiment . Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated .

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Figure 1Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.


Figure 2Treatment of HeLa cells with DFP ( 1mM for 24 h ) decreases levels of CHIP ( n = 8 , biological replicates ; * * * p < 0 . 001 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to decreased levels of CHIP mRNA ( n = 3 , biological replicates ; * p < 0 . 05 ; unpaired t - test ) . Treatment of DFP ( 1mM for 24 h ) - treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP . Histogram shows relative levels of cellular CHIP upon four different conditions ( n = 3 , biological replicates ; * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; repeated measures analysis of variance followed by Tukey ' s multiple comparisons test ) . CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells . Nsi or CHIPi ( Nsi , non - specific siRNA ; CHIPi , CHIP siRNA ; concentration , 20 nM ) was transfected into HeLa cells for 48 h . Lysate samples were blotted as indicated . CHIP knockdown prevents the DFP - induced increase in SENP3 levels . Nsi , SENP3i or CHIPi ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; CHIPi , CHIP siRNA ; siRNA concentration , 20nM ) was transfected into HeLa cells for 48 h . Two days post - transfection the cells were treated with DFP for a further 24 h .

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Figure 2Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).  Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.


Figure 3SENP3 knockdown abolishes the DFP - induced increase in LC3 - II in HeLa cells . Nsi or SENP3i ( I ) was transfected into HeLa cells . Two days post - transfection the cells were treated with DFP for a further 24 h . Whole cell lysate samples were blotted as indicated ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; siRNA concentration , 20nM ; DFP , 1mM for 24 h ; values normalized to the control value ; n = 3 , biological replicates ; * * * , p < 0 . 001 ; N . S . , not significant , p > 0 . 05 ; paired t - test ) . Nsi or SENP3i ( II ) was transfected into HeLa cells with stable expression of mito - Keima grown on 6 - well plates . Two days post - transfection the cells were treated with DFP ( 1mM ; 24 h ) . Cells transfected with Nsi and treated with CQ ( 50μM ; 24 h ) in the absence or presence of DFP ( 1mM ; 24 h ) were included as a negative control for mitophagy induction . The images show overlaid fluorescence and transmitted light images of unfixed , live cells obtained using an EVOS - fl inverted LED fluorescence microscope ( Red : mitophagic puncta ; Scale bar , 100 μm ) . Histogram shows the percentages of mitophagic cells under 6 different conditions ( n = 3 , biological replicates ; * * * * , p < 0 . 0001 ; Ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test ) . SENP3 knockdown abolishes DFP - induced mitophagic autolysosomes . Nsi or SENP3i ( I ) together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , and were analyzed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Magenta : SENP3 ; Scale bar , 10 μm ; n = 28 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - 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 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 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 3SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).


Figure 4Fis1 knockdown abolishes DFP - induced LC3 - II . Fis1 siRNA was introduced into HeLa cells for 48 h , and the cells were treated with DFP for further 24 h ( Nsi , non - specific siRNA ; Fis1i , Fis1 siRNA ; siRNA concentration , 20nM ; DFP , 1mM ) . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 3 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Fis1 knockout abolishes DFP - induced LC3 - II . Fis1 + / + or Fis1 - / - cells were treated with DFP for 24 h . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 6 , biological replicates ; * , p < 0 . 05 ; paired t - test ) . Fis1 knockdown prevents DFP - induced mitophagic autolysosomes . Nsi or Fis1i together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , which were analysed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Blue : Fis1 ; Scale bar , 10 μm ; n = 25 cells per condition from three individual experiments ; * * * p < 0 . 001 ; unpaired - t 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 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 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

Figure 4Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).


Figure 5Flag - Fis1 is deSUMOylated by SENP3 . SENP3 knockdown enhances Fis1 SUMOylation . Nsi or SENP3i was transfected into HEK293 cells expressing Flag - Fis1 , His - SUMO - 2 and Ubc9 for 48 h . His - pulldown and lysate samples were detected by immunoblotting for Flag , SENP3 or β - actin . Values are presented as mean ± SEM ( n = 3 , biological replicates ; * p < 0 . 05 ; paired - t test ) .

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Figure 5Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).


Figure 6The SUMOylation status of Fis1 influences its mitochondrial localization . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG was expressed in HeLa cells for 48 h . Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated ( the upper panel ) . Histograms ( the right panel ) show normalized levels of Flag - Fis1 WT or Flag - Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction ( Value are presented as mean ± SEM and are normalized to the control value ; n = 7 , biological replicates ; * , p < 0 . 05 ; paired t - test ) . The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum ( ER ) . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG along with GFP - Cb5 as an ER marker were expressed in HeLa cells for 48 h . Immunocytochemistry was then performed ( Green : ER labelled by GFP - Cb5 ; Blue : different Flag - Fis1 forms ; Scale bar , 10 μm )

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 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

Figure 6The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)


Figure 7Expressing a SUMOylation - deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP - induced LC3 - II induction . pcDNA3 , Flag - Fis1 WT or Flag - Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ( I ) ; siRNA concentration , 20nM ) , and the cells were treated with DFP for a further 24 h ( DFP , 1mM ) . Lysate samples were blotted as indicated . SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP - Fis1 K149R in the presence of DFP . CFP - Fis1 WT or CFP - Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h , and the cells were treated with DFP for a further 24 h ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Cyan / blue : CFP - Fis1 ; Magenta , SENP3 ; Scale bar , 10 μm ) ; Values are presented as mean ± SEM ( n = 21 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) . Knockdown of SENP3 was further confirmed by immunoblotting ( the lower right panel ) .

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 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

Figure 7Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).


Figure 1A ) oxLDL plasma concentration in ACM patients and HC ( n = 36 ; Mann - Whitney test ) . B ) oxLDL plasma concentration in mutated ACM ( n = 7 ) and NON ACM relatives , carriers of the same causative mutation ( n = 9 ; Mann - Whitney test ) . C ) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations , or gene elusive ( n = 10 vs . n = 26 ; One - Way ANOVA ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . D ) Representative images of MDA immunostaining ( green ) on ACM and HC ventricular tissue sections and relative quantification ( n = 4 biological replicates ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . E ) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification ( n = 4 biological replicates ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 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 1A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test). B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test). C) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.D) Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


Figure 2B ) Left panels : representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI . On the left a case with RV dilation , regional bulging of the RV wall without fat infiltration , and oxLDL levels below the cut - off ; in the center and on the right , two cases with fat infiltration in the right or left ventricle wall ( white arrow ) , respectively , and oxLDL levels above the cut - off . Right panel : quantification of the myocardial fat mass of the two ACM subpopulations ( above or below the cut - off ) whose MRI was available for re - analysis ( n = 14 oxLDL < 86ng / ml , n = 25 oxLDL > 86ng / ml ; Two - tailed Student ' s t - test ) . G ) Kaplan - Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5 - year follow - up ( n = 26 oxLDL < 86ng / ml vs . n = 41oxLDL > 86ng / ml ; Log - Rank p < 0 . 0001 ; HR = 0 . 223 [ 0 . 116 - 0 . 428 ] ) .

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 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

Figure 2B) Left panels: representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI. On the left a case with RV dilation, regional bulging of the RV wall without fat infiltration, and oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass of the two ACM subpopulations (above or below the cut-off) whose MRI was available for re-analysis (n=14 oxLDL<86ng/ml, n=25 oxLDL>86ng/ml; Two-tailed Student's t-test).G) Kaplan-Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5-year follow-up (n=26 oxLDL<86ng/ml vs. n=41oxLDL>86ng/ml; Log-Rank p<0.0001; HR=0.223[0.116-0.428]).


Figure 3B ) Left panels : representative images of MDA immunostaining ( green ) on ACM and HC C - MSC in GM . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : image quantification ( n = 4 biological replicates ACM , n = 5 biological replicates HC ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . D ) Top panel : representative images of Western Blot analysis of proteins extracted from ACM and HC C - MSC cultured in GM , hybridized with anti - CD36 and anti - PPARγ antibodies . Immunostaining of the housekeeping GAPDH is shown for normalization . Bottom panel : d . a . of PPARγ ( n = 18 biological replicates ) and CD36 ( n = 17 vs . n = 16 biological replicates ) levels , normalized on GAPDH ( Mann - Whitney test ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . F ) Top panels : representative images of internalization of oxLDL ( red ) in HC and ACM cells , cultured either in GM or in AM , and subjected to 10μg / ml DiI - oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Bottom panel : quantification of the relative mean DiI fluorescence for each sample , measured by FACS analysis ( n = 3 biological replicates ; Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 .

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

Figure 3B) Left panels: representative images of MDA immunostaining (green) on ACM and HC C-MSC in GM. Nuclei are counterstained with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05, ** p<0.01.D) Top panel: representative images of Western Blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARγ antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARγ (n=18 biological replicates) and CD36 (n=17 vs. n=16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test). Data information: mean ± SEM. * p<0.05, ** p<0.01.F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01.


Figure 4A ) Left panels : representative images of ORO staining on ACM and HC C - MSC in AM supplemented or not with 150µg / ml oxLDL . Middle panel : image quantification ( n = 11 biological replicates ; Two - Way Anova ) . Right panels : representative images of Western Blot of CD36 , PPARγ and GAPDH protein expression of ACM and HC C - MSC protein extracts in AM supplemented or not with 150µg / ml oxLDL ( n = 5 biological replicates ) and d . a . normalized on the house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B ) Left panels : representative images of ORO staining on ACM and HC C - MSC in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both . Right panel : image quantification ( n = 13 biological replicates ; Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C ) Left panel : representative images of Western Blot of CD36 , PPARγ and GAPDH protein expression of ACM and HC C - MSC protein extracts in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both ( n = 8 biological replicates ) . Right panels : d . a . normalized on the house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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

Figure 4A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


Figure 5D ) Left panel : representative images of internalization of oxLDL ( red ) in ACM C - MSC treated with scramble siRNA or CD36 siRNA , cultured in AM and subjected to 10μg / ml DiI oxLDL treatment . Right panel : quantification of the DiI fluorescence normalized on nuclei number ( n = 3 biological replicates , arbitrary units ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 . E ) Left panel : representative images of internalization of oxLDL ( red ) in ACM C - MSC cultured in AM or AM + 5μM GW9662 and subjected to 10μg / ml DiI oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : quantification of the DiI fluorescence normalized on nuclei number for each sample ( n = 3 biological replicates , arbitrary units ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 . F ) Left panel : representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C - MSC cultured in AM or AM + 5μM GW9662 . Right panels : d . a . normalized on the house - keeping GAPDH ( n = 7 biological replicates ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 .

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 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 5D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


Figure 6C ) Left panels : representative images of ORO staining of HFD - fed WT and Pkp2 + / - cardiac sections . Right panel : quantification of ORO positive area percentage ( n = 10 ) . For comparison , quantification of ORO positive area of cardiac sections of CD - fed WT and Pkp2 + / - mice ( n = 9 ; Two - Way Anova ) is shown ( Appendix Figure S3 ) . D ) Representative images of PPARγ ( green ) immunostaining on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Quantification of the PPARγ staining in HFD is shown relative to the values of CD ( n = 9 ) . E ) Representative images of MDA ( green ) immunostaining on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Quantification of the MDA staining in HFD is shown relative to the values of CD ( n = 9 ) . F ) Representative images of CD36 immunostaining ( green ) on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Quantification of the staining in HFD is shown relative to the values of CD ( n = 9 ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

Figure 6C) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


Figure 7C ) Left panel : representative images of ORO staining of cardiac sections of Pkp2 + / - mice , fed a 3 - month HFD plus atorvastatin . Right panel : quantification of the percentage of ORO positive area ( n = 9 ) is compared to that in HFD ( as in Figure 6C ; Two - tailed Student ' s t - test ) . D ) Representative images of PPARγ immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - in HFD and relative to WT in CD ( as in Figure 6D ; Two - tailed Student ' s t - test ) . E ) Representative images of MDA immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - in HFD and relative to WT in CD ( as in Figure 6E ; Two - tailed Student ' s t - test ) . F ) Representative images of CD36 immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - HFD and relative to WT in CD ( as in Figure 6F ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

Figure 7C) Left panel: representative images of ORO staining of cardiac sections of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test). D) Representative images of PPARγ immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test). E) Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test). F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD and relative to WT in CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


Figure 1 A ) Hek293T cells expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) and schematic of BRET assay , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm . B ) Lamplight - driven response to increasing intensity 1s 405nm light C ) 405nm irradiance response curve D ) Response to 1s 405nm ( blue line ) and 525nm light ( green line ) E ) Response to 405nm ( blue line ) followed by 525nm light of different intensities ( green line ) G ) Response to 405nm alone or with 525nm light H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) dark or ( I ) after 8s 405nm light ( blue bar ) J ) Sustained Lamplight - driven activity ( mean BRET ratio 20 - 80s after stimulus ) after exposure to mixed stimuli from dark ( ON ) or 405nm light - adapted state ( OFF ) . Lamplight data fit with linear trendline . Best fitting parameters for ON , slope = 0 . 311 , Y - intercept = 0 . 04 , R2 = 0 . 85 . For OFF , slope = 0 . 30 , Y - intercept = 0 . 07 , R2 = 0 . 69 ) .

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

Figure 1 A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line) G) Response to 405nm alone or with 525nm light  H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).


Figure 2 B ) Representative trace of hyperpolarisation to 405nm light in a Lamplight - expressing SCN neuron C ) Hyperpolarisation to 405nm was observed over a range of intensities . Recordings from 3 different cells across 2 SCN slices D ) Representative trace showing hyperpolarisation was sustained during dark after 405nm light ( 5s ) and reversed by 525nm light ( 15s ) E ) Hyperpolarisation and depolarisation of a Lamplight - expressing neuron by 405nm and 525nm light , respectively , across multiple stimulus presentations F ) Three example traces from a single cell exposed to 405nm followed by 525nm light . Individual traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2 cells G , H ) Parameters from best - fit curves used to calculate ( G ) latency ( ms ) and ( H ) half - life ( ms ) . I ) Simultaneous 525nm and 405nm light prevents spike silencing J ) Constant 405nm light ( 12 . 9 log photons ) presented with 525nm light of decreasing intensity from 14 . 9 - 12 . 9log photons in 6 steps of 10s ( left ) or 20s ( right ) . 525nm light antagonised the 405nm - driven hyperpolarisation for all intensities except 12 . 9log photons

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Figure 2 B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron C) Hyperpolarisation to 405nm was observed over a range of intensities. Recordings from 3 different cells across 2 SCN slices  D) Representative trace showing hyperpolarisation was sustained during dark after 405nm light (5s) and reversed by 525nm light (15s)  E) Hyperpolarisation and depolarisation of a Lamplight-expressing neuron by 405nm and 525nm light, respectively, across multiple stimulus presentations  F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells G, H) Parameters from best-fit curves used to calculate (G) latency (ms) and (H) half-life (ms). I) Simultaneous 525nm and 405nm light prevents spike silencing J) Constant 405nm light (12.9 log photons) presented with 525nm light of decreasing intensity from 14.9 - 12.9log photons in 6 steps of 10s (left) or 20s (right). 525nm light antagonised the 405nm-driven hyperpolarisation for all intensities except 12.9log photons


Figure 3 A ) L7Cre rd1 retina expressing Lamplight - T2A - mCherry AAV . Anti - mCherry staining ( white ) was widespread ( Left , scale bar = 500µm ) . Fluorescent cell bodies found in different focal planes consistent with expression in ganglion ( Top Right , scale bar = 50µm ) and bipolar cells ( Bottom Right , scale bar = 50µm ) . B ) Representative single unit showing characteristic ipRGC response . C ) Three Lamplight response categories - transient excitation ( N = 35 biological replicates ) , transient inhibition ( N = 6 biological replicates ) , and sustained inhibition ( N = 8 biological replicates ) . Left panel shows representative single unit response to 2s 405nm or 525nm light . Mean baseline normalised firing ( grey shading shows SEM ) is shown in centre left ( for entire recording , 500ms bins ) , centre right and far right panels ( during 405nm and 525nm stimuli , respectively 100ms bins , N as above , except N = 5 biological replicates for sustained response to 525nm ) . D ) Representative single unit showing light responses over 20 stimulus repeats . E ) Distribution of response latencies for 3 Lamplight - driven responses to 405nm , as well as reversal caused by 525nm in sustained inhibition units . Two outliers with slow latency ( > 2 . 5s ) in transient excitation group are likely responding to stimulus offset ( N = 35 for Transient Excitation , N = 6 for Transient Inhibition , N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation , all biological replicates ) . F ) Inhibition responses were smaller and less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) . G ) Excitation responses were attenuated for mixed stimuli compared to 405nm only . Left panel shows representative retinal single unit ( N = 35 biological replicates ) . Data information : For ( F - G ) Left panel shows representative single unit . Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus . All representative single units show perievent rasters ( first trial at top ) and firing rate histograms ( Bin size = 500ms ) . Timing of light stimuli shown by shaded vertical bars . All light stimuli are 16 log total Lamplight effective photons / cm2 / s . Error bars show SEM . * p < 0 . 05 , * * * p < 0 . 001 ( One - sample t - test compared to zero ) .

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

Figure 3 A) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV. Anti-mCherry staining (white) was widespread (Left, scale bar = 500µm). Fluorescent cell bodies found in different focal planes consistent with expression in ganglion (Top Right, scale bar = 50µm) and bipolar cells (Bottom Right, scale bar = 50µm). B) Representative single unit showing characteristic ipRGC response. C) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm).D) Representative single unit showing light responses over 20 stimulus repeats. E) Distribution of response latencies for 3 Lamplight-driven responses to 405nm, as well as reversal caused by 525nm in sustained inhibition units. Two outliers with slow latency (>2.5s) in transient excitation group are likely responding to stimulus offset (N = 35 for Transient Excitation, N = 6 for Transient Inhibition, N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation, all biological replicates).  F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates). G) Excitation responses were attenuated for mixed stimuli compared to 405nm only. Left panel shows representative retinal single unit (N = 35 biological replicates). Data information: For (F-G) Left panel shows representative single unit. Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus. All representative single units show perievent rasters (first trial at top) and firing rate histograms (Bin size = 500ms). Timing of light stimuli shown by shaded vertical bars. All light stimuli are 16 log total Lamplight effective photons/cm2/s. Error bars show SEM. * p<0.05, *** p < 0.001 (One-sample t-test compared to zero).


Figure 3A , B . Aggregate number of detected and undetected cysteines ( A ) and lysines ( B ) in 3 , 840 CpDAA - containing proteins .

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Figure 3A, B. Aggregate number of detected and undetected cysteines (A) and lysines (B) in 3,840 CpDAA-containing proteins.


Figure 4C . Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220 - 479 of CASP8 ( UniProt ID Q14790 ) . Dashed horizontal line marks deleterious threshold of 25 . E . Activity of recombinant caspase - 8 protein assayed using fluorogenic IETD - AFC substrate . Percentage activity shown relative to wild - type ( WT ) protein for three replicate experiments , bars and error bars as mean ± SD .

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Figure 4C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.


Figure 1 ( B - E ) Immunostaining of germaria from 7 day - old females with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ cell loss and tumor , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . ( F ) Quantification of mutant germaria with 0 to 1 GSC , or with GSC tumors shown in ( B - E ) . The number of scored germaria ( n ) is indicated on the right graph . * * * p - value < 0 . 001 , ns : non significant , using the χ2 test . ( G - H ' ) Germaria containing control ( G , G ' ) or aubHN2 mutant ( H , H ' ) clonal GSCs stained with anti - GFP ( green ) and anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain DNA . Clonal cells are marked by the lack of GFP . Clonal GSCs and cysts are outlined with dashed line . White arrowheads show clonal GSCs in the control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E and G - H ' . ( I ) Quantification of germaria containing at least one clonal GSC at 7 , 14 and 21 days after clonal induction . ( J ) Division rate of wild - type and aubHN2 clonal GSCs . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

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Figure 1(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.(F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test.(G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'.(I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction.(J) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student's t test.


Figure 2 ( A , B ) Immunostaining of germaria with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of mnkP6 and mnkP6 aubHN2 / QC42 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . ( C ) Quantification of the number of GSCs per non - tumorous germarium , and of germaria with GSC tumors , in the indicated genotypes . The number of scored germaria ( n ) is indicated . The two - tailed Student ' s t test and the χ2 test were used in the left and right panels , respectively . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 . ( D - F ) Immunostaining of germaria from 7 day - old females with anti - Vasa or anti - GFP ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . aubHN2 / QC42 ; nos - Gal4 / + was used as a negative control . Examples of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - Aub germarium ( E ) , and of lack of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - AubAA germarium ( F ) . White arrowheads indicate GSCs ; white arrows indicate GSC loss in ( D ) and germ cell loss in ( F ) . Scale bar : 10 μm in A , B and D - F . ( G ) Quantification of mutant germaria with 0 to 1 GSC shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , ns : non - significant , using the χ2 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 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 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

Figure 2(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.(C) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student's t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05.(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.(G) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.


Figure 3 ( A - B ' ' ) Immunostaining of wild - type ( A - A ' ' ) or nos - Gal / UASp - CCR4 - HA ( B - B ' ' ) germaria with anti - Aub ( red ) , and anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . White arrowheads in ( B - B ' ' ) indicate cytoplasmic accumulation of CCR4 - HA colocalized with Aub in GSCs . ( C ) Co - immunoprecipitation ( IP ) of NOT1 , NOT3 and CCR4 with GFP - Aub in ovaries . Wild - type ( WT , mock IP ) or nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) ovarian extracts were immunoprecipitated with anti - GFP , either in the absence or the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 and anti - CCR4 . Inputs are extracts prior to IP . ( D ) Co - IP of NOT1 and NOT3 with GFP - Aub or GFP - AubAA in ovaries . Wild - type ( WT , mock IP ) , nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) or nos - Gal4 / UASp - GFP - AubAA ( GFP - AubAA ) ovarian extracts were immunoprecipitated with anti - GFP in the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 and anti - NOT3 . Inputs are extracts prior to IP . ( E - G ) Genetic interaction between aub and twin in Vasa self - renewal . Immunostaining of germaria with anti - DNA ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of aubHN2 / + , twinDG24102 and aubHN2 / + ; twinDG24102 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . Scale bar : 10 μm in A - B ' ' and E - G . ( H ) Quantification of mutant germaria with no GSC in 3 , 7 and 14 day - old females of the genotypes shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 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 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 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 O O 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

Figure 3(A-B'') Immunostaining of wild-type (A-A'') or nos-Gal/UASp-CCR4-HA (B-B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A''). White arrowheads in (B-B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs.(C) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP.(D) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP.(E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G.(H) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.