( D ) Representative of conventional of RPE of both 2 - month - old 3 - month - old WT miR - 211 - / - mice . miR - 211 - / - mice show accumulation of phagolysosomes ( red arrows ) in RPE . Scale bar 2 μm . Enlarged boxes highlight phagolysosome - like structures containing poorly processed POS . Scale bar 1 . 5 μm . Representative of conventional of RPE shown at higher magnification also highlighted accumulation of lipofuscin - like granules ( blue arrows ) in 3 - month - old miR - 211 - / - compared to control WT mice . Melanosomes are indicated by asterisks . Scale bar 500 nm ( n = 3 mice ) . ( E ) Graphs showing number of lipofuscin - like granules ( n / 100 μm2 ) from RPE of 3 - month - old miR - 211 - / - mice as in ( D ) . Bar graphs represent mean values ± s . e . m . Mann Whitney ( miR - 211 - / - vs WT ) * * * p ≤ 0 . 005 ( n = 6 mice ) . Data information : ( RPE ) Retinal Pigment

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

(D) Representative images of conventional TEM analysis of RPE of both 2-month-old and 3-month-old WT and miR-211-/- mice. miR-211-/- mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome-like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin-like granules (blue arrows) in 3-month-old miR-211-/- compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm (n=3 mice). (E) Graphs showing number of lipofuscin-like granules (n/100 μm2) from the RPE of 3-month-old miR-211-/- mice as in (D). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT) ***p ≤ 0.005 (n=6 mice). Data information: (RPE) Retinal Pigment Epithelium


( A ) Representative confocal of eye cryosections immunostained with anti - Ezrin ( green ) anti - ZO - 1 ( red ) antibodies from 2 - month - old WT mice sacrificed 3h after on at 10 AM ( diurnal condition - ) 3h after off at 10 PM ( nocturnal condition - ) , nuclei were counterstained with DAPI ( blue ) . Enlarged boxes highlight Ezrin in both basal infoldings ( BI ) apical extension ( AE ) of RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

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

(A) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( B ) Graph representing kinetic expression pattern of Ezrin in RPE from 2 - month - old WT mice during / / transitions . Mice were sacrificed at determined time points as indicated on X axis . Scheme at top describes / adaptation regime . Values normalized to Hprt , represent means ± s . e . m . Mann Whitney * p ≤ 0 . 05 ( n = 3 mice for each time point ) . Values from 12h - adapted mice ( T0 ) were set to one .

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

(B) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated on the X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. Mann and Whitney test *p ≤ 0.05 (n=3 mice for each time point). Values from 12h dark-adapted mice (T0) were set to one.


( C ) Confocal of representative eye cryosections immunostained with anti - Ezrin ( green ) anti - ZO - 1 ( red ) antibodies from 2 - month - old miR - 211 - / - WT control mice sacrificed 3h after on at 10 AM ( diurnal condition ) . Enlarged boxes highlight Ezrin in both basal infoldings ( BI ) apical extension ( AE ) of RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

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

(C) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old miR-211-/- and WT control mice sacrificed 3h after light on at 10 AM (diurnal condition). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( D ) qRT - PCR for Ezrin from RPE 2 - month - old WT miR - 211 - / - mice sacrificed 3h after on at 10 AM . graph shows expression level of Ezrin normalized to Hprt . Bar graphs represent mean values ± s . e . m . Mann Whitney ( miR - 211 - / - vs WT ) , * * * p ≤ 0 . 005 ( n = 6 mice ) .

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

(D) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).


( E ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT miR - 211 - / - mice . Representative image at low exposure of Western blot of Ezrin from WT miR - 211 - / - mice . graph shows of Ezrin normalized to Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 5 mice ) Mann Whitney ( miR - 211 - / - vs WT ) , * * p ≤ 0 . 01 .

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

(E) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR-211-/- mice. The graph shows the quantification of Ezrin normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=5 mice) Mann and Whitney test (miR-211-/- vs WT), **p ≤ 0.01.


( F ) Graph representing kinetic expression pattern of Ezrin in RPE from 2 - month - old miR - 211 - / - mice during / / transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at top describes / adaptation regime . Values normalized to Hprt , represent means ± s . e . m . ( n = 3 mice for each time point ) . Values from 12h adapted mice ( T0 ) were set to one . ( G ) Graph representing kinetic expression pattern of Trpm1 in RPE from 2 - month - old WT mice during / / transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at top describes / adaptation regime . Values normalized to Hprt , represent means ± s . e . m ( n = 3 mice for each time point ) . Values from 12h adapted mice ( T0 ) were set to one .

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

(F) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old miR-211-/- mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one. (G) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one.


( H ) qRT - PCR for Mtmr10 Klf13 from RPE 2 - month - old WT miR - 211 - / - mice sacrificed 3h after on at 10 AM . graph shows expression level of these genes normalized to Hprt . Bar graphs represent mean values ± s . e . m . ( n = 3 mice )

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

(H) qRT-PCR assay for Mtmr10 and Klf13 from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after on at 10 AM. The graph shows the expression level of these genes normalized to the Hprt. Bar graphs represent mean values ± s.e.m. (n=3 mice)


( A ) Representative from ARPE - 19 treated with DMSO NSC668394 . were fixed stained with anti - LC3 ( green ) anti - LAMP1 ( red ) antibodies nuclei were counterstained with DAPI ( blue ) . Scale bar 5 μm . ( B ) plot shows of numbers of LC3 / LAMP1 - positive vesicles per ( n ≥ 100 ) . Values represent means ± s . e . m from 3 independent experiments . Student ' s t - ( NSC668394 vs DMSO ) * * * p ≤ 0 . 005 .

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

(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005.


( C ) Representative Western blots of LAMP1 , EZRIN , EZRIN - pT567 , CLN5 , TRPML1 , SQSTM1 / p62 , CTSD LC3 from DMSO or NSC668394 treated . Note an increase of both proCTSD its maturation CTSD heavy chain ( hc ) . plot shows of indicated normalized to GAPDH loading control . Bar graphs represent mean values ± s . e . m . of at least n = 6 independent experiments . Mann Whitney ( NSC668394 vs DMSO ) * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.


( D ) Representative of RFP - GFP - LC3 in ARPE - 19 transiently transfected with RFP - GFP - LC3 treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI ( blue ) . Scale bars : 5 μm . ( E ) Box plots showing quantitative of RFP + GFP + ( Autophagosome ) RFP + GFP - ( Autolysosome ) ( n ≥ 100 ) from 3 independent experiments . Box limits represent 25th percentile 75th percentile ; horizontal lines represent medians ; whiskers display min . to max . values . Student ' s t - ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.


( F ) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B ( CTSB ) Magic Red substrate for 20 min of living collected on HC . Scale bar 10 μm . ( G ) Graph showing mean values of of cresyl violet ± s . e . m . of representative data ( n ≥ 100 ) from 3 independent experiments . Mann Whitney ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( H ) Cathepsin B ( CTSB ) activity in lysates from NSC668394 - treated DMSO - control ARPE - 19 . Graph showing of values ± s . e . m . of CTSB activity from 4 independent experiments . Mann Whitney ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( A ) Representative from HC of HeLaTFEB - GFP transfected with control ( siCTRL ) or siPPP3CB , subjected to indicated conditions . of TFEB translocation in HeLaTFEB - GFP transfected with siCTRL or siPPP3CB treated with DMSO or NSC668394 , silenced for EZRIN ( siEZR ) or serum - starved ( stv ) . Both pharmacological inhibition silencing of Ezrin induced TFEB localization in stable HeLaTFEB - GFP . Starvation ( stv ) was used as control . translocation of TFEB in stable HeLaTFEB - GFP subjected to indicated conditions is rescue after silencing of PPP3CB . Scale bar 5 μm .

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

(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.


( B ) graph shows mean ± s . e . m . of of TFEB translocation in Ezrin - inhibited compared to control ; n = 3 independent experiments were performed . Mann Whitney ( NSC668394 stv vs DMSO ; siEZR vs siCTRL ) * p ≤ 0 . 05 .

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

(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.


( C ) graph shows mean ± s . e . m . of of TFEB translocation in Ezrin - inhibited subjected to silencing of PPP3CB . At least 3 independent experiments were performed . Mann Whitney ( siPPP3CB vs siCTRL ) * p ≤ 0 . 05 .

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

(C) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells and subjected to the silencing of PPP3CB. At least 3 independent experiments were performed. Mann and Whitney test (siPPP3CB vs siCTRL) *p ≤ 0.05.


( D ) qRT - PCR for PPP3CB from HeLaTFEB - GFP transfected with siPPP3CB siCTRL . graph shows reduction in PPP3CB expression level normalized to Hprt . Bar graphs represent mean values ± s . e . m . Mann Whitney ( siPPP3CB vs siCTRL ) , * * * p ≤ 0 . 005 ( n = 6 independent experiments ) .

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

(D) qRT-PCR assay for PPP3CB from HeLaTFEB-GFP cells transfected with siPPP3CB and siCTRL. The graph shows the reduction in PPP3CB expression level normalized to the Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (siPPP3CB vs siCTRL), ***p ≤ 0.005 (n=6 independent experiments).


( E ) Both pharmacological inhibition silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot . Torin treatment was used as control .

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

(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.


( F ) Representative traces of ML1 - GCaMP3 normalized recorded in transiently transfected ARPE - 19 . During time - lapse recording , were stimulated with ML - SA1 ( 20 µM ) after addition ( arrowhead ) of DMSO or NSC668394 . Where indicated , specific ML1 inhibitor ML - SI3 ( 10 µM ) was added . Bar graph reports mean values ± s . e . m . of time required by to decay to half of ML - SA1 - induced peak . n = 50 from 3 independent experiments . * * * p < 0 . 005 by Mann Whitney .

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

(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.


( G ) translocation of TFEB in stable HeLaTFEB - GFP subjected to indicated conditions is reduced after Ca2 + chelator BAPTA treatment . Scale bar 5 μm .

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

(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.


H ) graph shows mean ± s . e . m . of of TFEB translocation in Ezrin - inhibited compared with DMSO under Ca2 + chelator BAPTA treatment . At least 3 independent experiments were performed . Mann Whitney ( stv , stv + BAPTA , BAPTA , NSC668394 , NSC668394 + BAPTA vs DMSO ) * p ≤ 0 . 05 .

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

H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.


( I ) qRT - PCR for TFEB target genes ( TRPML1 , BECLIN , MAPLC3B LAMP1 ) was performed on ARPE - 19 treated with DMSO or NSC668394 . Bar graphs represent mean values ± s . e . m . of at least 3 independent experiments . Mann Whitney * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.


( A ) Confocal of representative eye cryosections immunostained with anti - Ezrin ( green ) / anti - ZO - 1 ( red ) , anti - Ezrin - pT567 ( green ) / anti - ZO - 1 ( red ) , anti - Lamp1 ( green ) anti - Sqstm1 / p62 ( green ) antibodies from 6 - month - old miR - 211 - / - sacrificed five months after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer layer .

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

(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.


( B ) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6 - month - old miR - 211 - / - mice . Representative Western blots from these tissues were performed to determine expression of Lamp1 , Ezrin , Ezrin - pT567 , Sqstm1 / p62 LC3 . plots show of indicated normalized to β - Actin loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) . Mann Whitney ( DMSO vs WT ; NSC668394 vs DMSO ) , * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.


( C ) Cathepsin B ( CtsB ) activity in RPE lysates from miR - 211 - / - WT mice sacrificed 3h after on at 10 AM ( diurnal condition ) . RPE was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . CtsB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann Whitney ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) , * p ≤ 0 . 05 . ( D ) GUSB activity from retina from miR - 211 - / - control mice sacrificed 3h after on at 10 AM ( diurnal condition ) . Retina was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . GUSB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent of GUSB activity ± s . e . m . of independent experiments ( n = 3 mice ) . Mann Whitney ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) .

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

(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).


( A ) Representative of conventional of RPE of WT , DMSO - treated miR - 211 - / - mice NSC668394 - treated miR - 211 - / - three - month - old mice . NSC668394 - treated miR - 211 - / - mice show rescue of accumulation of double - phagolysosome - like structures containing poorly processed POS ( red arrows ) compared to DMSO - treated miR - 211 - / - mice . Scale bar 1 mm . Data information : ( OS ) outer segment ( RPE ) Retinal Pigment Epithelium .

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

(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium.


( B ) Confocal of representative eye cryosections immunostained with anti - Rhodopsin ( green ) antibody from WT miR - 211 - / - mice at three months of after DMSO or NSC668394 treatment , which are sacrificed 3h after on at 10 AM ( diurnal condition ) . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . enlarged box highlights rhodopsin accumulation in RPE ( green spots ) . Scale bar 5 μm . Autofluorescence from lipofuscin granules from WT miR - 211 - / - mice at three months of after DMSO or NSC668394 treatment . Representative of retina cryosections immunostained with anti - Cone Arrestin ( green ) antibody from WT miR - 211 - / - mice at three months of after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . N = at least 6 mice per group . Scale bar 100 μm . Data information : ( OS ) outer segment ; ( ONL ) outer layer ; ( RPE ) Retinal Pigment Epithelium .

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

(B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.


( C ) Graphs showing number of lipofuscin granules from RPE of mice as in ( B ) . Bar graphs represent mean values ± s . e . m . Mann Whitney ( miR - 211 - / - DMSO vs WT miR - 211 - / - NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 . * p ≤ 0 . 05 ( n = 6 mice ) .

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

(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).


( D ) Graphs show cone ( cones / ) from retina of mice treated as in ( B ) . Error bars represent s . e . m . Mann Whitney ( DMSO vs WT NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 ( n = 6 mice ) .

O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O

(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).


( E ) Representative flicker traces at three months of show rescue of flicker responses of NSC668394 - treated miR - 211 - / - mice ( green lines ) compared to DMSO - treated miR - 211 - / - control mice ( red lines ) . WT mice were used as a control ( black lines ) . Flicker recordings were performed with intensities ranging from 10 − 4 to 15 cd s / m2 in steps of 0 . 6 logarithmic units at 6 Hz frequency . ( F ) Flicker responses , plotted as a function of stimulus , from WT ( black lines ) , DMSO - treated miR - 211 - / - ( red lines ) NSC668394 - treated miR - 211 - / - ( green lines ) mice , at three months of . amplitude of recordings from NSC668394 miR - 211 - / - treated mice is significantly rescued compared to DMSO - treated miR - 211 - / - mice . WT mice were used as a control . Error bars represent s . e . m . ANOVA ( DMSO vs WT NSC668394 vs DMSO treated mice ) * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.


( B ) Met - Phe formation monitored upon rapidly mixing initiation complexes ( 80S IC ) with ternary complexes eEF1A - GTP - [ 14C ] Phe - tRNAPhe in absence ( orange , 0 . 13 ± 0 . 02 s - 1 ) or presence ( cyan , 0 . 15 ± 0 . 02 s - 1 ) of eEF3 ( 2 µM ) in a quench - flow apparatus , extent of peptide formation was analyzed by HPLC radioactivity counting . Data are normalized to Met - Phe formation in absence of eEF3 with maximum value in dataset set to 1 . Data are presented as mean ± of n = 3 biological replicates . ( C ) Met - Phe - Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe - tRNAPhe ( 80S 2C ) with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in presence of eEF2 eEF3 ( green , 0 . 15 ± 0 . 01 s - 1 ) , eEF2 ( red ) or eEF3 ( blue ) . Data presented as mean ± of n = 3 biological replicates . ( D ) Met - Phe - Val formation monitored with 80S 2C incubated with eEF2 eEF3 ( green , 0 . 4 ± 0 . 14 s - 1 ) , eEF2 ( red , 1 . 0 ± 0 . 5 s - 1 ) or eEF3 ( blue ) , or in absence of eEF2 eEF3 ( brown ) for 15 min before adding with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal monitoring kinetics of Val incorporation . Data presented as mean ± of n = 3 . Data information : Datasets in ( C ) ( D ) are normalized to Met - Phe - Val formation in presence of eEF2 eEF3 with maximum value set to 1 .

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

(B) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates. (C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates. (D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1.


( B ) mRNA - tRNA translocation monitored in a time - resolved Pmn upon mixing 80S 2C with Pmn in absence ( brown triangles ) or presence of eEF2 eEF3 ( green , 0 . 13 ± 0 . 01 min - 1 ) , eEF2 ( red , 0 . 030 ± 0 . 005 min - 1 ) or eEF3 ( blue , 0 . 10 ± 0 . 01 min - 1 ) . Data are normalized to Met - Phe - Pmn formation in presence of eEF2 with maximum value set to 1 . Data presented as mean ± of n = 3 .

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

(B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3.


( C ) Dissociation of deacylated tRNA from E site by change of tRNAfMet ( Flu ) upon rapidly mixing of 80S 2C with buffer ( grey ) , or with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in presence of eEF2 eEF3 ( green ) , eEF2 ( red ) , eEF3 ( blue ) , or eEF2 eEF3 without ternary ( magenta ) in a stopped - flow apparatus . Each trace is an average of 5 - 7 time courses normalized at 1 for at start of reaction .

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

(C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.


( D ) tRNAfMet ( Flu ) co - eluting with 80S 2C incubated without factors ( orange ) , with eEF2 ( red ) , with eEF2 eEF3 ( magenta ) , or with eEF2 , eEF3 TC - Val ( green ) for 15 min before loading on a BioSuite 450 - exclusion column to separate ribosome - bound - unbound tRNA . Data are normalized to tRNAfMet ( Flu ) bound to 80S 2C in absence of eEF2 / eEF3 with maximum value set to 100 % . Data presented as mean ± of n = 3 .

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

(D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3.


( E ) Dissociation of tRNAfMet ( Flu ) from 80S 2C deacylated tRNA incubated with eEF2 eEF3 ( green ) or eEF2 ( red ) for 15 min before adding with ternary complexes . Each trace is an average of 5 - 7 time courses normalized at 1 for at start of reaction .

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

(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.


( F ) Codon - specific ribosome occupancies for 21 nt RPFs from WT eEF3d ( two biological replicates ) . CGA AGG codons are shown in red purple , respectively . Variance for each dataset is indicated .

O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

(F) Codon-specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells (two biological replicates). CGA and AGG codons are shown in red and purple, respectively. Variance for each dataset is indicated.


( G ) Scatter plot of codon - specific occupancies for 28 nt RPFs comparing eEF3d to WT from libraries prepared with CHX + ANS . Trend ( blue ) its slope are shown . Codons that encode proline glycine are colored in purple green , respectively .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

(G) Scatter plot of codon-specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX+ANS. Trend line (blue) and its slope are shown. Codons that encode proline and glycine are colored in purple and green, respectively.


Telomeric lncRNA in indicated deletion strains as analyzed by dot blot ( top ) or Northern blot hybridization ( bottom ) using a double ‐ stranded telomeric probe . Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA . Bars error bars are averages s . d . from 4 independent experiments . U6 18S rRNA are shown as loading controls for Northern blotting . asterisk indicates unspliced U6 . Molecular weights are on left in kilobases .

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

Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot (top) or Northern blot hybridization (bottom) using a double‐stranded telomeric probe. Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA. Bars and error bars are averages and s.d. from 4 independent experiments. U6 and 18S rRNA are shown as loading controls for Northern blotting. The asterisk indicates unspliced U6. Molecular weights are on the left in kilobases.


ARRET αARRETNorthern blot of indicated strains using strand ‐ specific probes .

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

ARRET and αARRETNorthern blot analysis of the indicated strains using strand‐specific probes.


Telomere of ApaI ‐ digested from indicated strains .

Telomere length analysis of ApaI‐digested DNA from the indicated strains.


of subtelomeric from ChIP experiments using strains carrying indicated Myc ‐ tagged ( 2A9 8B7 : SPBC2A9 . 02 ‐ Myc SPBP8B7 . 08c ‐ Myc , respectively ) . Immunoprecipitated is expressed as fraction of input . An untagged ( − ) served as a negative control . Bars error bars are averages s . d . from three independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * * P 0 . 001 relative to untagged .

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

Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc‐tagged proteins (2A9 and 8B7: SPBC2A9.02‐Myc and SPBP8B7.08c‐Myc, respectively). Immunoprecipitated DNA is expressed as fraction of input DNA. An untagged strain (−) served as a negative control. Bars and error bars are averages and s.d. from three independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to untagged strain.


Chromosome maps of differentially expressed genes in cay1Δ shown as fold enrichment over wt . Red dots are overexpressed genes Tf2 retrotransposons , blue dots are overexpressed LTR sequences .

B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

Chromosome maps of differentially expressed genes in cay1Δ cells shown as fold enrichment over wt. Red dots are overexpressed genes and Tf2 retrotransposons, blue dots are overexpressed LTR sequences.


Northern blot of tlh1 / 2 + , Tf2 retrotransposons , 18S rRNA ( loading control ) in wt , cay1Δ , trichostatin A ( TSA ) ‐ treated wt . Black arrowhead indicates tlh1 / 2 + mRNA ; red arrowhead unprocessed Tf2 transcripts ; blue arrowhead processed Tf2 transcripts .

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

Northern blot analysis of tlh1/2+, Tf2 retrotransposons, and 18S rRNA (loading control) in wt, cay1Δ, and trichostatin A (TSA)‐treated wt cells. Black arrowhead indicates tlh1/2+ mRNA; red arrowhead unprocessed Tf2 transcripts; and blue arrowhead processed Tf2 transcripts.


ChIP of Cay1 ‐ Myc binding to indicated genomic loci . subtel : subtelomeres ; Tf2 : Tf2 retrotransposon genes ; LTR : Tf2 LTR sequences ; Tlh : tlh1 / 2 + genes ; cen : centromeres . Immunoprecipitated is normalized to input expressed as fold increase over untagged strains ( unt ) .

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

ChIP analysis of Cay1‐Myc binding to the indicated genomic loci. subtel: subtelomeres; Tf2: Tf2 retrotransposon genes; LTR: Tf2 LTR sequences; Tlh: tlh1/2+ genes; cen: centromeres. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains (unt).


ChIP of H3K9ac , H3K9me3 , total H3 at indicated genomic loci . Immunoprecipitated is normalized to input expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only .

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

ChIP analysis of H3K9ac, H3K9me3, and total H3 at the indicated genomic loci. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only.


Western blot of endogenous Rap1 Taz1 in indicated strains . Act1 was used as a loading control . Asterisks indicate cross ‐ reacting bands .

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

Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains. Act1 was used as a loading control. Asterisks indicate cross‐reacting bands.


RT - PCR of splicing efficiencies of indicated pre ‐ mRNAs in wt cay1 ∆ . prp1 ‐ 1 carries a thermosensitive allele of splicing factor Prp1 was used as a positive control for splicing impairment . prp1 ‐ 1 were grown at 36 ° C for 4 h before harvesting . Amplification products corresponding to unspliced spliced mRNAs are indicated by asterisks gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 + exon 3 thereby amplifying both spliced unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at bottom of each panel are ratios between spliced unspliced forms ( s / u ) spliced act1 + ( s / act1 ) . Values are expressed as fold increase over wt .

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

RT-PCR analysis of splicing efficiencies of the indicated pre‐mRNAs in wt and cay1∆ cells. The prp1‐1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1+ exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.


Western blot of Rap1 - YFP in indicated strains upon nmt1 promoter shutoff by thiamine addition . Membranes were probed with antibodies against GFP Act1 ( loading control ) . of Rap1 - YFP in experiments as in ( C ) . Rap1 - YFP are expressed as fold increase over time 0 after normalization through Act1 . Data points error bars are averages s . d . from at least four independent experiments . Statistical significance was assayed using unpaired , two - Student ' s t - . * P 0 . 05 , * * P 0 . 01 for cay1 + versus cay1 ∆ samples .

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

Western blot analysis of Rap1-YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).Quantification of Rap1-YFP protein levels in experiments as in (C). Rap1-YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t-test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.


Telomere of ApaI ‐ digested from cay1 ∆ trt1 ∆ harvested at increasing generation doublings ( gen ) .

B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).


Telomere of ApaI ‐ digested from indicated strains . dh : centromeric dh repeats shown as loading control .

B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O

Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control.


PFGE of telomeric fusions in strains grown to logarithmic phase ( log ) or G1 ‐ arrested by nitrogen starvation ( G1 ) . Genomic was digested with NotI hybridized to C , I , L , M probes detecting terminal fragments of chromosomes I II . Bands corresponding to chromosome end fusions are indicated ( fused ) .

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

PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).


Northern blot of ARIA , ARRET , Tf2 retrotransposons , 18S rRNA ( loading control ) in indicated strains .

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

Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains.


qRT - PCR of TERRA expressed as fold increase over wt after normalization through act1 + mRNA . Bars error bars are averages s . d . from 3 independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * P 0 . 01 relative to wt .

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

qRT-PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt.


ChIP of H3K9ac total H3 for indicated strains . Immunoprecipitated is normalized to input expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars error bars are averages s . d . from at least four independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * * P 0 . 001 relative to wt .

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

ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.


Serial dilutions of indicated strains were spotted on complete medium grown at 30 ° C or 20 ° C . Otrt1 ∆ Otrt1 ∆ cay1 ∆ are strains with circularized chromosomes lacking telomeric ( Supplementary Fig S7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O

Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7).


Representative of wt cay1 ∆ grown for 24 h at 30 ° C or 20 ° C . Scale bar , 20 μm .

O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm.


of for strains grown in complete medium for 24 h at 30 ° C or 20 ° C . Each dot represents a single median ( m ) is indicated for each sample . Data are from three independent experiments at least 100 were scored per sample in each experiment .

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

Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment.


Top : Examples of DAPI ‐ stained wt cay1 ∆ grown in complete medium for 24 h at 30 ° C or 20 ° C . arrowhead indicates a chromosome bridge . Scale bar , 10 μm . Bottom : of chromosome bridges . Bars error bars are averages s . d . from three independent experiments at least 200 were scored per sample in each experiment . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * * P 0 . 001 relative to 30 ° C .

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Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.


RT - PCR of pre ‐ mRNA splicing efficiencies of rap1 + poz1 + pre ‐ mRNAs in cay1 + cay1Δ expressing Cay1 ‐ YFP or Rap1 ‐ YFP . Amplification products corresponding to unspliced spliced mRNAs are indicated by asterisks gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 thereby amplifying both spliced unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at bottom of each panel are ratios between spliced unspliced forms ( s / u ) spliced act1 + ( s / act1 ) . Values are expressed as fold increase over wt .

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RT-PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1Δ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.


Western blot of total from early generation cay1 + cay1Δ expressing Cay1 ‐ YFP , Rap1 ‐ YFP , Taz1 ‐ YFP using antibodies against Rap1 , Taz1 , Act1 ( loading control ) . Numbers at bottom are ratios between Rap1 ( endogenous plus ectopic ) Act1 ( Rap1 / Act1 ) . Values are expressed as fold increase over wt .

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Western blot analysis of total proteins from early generation cay1+ and cay1Δ cells expressing Cay1‐YFP, Rap1‐YFP, and Taz1‐YFP using antibodies against Rap1, Taz1, and Act1 (loading control). Numbers at the bottom are ratios between Rap1 proteins (endogenous plus ectopic) and Act1 (Rap1/Act1). Values are expressed as fold increase over wt.


Left : Representative YFPfluorescence microscopy DIC of cay1 ∆ strains expressing Cay1 ‐ YFP . Scale bar , 10 μm . Right : Representative YFP RFP of Cay1 ‐ YFP ‐ Pot1 ‐ RFP ‐ expressing . Arrows point to YFP RFP foci within same nucleus demonstrating lack of co ‐ localization . Scale bar , 5 μm .

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

Left: Representative YFPfluorescence microscopy and DIC images of cay1∆ strains expressing Cay1‐YFP. Scale bar, 10 μm. Right: Representative YFP and RFP fluorescence images of Cay1‐YFP‐ and Pot1‐RFP‐expressing cells. Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co‐localization. Scale bar, 5 μm.


Representative YFPfluorescence microscopy DIC of cay1 + cay1 ∆ strains expressing Rap1 ‐ YFP . Scale bar , 5 μm .

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Representative YFPfluorescence microscopy and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.


Telomere of ApaI ‐ digested from harvested at early ( left ) late generations ( right ) after expression of YFP ‐ tagged .

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Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.


Northern blot of ARIA , ARRET , 18S rRNA ( loading control ) at early generations after expression of YFP ‐ tagged . Numbers at bottom are ratios between ARRET 18S rRNA signals , expressed as fold increase over wt .

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Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt.


ChIP of subtelomeric H3K9ac , H3K9me3 , total H3 in indicated strains at early generations . Immunoprecipitated is normalized to input expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars error bars are averages s . d . from 3 independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 relative to wt .

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ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.


Serial dilutions of early generation cay1 + cay1 ∆ expressing Cay1 ‐ YFP Rap1 ‐ YFP were spotted on complete medium grown at 30 ° C 20 ° C .

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Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C.


of in strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Each dot represents a single median ( m ) is indicated for each sample . Data are from 3 independent experiments at least 100 were scored per sample in each experiment .

Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment.


of chromosome bridges of strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Bars error bars are averages s . d . from three independent experiments at least 200 were scored per sample in each experiment .

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Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.


Northern blot of Tf2 retrotransposons , U6 snRNA , act1 + mRNA ( loading control ) in wt , cay1Δ , prp1 ‐ 1 . prp1 ‐ 1 were grown at 36 ° C for 4 h before harvesting . Black arrowhead indicates unprocessed Tf2 transcripts . asterisk indicates unspliced U6 . Numbers at bottom are ratios between Tf2 act1 + signals ( Tf2 / Act1 ) expressed as fold increase over wt .

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Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1Δ, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.


Northern blot as in ( A ) in mpn1 + mpn1 ∆ strains carrying a stably integrated extra copy of snu6 + gene or empty vector control plasmids ( ev ) .

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Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).


A Dose - response viability curves in presence of increasing concentrations of IMT1 for one - week , viable were counted normalized to DMSO - treated controls . Data are expressed as mean ± SD of n = 3 independent experiments with four technical replicates . IC50 values were calculated with non - linear least square fit , RKO : 521 . 8 nM , MiaPaCa - 2 : 291 . 4 nM HeLa : 29 . 9 nM .

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A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.


B Mitochondrial transcript measured by qRT - PCR in RKO after 96 hours of 1 μM IMT1 treatment . Data are relative to DMSO - treated controls are expressed as mean ± SD of n = 4 independent experiments . Ordinary one - way ANOVA was used for comparisons to DMSO - treated controls ( RNR1 , RNR2 , MT - COX2 , MT - ATP6 , MT - ND1 : * * * p < 0 , 0001 ) .

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B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).


D Volcano plot showing proteomic changes of RKO treated with 1 μM IMT1 for 96 hours . Data are plotted as average log2 - fold change versus log10 of adjusted p - value of DMSO - treated controls ; mitochondrial are highlighted in red .

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D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.


F CRISPR guides enriched ( positive hits ) in IMT1 - treated RKO plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value . mTORC1 VHL pathway - related genes are reported in green blue , respectively .

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F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.


B - D Viable in presence of DMSO , IMT1 alone or in combination with rapamycin in RKO ( B ) , MiaPaCa - 2 ( C ) HeLa ( D ) . Data are expressed as mean values ± SD of n = 6 independent experiments each including four technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs . IMT1 + RAPA comparisons : RKO : * * * p < 0 , 0001 , MiaPaCa - 2 : * * p = 0 , 0014 , HeLa : * p = 0 , 0238 .

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B-D Viable cells count in presence of DMSO, IMT1 alone or in combination with rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.


F - H Viability assessment of RKO ( F ) , MiaPaCa - 2 ( G ) HeLa ( H ) treated with DMSO , IMT1 alone in combination with FG4592 . Data are expressed as mean values ± SD of n = 6 , 5 , 6 independent experiments for RKO , HeLa MiaPaCa - 2 , respectively . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs IMT1 + FG4592 comparisons : RKO : * * p < 0 , 0028 , MiaPaCa - 2 : * * p = 0 , 0011 , HeLa : * p = 0 , 0029 .

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F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.


C Western blot analyses of OXPHOS steady state of RKO after treatment with DMSO , IMT1 , rapamycin , rapamycin with IMT1 , FG4592 FG4592 with IMT1 . ACTINB is shown as loading control .

C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control.


F , G OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function . RKO were treated with DMSO , rapamycin ( F ) or FG4592 ( G ) in presence or absence of IMT1 . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA . DMSO - treated controls vs : IMT1 : p = 0 , 0008 ; RAPA : p = 0 , 1578 ; RAPA + IMT1 : p = 0 , 0034 ; FG4592 : p = 0 , 872 ; FG4592 + IMT1 : p = 0 , 0029 . Paired t - of IMT1 vs RAPA + IMT1 : p = 0 , 034 ; IMT1 vs FG4592 + IMT1 : non - significant .

O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.


A Differences in sensitivity to increasing IMT1 doses between RKO resistant RKO . graph shows mean values ± of n = 3 independent experiments .

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A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.


B Western blot analyses of OXPHOS steady state at increasing concentrations of IMT1 of RKO ( left ) IMT1 - resistant RKO ( right ) . samples were collected analyzed after eight of chronic IMT1 treatment of resistant ; GAPDH is shown as loading control .

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B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.


G Relative mtDNA copy number measured in RKO IMT1 - resistant treated with IMT1 for 96 hours . Data are expressed as average from n = 3 experiments ± . Statistical significance was calculated with one - way ANOVA . RKO + IMT1 vs Resistant + IMT1 : MT - ND1 * * p = 0 , 0035 , MT - ATP6 * * * p = 0 , 0002 , MT - CYTB * * p = 0 , 0028 .

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G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.


H OCR measured by Seahorse extracellular flux after sequential addition of different modulators of mitochondrial function in RKO IMT1 - resistant RKO treated with DMSO or IMT1 . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA . RKO DMSO vs RKO + IMT1 : p = 0 , 0008 ; RKO DMSO vs resistant DMSO : p = 0 , 0343 ; RKO DMSO vs resistant + IMT1 : p = 0 , 0092 . Paired t - of RKO + IMT1 vs resistant + IMT1 : p = 0 , 135 .

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H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.


A ECAR measured by Seahorse extracellular flux in DMSO IMT1 - treated RKO IMT1 - resistant . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA for multiple comparisons . RKO DMSO vs RKO + IMT1 : * * p = 0 , 0019 ; RKO + IMT1 vs resistant + IMT : * p = 0 , 0214 ; RKO DMSO vs resistant DMSO : non - significant .

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A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.


D , E IMT1 extracellular ( D ) extracellular ( E ) concentrations measured in medium at 0 , 2 24 hours of IMT1 treatment in RKO IMT1 - resistant RKO . Mean values ± of n = 3 experiments .

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D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.


B Comparison between proteomic analyses of parental RKO resistant treated with 1 μM IMT1 for 96 hours ( r : sample correlation coefficient ) . Data are expressed as average log2 - fold change of controls ( DMSO - treated RKO ) . Mitochondrial are reported in red .

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

B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.


A Depleted CRISPR guides ( negative hits ) in IMT1 - treated RKO plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value .

O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O

A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.


C Representative related plot of IMT1 - resistant RKO spheroid areas in presence of aforementioned compounds for two ( IMT1 : 1 μM , CAP : 1 μg / ml ) ; Scale bar : 1mm . Data show mean values ± SD of n = 4 experiments . Statistical significance was calculated with paired t - : IMT1 vs IMT1 + CAP : * p = 0 , 0242 .

O O O O O B-SMALL_MOLECULE O O B-CELL_LINE B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.


G Viable of IMT1 - resistant RKO treated for one - week with either DMSO or IMT1 in presence of controls ( control # 1 , # 2 ) or TFAM ( TFAM # 1 , TFAM # 2 ) siRNAs . Data are expressed as mean values ± SD of n = 5 independent experiments each including six technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA . Controls + IMT1 vs TFAM # 1 + IMT1 : p = 0 , 2148 ; Controls + IMT1 vs TFAM # 2 + IMT1 : * p = 0 , 0415 .

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

G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.


( B ) Detection of soluble , NrCAM ( sNrCAM ) full - , mature NrCAM ( mNrCAM ) soluble APPα ( sAPPα ) in neuronal supernatants lysates ( prepared from E16 neurons ) , treated with GI254023x ( 5 µM ) , or solvent for 48h . Densitometric quantifications of Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 6 - 8 ) .

O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).


( C ) Detection of sAPPα , sNrCAM mNrCAM in ADAM10 cKO neuronal supernatants lysates at 7 days in vitro ( DIV7 ) . neurons prepared from floxed ADAM10 ( ADAM10 fl / fl ) mice were infected with a lentivirus encoding improved Cre recombinase ( iCre ) or a control GFP lentivirus at DIV2 . Conditioned media were collected for 48 h . Densitometric quantifications of Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 6 - 8 ) .

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

(C) Detection of sAPPα, sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro (DIV7). The neurons prepared from floxed ADAM10 (ADAM10 fl/fl) mice were infected with a lentivirus encoding improved Cre recombinase (iCre) or a control GFP lentivirus at DIV2. Conditioned media were collected for 48 h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).


HEK 293 were transfected with a C - terminally VSV - tagged NrCAM construct or empty vector . were then treated with DAPT ( 1 µM ) , GI254023x ( 5 µM ) , either substances or solvent for 24 h . C - terminal NrCAM - fragment was detected with an antibody against VSV - tag . Representative Western Blots are shown .

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

HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.


( A ) ADAM10 fl / fl neurons were treated with an iCre ( to induce an ADAM10 KO ) , or a control lentivirus at DIV2 . At DIV7 , surface were labeled with biotin enriched by streptavidin pull - down . biotinylated were detected by immunoblotting . Total lysates were analyzed to compare mNrCAM ' s surface / lysate . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * * * p < 0 . 0001 , n = 7 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 .

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

(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1.


( B ) murine neurons were treated with GI254023x ( 5 µM ) or solvent , for 48h . surface were enriched like in A . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * * * p < 0 . 0001 , n = 11 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

O O O B-ORGANISM B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

(B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.