Figure 7B . Heatmap comparing ZGA genes expression between WT - 2 and NT - 2 and si6B - NT embryos ( FC > 5 , FPKM > 5 in each replicate ; left ) . A total of 1 , 813 DEGs are classified into two groups by unsupervised hierarchical clustering . KEGG and GO analysis of the two groups by unsupervised hierarchical clustering ( right ) . E . Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2 - cell embryos and derepressed in knock - down KDM6B ( left ) . Heat - map , KEGG and GO enrichment showing the expression pattern of 319 overlap genes ( FC > 5 , FPKM > 5 ; right ) . G . Representative localization of Xist expression in the nuclei of SCNT embryos injected with siRNA - 6A , siRNA - 6B or siRNA - Xist ( left ) . Arrows indicate the blastomeres enlarged in the bottom panels . The ratios of blastomeres classified according to the positive or negative expression of Xist analyzed by RNA - FISH ( right ) . Each bar represents a single embryo . H . Large - scale qPCR analysis of Xist and eight X - linked genes in 4 - cell stage male embryo . Each rectangle bar represents the expression value detected by single embryo RT - qPCR . The number of embryos in each group is indicated . Coloured bars indicate expression levels . I . Image of full - term cloned pups derived from NT Sertoli cells injected with siRNA - control , siRNA - Xist , or siRNA - Xist - 6B .

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

Figure 7B. Heatmap comparing ZGA genes expression between WT-2 and NT-2 and si6B-NT embryos (FC > 5, FPKM > 5 in each replicate; left). A total of 1,813 DEGs are classified into two groups by unsupervised hierarchical clustering. KEGG and GO analysis of the two groups by unsupervised hierarchical clustering (right).E. Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in knock-down KDM6B (left). Heat-map, KEGG and GO enrichment showing the expression pattern of 319 overlap genes (FC > 5, FPKM >5; right).G. Representative localization of Xist expression in the nuclei of SCNT embryos injected with siRNA-6A, siRNA-6B or siRNA-Xist (left). Arrows indicate the blastomeres enlarged in the bottom panels. The ratios of blastomeres classified according to the positive or negative expression of Xist analyzed by RNA-FISH (right). Each bar represents a single embryo.H. Large-scale qPCR analysis of Xist and eight X-linked genes in 4-cell stage male embryo. Each rectangle bar represents the expression value detected by single embryo RT-qPCR. The number of embryos in each group is indicated. Coloured bars indicate expression levels.I. Image of full-term cloned pups derived from NT Sertoli cells injected with siRNA-control, siRNA-Xist, or siRNA-Xist-6B.


Figure 1B : Double - IHC of coronal brain sections ( 40 μm ) of a littermate ( LM ) and a NexCre cTKO mouse ( age : 5 months ) stained for Ctip2 ( green , expressed in layer V / VI ) and Calbindin ( red , expressed in layer II / III and to some extent in layer V ) . Note that no gross abnormalities in cortical lamination were observed . Layers of DAPI stained cell nuclei are denoted on the right ( I - VI ) , scale bar : 100 µm . C : Giemsa - stained , glycolmethacrylate - embedded coronal brain sections ( bregma - 1 . 9 ) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype ( WT , top ) , littermate control ( LM , middle ) and a NexCre cTKO ( cTKO , bottom ; age : 5 - 6 months ) . Note the agenesis of the corpus callosum ( no CC ) in NexCre cTKO mice , which should always be present at this bregma and in sections in which the medial habenula ( mHb ) is present ( CC in WT and LM ) . While WTs and LMs show a compact layer of CA1 cells , NexCre cTKOs display a bilaminar cytoarchitecture ( boxed regions marking higher magnification to the right ) with scattered ectopic cells in the stratum radiatum ( white arrowheads ) . In addition , the CA3 appears less compact ( red arrowheads ) . Layer identity visualized via Calbindin staining ( right panel , scale bar : 50 µm ) .

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

Figure 1B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm.C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm).


Figure 2C : Representative 3D reconstructions of CA1 pyramidal neurons from deep ( left , dark blue ) and superficial ( right , light blue ) radial layers . D : Sholl analysis reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons ( dCA1 ; LM : n = 21 , N = 7 ; cTKO : n = 21 / N = 6 ) . E : Compared to littermate controls , dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites ( unpaired Student ' s t - test , nsp = 0 . 4979 ) . F : The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in dCA1 ( Mann - Whitney test , * * p = 0 . 0032 ) . G :

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

Figure 2C: Representative 3D reconstructions of CA1 pyramidal neurons from deep (left, dark blue) and superficial (right, light blue) radial layers.D: Sholl analysis reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons (dCA1; LM: n=21, N=7; cTKO: n=21/N=6). E: Compared to littermate controls, dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites (unpaired Student's t-test, nsp = 0.4979). F: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in dCA1 (Mann-Whitney test, **p = 0.0032). G:


Figure 3A , B : Spine density of deep CA1 pyramidal cells is significantly reduced in basal dendritic segments ( A ; unpaired Student ' s t - test , * * p = 0 . 0067 ) , while it is unchanged in midapical dendritic segments ( B ; unpaired Student ' s t - test , nsp = 0 . 4949 ) . C : Representative images of basal and midapical dendritic segments from both radial layers . Brightness and contrast were adjusted to ensure a uniform appearance . Scale bar : 5 µm . D

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Figure 3A, B: Spine density of deep CA1 pyramidal cells is significantly reduced in basal dendritic segments (A; unpaired Student's t-test, **p = 0.0067), while it is unchanged in midapical dendritic segments (B; unpaired Student's t-test, nsp = 0.4949). C: Representative images of basal and midapical dendritic segments from both radial layers. Brightness and contrast were adjusted to ensure a uniform appearance. Scale bar: 5 µm. D


Figure 4A : mEPSC and mIPSC sample traces of recordings from deep CA1 pyramidal cells . B - E : Bar graphs of mEPSC and mIPSC frequencies and amplitudes in deep CA1 pyramidal cells . mEPSC frequency was not different in deep CA1 pyramidal neurons of adult ( 4 months ) NexCre cTKO versus littermate controls ( B , p = 0 . 6415 ) . mEPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM control cells ( C , * p = 0 . 0363 ) . mIPSC frequency was not different in deep CA1 pyramidal neurons of adult ( 4 months ) NexCre cTKO versus littermate controls ( D , p = 0 . 0564 ) and mIPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM ( E , * p = 0 . 0356 ) . F : mEPSC and mIPSC sample traces of recordings from superficial CA1 pyramidal cells . G

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

Figure 4A: mEPSC and mIPSC sample traces of recordings from deep CA1 pyramidal cells. B - E: Bar graphs of mEPSC and mIPSC frequencies and amplitudes in deep CA1 pyramidal cells. mEPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (B, p = 0.6415). mEPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM control cells (C, *p = 0.0363). mIPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (D, p = 0.0564) and mIPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM (E, *p = 0.0356). F: mEPSC and mIPSC sample traces of recordings from superficial CA1 pyramidal cells. G


Figure 5A : Diurnal behavior in the home cage ( HC ) . NexCre cTKOs show overshooting activity in the dark phase . Line graphs illustrate the average locomotion per hour . [ Sidak ' s multiple comparisons test , geno F ( 1 , 32 ) = 9 . 503 , p = 0 . 0042 ; timepoint F ( 6 . 102 , 195 . 3 ) = 10 . 51 , p < 0 . 0001 ; timepoint × geno F ( 47 , 1504 ) = 5 . 874 , p < 0 . 0001 ] . I : Sociability in the three chambers test ( 3CT ) . LM controls spend more time with mouse stranger 1 ( S1 ) whereas NexCre cTKOs spend comparable time exploring the novel object ( NO ) or S1 . Bottom : schematic representation of test . [ Paired Student ' s t - test comparison between chambers LM * * * p = 0 . 0002 ; Wilcoxon test between chambers NexCre cTKO ns p = 0 . 5412 ] .

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

Figure 5A: Diurnal behavior in the home cage (HC). NexCre cTKOs show overshooting activity in the dark phase. Line graphs illustrate the average locomotion per hour. [Sidak's multiple comparisons test, geno F(1,32) = 9.503, p = 0.0042; timepoint F(6.102,195.3) = 10.51, p < 0.0001; timepoint × geno F(47,1504) = 5.874, p < 0.0001].I: Sociability in the three chambers test (3CT). LM controls spend more time with mouse stranger 1 (S1) whereas NexCre cTKOs spend comparable time exploring the novel object (NO) or S1. Bottom: schematic representation of test. [Paired Student's t-test comparison between chambers LM ***p = 0.0002; Wilcoxon test between chambers NexCre cTKO ns p = 0.5412].


Figure 6C : T - Maze alternation test shows that spontaneous alternation is absent in NexCre cTKOs . [ geno F ( 1 , 16 ) = 18 . 29 , p = 0 . 0006 ] . Chance level is indicated by a dotted line . N : Morris Water Maze ( MWM ) , escape latency . During place navigation training , NexCre cTKO animals showed no evidence of learning . [ geno F ( 1 , 18 ) = 30 . 53 , p < 0 . 0001 ; day F ( 4 , 72 ) = 13 . 35 , p < 0 . 0001 ; day × geno F ( 4 , 72 ) = 11 . 77 p < 0 . 0001 ] .

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

Figure 6C: T-Maze alternation test shows that spontaneous alternation is absent in NexCre cTKOs. [geno F(1,16) = 18.29, p = 0.0006]. Chance level is indicated by a dotted line.N: Morris Water Maze (MWM), escape latency. During place navigation training, NexCre cTKO animals showed no evidence of learning. [geno F(1,18) = 30.53, p < 0.0001; day F(4,72) = 13.35, p < 0.0001 ; day × geno F(4,72) = 11.77 p < 0.0001].


Figure 1All liposome permeabilization assays were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( a - d ) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes . ( a ) Pore formation of 50 nM BakΔTM ( red ) is activated by 20 nM cBid ( blue ) . 20 nM cBid alone ( orange ) shows no liposome permeabilization . Addition of 25 nM BclxLΔTM ( green ) inhibits pore - formation . ( b ) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence ( blue ) and presence ( green ) of 25 nM BclxLΔTM . ( c , d ) BakΔTM becomes autoactive at increasing concentrations . The concentrations are color coded as indicated in ( d ) . ( c ) The averaged kinetics for increasing BakΔTM concentrations . Bar diagrams in ( b ) and ( d ) represent data from the kinetic experiments averaged between 9000 - 10000 s . The standard deviation was calculated from three measurements . ( All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( e ) Far - UV - CD spectra of BakΔTM + / - liposomes and the activators cBid or Puma - BH3 peptide as indicated . All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( f ) CD - detected thermal melting experiments of BakΔTM ( black ) with liposomes ( red ) and Puma - BH3 ( blue ) .

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

Figure 1All liposome permeabilization assays were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. ( All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated.All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (f) CD-detected thermal melting experiments of BakΔTM (black) with liposomes (red) and Puma-BH3 (blue).


Figure 6 ( c , d ) Liposome permeabilization assay comparing the variants in ( a , green ) and ( b , blue ) to wild - type BakΔTM ( black ) . ( c ) Comparison of 50 nM of wild - type BakΔTM ( black ) to the two variants after activation by 25 nM cBid . ( d ) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM . The data derived from the liposome permeabilization kinetics shown in panel ( c ) is averaged for the time interval of 9000 - 10000 s and the standard deviations were derived from three technical replicates . (

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

Figure 6(c, d) Liposome permeabilization assay comparing the variants in (a, green) and (b, blue) to wild-type BakΔTM (black). (c) Comparison of 50 nM of wild-type BakΔTM (black) to the two variants after activation by 25 nM cBid. (d) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM. The data derived from the liposome permeabilization kinetics shown in panel (c) is averaged for the time interval of 9000-10000 s and the standard deviations were derived from three technical replicates. (


Figure 1 ( B - C ) Representative confocal images and their quantification of Gb3 accumulation within the lysosome , detected by fluorescent - conjugated Shiga toxin , from WT , CLN3 - KO and CLN7 - KO ARPE - 19 cells treated with PDMP or silenced for Gb3 synthase ( siGb3S ) ( * * * vs WT , ° ° ° vs DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

Figure 1(B-C) Representative confocal images and their quantification of Gb3 accumulation within the lysosome, detected by fluorescent-conjugated Shiga toxin, from WT, CLN3-KO and CLN7-KO ARPE-19 cells treated with PDMP or silenced for Gb3 synthase (siGb3S) (*** vs WT, °°° vs DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Figure 2 ( A - B ) Representative confocal images of Gb3 accumulation , revealed by STX staining , in brain sections from CLN7Δex2 mice at 3 and 7 . 5 months of age compared with CLN7 WT mice . Scale bars : 80 µm . ( E ) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain . Data are presented as mean ± SD , * * : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas )

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Figure 2(A-B) Representative confocal images of Gb3 accumulation, revealed by STX staining, in brain sections from CLN7Δex2 mice at 3 and 7.5 months of age compared with CLN7 WT mice. Scale bars: 80 µm.(E) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain. Data are presented as mean ± SD, **: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (n= 3 biological replicas)


Figure 3 ( A ) Representative confocal images and quantification of SCMAS staining within the lysosome of ARPE - 19 WT and CLN3 KO upon depletion of Gb3S ( siGb3S ) , LacCer ( siLCS ) , and GM3s ( siGM3S ) . ( * * * vs WT , ° ° ° vs CLN3 KO siSCR ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm . ( D ) Representative confocal images and quantification of STX staining within the lysosome of ARPE - 19 WT and CLN7 KO in DMSO or treated 48h with Tamoxifen . ( * * * vs WT , ° ° ° vs CLN3 KO DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm . ( E ) Representative confocal images and quantification of STX staining within the lysosome of NPCs WT and derived from CLN7Pa474 patient IPSCs in DMSO or treated with Tamoxifen for 48h . ( * * * vs WT , ° ° ° vs CLN7Pa474 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

Figure 3(A) Representative confocal images and quantification of SCMAS staining within the lysosome of ARPE-19 WT and CLN3 KO upon depletion of Gb3S (siGb3S), LacCer (siLCS), and GM3s (siGM3S). (*** vs WT, °°° vs CLN3 KO siSCR). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.(D) Representative confocal images and quantification of STX staining within the lysosome of ARPE-19 WT and CLN7 KO in DMSO or treated 48h with Tamoxifen. (*** vs WT, °°° vs CLN3 KO DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.(E) Representative confocal images and quantification of STX staining within the lysosome of NPCs WT and derived from CLN7Pa474 patient IPSCs in DMSO or treated with Tamoxifen for 48h. (*** vs WT, °°° vs CLN7Pa474 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Figure 4 ( A ) Representative confocal images and Quantification of STX within the lysosome in U2 - OS and HeLa cells after acute silencing of CLN3 ( siCLN3 ) in DMSO or treated 48h with Tamoxifen . ( * * * vs siSCR DMSO , ° ° ° vs siCLN3 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm . ( B ) Representative confocal image and quantification of Gb3 in ARPE - 19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB ( siTFEB ) for 72h and treated for the last 48h with DMSO or Tamoxifen . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm . ( C ) Representative confocal image and quantification of TFEB in U2 - OS cells silenced with siRNA against scramble sequence and CLN3 ( siCLN3 ) for 72h and treated for the last 48h with DMSO or Tamoxifen ( 5 µM and 10 µM ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

Figure 4(A) Representative confocal images and Quantification of STX within the lysosome in U2-OS and HeLa cells after acute silencing of CLN3 (siCLN3) in DMSO or treated 48h with Tamoxifen. (*** vs siSCR DMSO, °°° vs siCLN3 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.(B) Representative confocal image and quantification of Gb3 in ARPE-19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB (siTFEB) for 72h and treated for the last 48h with DMSO or Tamoxifen. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.(C) Representative confocal image and quantification of TFEB in U2-OS cells silenced with siRNA against scramble sequence and CLN3 (siCLN3) for 72h and treated for the last 48h with DMSO or Tamoxifen (5 µM and 10 µM). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Figure 5 ( C ) Representative confocal image and quantification of TFEB localization in HeLa TFEB - GFP transfected with RagC for 48h and treated for the last 3h with DMSO , Torin1 , Tamoxifen or Ospemifene . Ratios of nuclear to cytosolic TFEB localization in RagC non - expressing ( RagC - ) and RagC - expressing cells ( RagC + ) are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm . D ) Representative confocal images and quantification of STX in ARPE - 19 CLN3 KO cells transfected with empty vector or HA - RagC for 48h and treated for 48h with DMSO or Tamoxifen . STX average spot area presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

Figure 5(C) Representative confocal image and quantification of TFEB localization in HeLa TFEB-GFP transfected with RagC for 48h and treated for the last 3h with DMSO, Torin1, Tamoxifen or Ospemifene. Ratios of nuclear to cytosolic TFEB localization in RagC non-expressing (RagC-) and RagC-expressing cells (RagC+) are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.D) Representative confocal images and quantification of STX in ARPE-19 CLN3 KO cells transfected with empty vector or HA-RagC for 48h and treated for 48h with DMSO or Tamoxifen. STX average spot area presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Figure 6Representative confocal images of STX in the Cortex , Hippocampus , and Cerebellum brain section derived from 7 . 5 months - old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen ( Tamox ) . Quantification of confocal images , the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° / ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * / oo : P ≤ 0 . 001 , * * * / ooo : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 60 µm .

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

Figure 6Representative confocal images of STX in the Cortex, Hippocampus, and Cerebellum brain section derived from 7.5 months-old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen (Tamox). Quantification of confocal images, the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°°/°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, **/oo: P ≤ 0.001, ***/ooo: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 60 µm.


Figure 7Representative confocal images of SCMAS in the Cortex , Hippocampus , and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen . Quantification of confocal images , the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological ) . Scale bars : 60 µm .

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

Figure 7Representative confocal images of SCMAS in the Cortex, Hippocampus, and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen. Quantification of confocal images, the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological). Scale bars: 60 µm.


Figure 8 ( A - B ) Representative confocal images and quantification of IBA - 1 in the Cortex , Hippocampus , and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen . ( * * * / * * / * vs WT , ° ° ° / ° ° / ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * / o : P ≤ 0 . 01 , * * / oo : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 50 µm . ( D ) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen .

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

Figure 8(A-B) Representative confocal images and quantification of IBA-1 in the Cortex, Hippocampus, and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm.(D) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.


Figure 1A HeLa - H2B / tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine ( TT ) block . After release from the block , mitotic cells were counted by fluorescence microscopy ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . B Time from S - to M - phase was measured in si - RNA transfected HeLa - H2B / tub cells by time - lapse microscopy following release from TT block ( 4 repeat experiments ; all measurements depicted ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . Western blot analysis of unsynchronized HeLa - H2B / tub cells was carried out to monitor expression of p ( S10 ) - H3 , total H3 and TIAR in control and TIAR - depleted cells ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . D HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA . 72 hours after transfection , p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . HeLadox - YFP - TIARr cells were transfected with control or TIAR siRNAs , and 24h hours later cell were cultured in the abscence or presence of 1 μg / ml doxocycline for 48 hours . The expression of p ( S10 ) - H3 , total H3 and TIAR was measured by Westen blot analysis . The graph shows the quantification of p - H3 levels normalized to total H3 ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . F HeLadox - YFP - TIARr - RRM123m cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . G HeLadox - YFP - TIARr - dQRD cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Figure 1A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.B Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Figure 2A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours , mitotic cells were analyzed by IF microscopy and scored for chromatin ( mean ± SE , n = 3 ) . B HeLa cells were analyzed as in ( A ) and scored for spindle defects ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . C HeLa cells were transfected with control or TIAR siRNAs for 4 days , and subjected to colcemid for 2 hours prior to preparation of metaphase spreads . Chromosomes were stained with Hoechst ( red ) and anti - CENP - A antibody ( green ) ; the yellow arrowhead marks a chromosome break . D Metaphase spreads were prepared as in ( C ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . E Metaphase spreads were prepared as in ( C ) , and the frequency of cells with scattered chromosomes was quantified ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . F HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA , and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads . The frequency of cells with scattered chromatids was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . G Metaphase spreads were prepared as in ( F ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 ) . Data information , statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Figure 2A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3). B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.F HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Figure 3A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0 . 4 µM APH . Cells were fixed at regular time intervals , and p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SE , n = 3 ) . B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 2000 cells examined per experiment and condition ) . Each dot represents the signal from one cell , horizontal lines indicate mean values , and the blue area delineates cells above an arbitrarily chosen threshold . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . C Chromatin - bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs ( n = 3 , 600 cells examined per experiment and condition ) . D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi ( 5 μM . ) alone or together with Ro3306 ( 5 μM ) for 12 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 1000 cells examined per experiment and condition ) . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours , and the sub - G1 population was quantified by flow cytometry following propidium iodide staining ( mean ± SD , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . F HeLa cells transfected with control or TIAR siRNAs were treated with APH , alone or in combination with Gö6976 ( 1µg / ml ) or UCN - 01 ( 300 nM ) . After 24 hours , p ( S10 ) - H3 - positive cells were quantified by flow cytometry , and expressed relative to the values in the untreated condition ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

Figure 3A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.C Chromatin-bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs (n = 3, 600 cells examined per experiment and condition).D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.F HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


Figure 4A HeLa cells , untreated or treated for 24 hours with 0 . 4 µM APH , were fixed with methanol and analyzed by IF microscopy after staining with anti - p ( S10 ) - H3 antibody , anti - TIAR antibody and DAPI . Yellow arrows mark focal accumulation of TIAR in GMGs . B IF microscopy was performed as in ( A ) to determine the intensity of the TIAR signal within GMGs ( mean ± SD , n = 3 independent experiments , approximately 100 GMGs in 25 cells were analyzed per experiment and condition ) . C IF microscopy was performed as in ( A ) to determine the area of the TIAR signal within GMGs ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . D IF microscopy was performed as in ( A ) to quantify the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . E HeLa cells were processed for IF microscopy as in ( A ) , images depict different cell cycle phases . HeLa cell were treated for 16 hours with ( F ) the CDK1 inhibitor Ro3306 ( 9 µM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . HeLa cell were treated for 16 hours with ( G ) ICFR - 193 ( 1 μM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) .

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Figure 4A HeLa cells, untreated or treated for 24 hours with 0.4 µM APH, were fixed with methanol and analyzed by IF microscopy after staining with anti-p(S10)-H3 antibody, anti-TIAR antibody and DAPI. Yellow arrows mark focal accumulation of TIAR in GMGs.B IF microscopy was performed as in (A) to determine the intensity of the TIAR signal within GMGs (mean ± SD, n = 3 independent experiments, approximately 100 GMGs in 25 cells were analyzed per experiment and condition). C IF microscopy was performed as in (A) to determine the area of the TIAR signal within GMGs (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.D IF microscopy was performed as in (A) to quantify the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.E HeLa cells were processed for IF microscopy as in (A), images depict different cell cycle phases.HeLa cell were treated for 16 hours with (F) the CDK1 inhibitor Ro3306 (9 µM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.HeLa cell were treated for 16 hours with (G) ICFR-193 (1 μM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition).


Figure 5A - G HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR antibody , DAPI in combination with ( A ) anti - PCNA - 1 antibody , ( B ) anti - FANCD2 antibody , ( C ) anti - BRCA1 antibody , ( D ) anti - PRP19 antibody , ( E ) anti - Sm ( Y12 ) antibody , ( F ) anti - HuR antibody and ( G ) pS2 - RPOL2 ( H5 ) antibody . Intensity profiles along the white line in the merged image are presented on the right side ; yellow and green arrows mark GMGs .

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

Figure 5A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.


Figure 6A HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR and anti - CDK1 antibodies . Intensity profiles along the white line in the merged image are presented on the right side ; yellow arrows mark GMGs . B IF microscopy analysis as in ( A ) using anti - Cyclin B antibody . C HeLa cells transfected with control or TIAR siRNAs were treated with 0 . 4 µM APH 24 hours prior to fixation . Cells were processed for IF microscopy after staining with anti - PRP19 and anti - CDK1 antibodies . Intensity profiles from staining of the same cells with anti - TIAR and anti - CDK1 antibodies are depicted on the right side . D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs , tested for in vitro kinase activity using recombinant histone H1 as substrate , and visualized by Western blot analysis and autoradiography . The mean CDK1 activity ± SE was quantified from n = 5 experiments . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 . E CDK2 activity was analyzed following immunoprecipitation as in ( D ) ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . * , p < 0 . 05 ; * * * , p < 0 . 001 . F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p ( S22 ) - Lamin A by Western blot analysis ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 . G p ( S22 ) - Lamin A levels were analyzed by HTM in si - RNA transfected HeLa cells ( n = 3 , 1000 cells examined per experiment and condition ) . Data information statistical significance was determined by Wilcoxon rank - sum test ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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Figure 6A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs.B IF microscopy analysis as in (A) using anti-Cyclin B antibody.C HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side.D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SE was quantified from n = 5 experiments. Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.E CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. *, p < 0.05; ***, p < 0.001.F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p(S22)-Lamin A by Western blot analysis (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.


Figure 1HeLa cells were co - transfected with Myc empty vector ( Myc ) , Myc - tagged FUNDC1 ( F1 - Myc ) or Myc - tagged FUNDC1 - ΔLIR mutant ( ΔLIR - Myc ) and Mito - OM - cherry , which localizes to the mitochondrial outer membrane , and immunostained with fluorescent anti - p62 and anti - Myc antibodies . Scale bar = 10 μm . HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - Ubiquitin ( Ub ) and anti - Myc antibodies . Scale bar = 10 µm . Fractionation analysis of HeLa cells transfected with Myc , F1 - Myc or ΔLIR - Myc . The loading controls ( L . C . ) were α - Tubulin for total cell lysate ( TCL ) and cytosolic fraction ( Cyto ) , and HSP60 for mitochondrial fraction ( Mito ) . HeLa cells transfected with Myc or Myc / His6 double - tagged F1 - Myc - His were lysed and precipitated with Ni - NTA beads . Samples were then subjected to SDS - PAGE . The differential band ( upper arrow ) was isolated and subjected to mass spectral analysis . The identified proteins with a score higher than 50 are listed . Co - IP of endogenous HSC70 with F1 - Myc or ΔLIR - Myc . α - Myc , anti - Myc antibodyCo - IP of HSC70 with FUNDC1 . α - F1 , anti - FUNDC1 antibody . rIgG , control rabbit IgG . Co - IP analysis of endogenous FUNDC1 with Flag - HSP70 or Flag - HSC70 . α - Flag , anti - Flag antibody . Flag ， empty vector . HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - HSC70 and anti - Myc antibodies . Scale bar = 10 µm . Fractionation analysis of HeLa cells transfected with Myc or F1 - Myc . HeLa cells were co - transfected with Ub - R - GFP and Myc or F1 - Myc and immunostained with fluorescent anti - Myc and anti - GFP ( α - GFP ) antibodies . The α - GFP signals in F1 - Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R - GFP , making such signals of the equally distributed R - GFP invisible . Green arrowheads show the mitochondrially localized R - GFP proteins , which were more abundant than the equally distributed R - GFP and showed stronger R - GFP fluorescence , white arrowheads show the mitochondrially localized R - GFP proteins without stronger R - GFP fluorescence , although abundant . Scale bar = 10 µm . Fractionation analysis of HeLa cells co - transfected with Ub - R - GFP and Myc or F1 - Myc . HeLa cells were co - transfected with Ub - R - GFP and F1 - Myc . Mitochondria were isolated and then treated with or without proteinase K ( Pro . K ) , or proteinase K in combination with digitonin or Triton X - 100 ( TX - 100 ) . Samples were then subjected to Immunoblot analysis . HSP60 , TIM23 and TOM20 are representatives of mitochondrial matrix - , mitochondrial inner membrane - and mitochondrial outer membrane - localized proteins , respectively . Fractionation analysis of HeLa cells stably transfected with control ( Ctrl ) shRNA vector or HSC70 shRNA and transiently co - transfected with Ub - R - GFP , Flag or Flag - HSC70 ( shRNA - resistant ) and Myc , or F1 - Myc , as indicated .

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Figure 1HeLa cells were co-transfected with Myc empty vector (Myc), Myc-tagged FUNDC1 (F1-Myc) or Myc-tagged FUNDC1-ΔLIR mutant (ΔLIR-Myc) and Mito-OM-cherry, which localizes to the mitochondrial outer membrane, and immunostained with fluorescent anti-p62 and anti-Myc antibodies. Scale bar=10 μm.HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-Ubiquitin (Ub) and anti-Myc antibodies. Scale bar=10 µm.Fractionation analysis of HeLa cells transfected with Myc, F1-Myc or ΔLIR-Myc. The loading controls (L.C.) were α-Tubulin for total cell lysate (TCL) and cytosolic fraction (Cyto), and HSP60 for mitochondrial fraction (Mito).HeLa cells transfected with Myc or Myc/His6 double-tagged F1-Myc-His were lysed and precipitated with Ni-NTA beads. Samples were then subjected to SDS-PAGE. The differential band (upper arrow) was isolated and subjected to mass spectral analysis. The identified proteins with a score higher than 50 are listed.Co-IP of endogenous HSC70 with F1-Myc or ΔLIR-Myc. α-Myc, anti-Myc antibodyCo-IP of HSC70 with FUNDC1. α-F1, anti-FUNDC1 antibody. rIgG, control rabbit IgG.Co-IP analysis of endogenous FUNDC1 with Flag-HSP70 or Flag-HSC70. α-Flag, anti-Flag antibody. Flag，empty vector.HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-HSC70 and anti-Myc antibodies. Scale bar=10 µm.Fractionation analysis of HeLa cells transfected with Myc or F1-Myc.HeLa cells were co-transfected with Ub-R-GFP and Myc or F1-Myc and immunostained with fluorescent anti-Myc and anti-GFP (α-GFP) antibodies. The α-GFP signals in F1-Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R-GFP, making such signals of the equally distributed R-GFP invisible. Green arrowheads show the mitochondrially localized R-GFP proteins, which were more abundant than the equally distributed R-GFP and showed stronger R-GFP fluorescence, white arrowheads show the mitochondrially localized R-GFP proteins without stronger R-GFP fluorescence, although abundant. Scale bar=10 µm.Fractionation analysis of HeLa cells co-transfected with Ub-R-GFP and Myc or F1-Myc.HeLa cells were co-transfected with Ub-R-GFP and F1-Myc. Mitochondria were isolated and then treated with or without proteinase K (Pro.K), or proteinase K in combination with digitonin or Triton X-100 (TX-100). Samples were then subjected to Immunoblot analysis. HSP60, TIM23 and TOM20 are representatives of mitochondrial matrix-, mitochondrial inner membrane- and mitochondrial outer membrane-localized proteins, respectively.Fractionation analysis of HeLa cells stably transfected with control (Ctrl) shRNA vector or HSC70 shRNA and transiently co-transfected with Ub-R-GFP, Flag or Flag-HSC70 (shRNA-resistant) and Myc, or F1-Myc, as indicated.


Figure 2Fractionation analysis of the HeLa cells transfected with Ub - R - GFP and treated with DMSO ( control vehicle ) or 10 μM MG132 for 4 hours . Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub - R - GFP and treated with MG132 . Proteinase K protection analysis of the mitochondria isolated from HeLa cells co - transfected with Ub - R - GFP and Ctrl , TOM20 , TOM22 or TOM70 shRNA and treated with MG132 . Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co - transfected with Ub - R - GFP and Myc or LONP1 ( shRNA - resistant ) and then treated with DMSO or MG132 . Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA , F1 shRNA , or F1 shRNA together with shRNA - resistant F1 - Myc or ΔLIR - Myc , and transiently transfected with Ub - R - GFP and then treated with DMSO or MG132 . Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSC70 and then treated with DMSO or MG132 . Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSP70 and then treated with DMSO or MG132 . Mitochondria were isolated from the untransfected cells , cytosols were isolated from the cells transfected with GFP or Ub - R - GFP and treated with DMSO or MG132 for 8 hours , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis . Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub - R - GFP and incubated at RT for I hour , the mitochondria were then isolated from the in vitro system , then treated with or without proteinase K and subjected to immunoblot analysis . Mitochondria were isolated from the cells transfected with ctrl shRNA , F1 shRNA or F1 shRNA together with F1 - Myc , cytosols were isolated from the cells transfected with Ub - R - GFP , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis . Cytosol isolated from the cells transfected with Ub - R - GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA , HSC70 shRNA or HSC70 shRNA together with Flag - HSC70 , mitochondria were isolated from the untransfected cells , the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis .

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Figure 2Fractionation analysis of the HeLa cells transfected with Ub-R-GFP and treated with DMSO (control vehicle) or 10 μM MG132 for 4 hours.Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub-R-GFP and treated with MG132.Proteinase K protection analysis of the mitochondria isolated from HeLa cells co-transfected with Ub-R-GFP and Ctrl, TOM20, TOM22 or TOM70 shRNA and treated with MG132.Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co-transfected with Ub-R-GFP and Myc or LONP1 (shRNA-resistant) and then treated with DMSO or MG132.Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA, F1 shRNA, or F1 shRNA together with shRNA-resistant F1-Myc or ΔLIR-Myc, and transiently transfected with Ub-R-GFP and then treated with DMSO or MG132.Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSC70 and then treated with DMSO or MG132.Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSP70 and then treated with DMSO or MG132.Mitochondria were isolated from the untransfected cells, cytosols were isolated from the cells transfected with GFP or Ub-R-GFP and treated with DMSO or MG132 for 8 hours, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub-R-GFP and incubated at RT for I hour, the mitochondria were then isolated from the in vitro system, then treated with or without proteinase K and subjected to immunoblot analysis.Mitochondria were isolated from the cells transfected with ctrl shRNA, F1 shRNA or F1 shRNA together with F1-Myc, cytosols were isolated from the cells transfected with Ub-R-GFP, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.Cytosol isolated from the cells transfected with Ub-R-GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA, HSC70 shRNA or HSC70 shRNA together with Flag-HSC70, mitochondria were isolated from the untransfected cells, the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis.


Figure 3Co - IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated . Immunoblot analysis of the HeLa cells transfected with or without HA - Bcl - xL and treated with DMSO or MG132 for 6 hours . Ni2 + - NTA precipitation of HeLa cells transfected with indicated vectors . S13A - Myc - His , Myc / His6 double - tagged FUNDC1 - S13A mutant . D , E . Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132 . Quantification is shown in ( E ) .

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Figure 3Co-IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated.Immunoblot analysis of the HeLa cells transfected with or without HA-Bcl-xL and treated with DMSO or MG132 for 6 hours.Ni2+-NTA precipitation of HeLa cells transfected with indicated vectors. S13A-Myc-His, Myc/His6 double-tagged FUNDC1-S13A mutant.D, E. Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132. Quantification is shown in (E).


Figure 4HeLa cells were transfected with GFP - p62 , treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti - Ub and anti - γ - Tubulin antibodies . Note that the aggresome ( blue arrowhead ) , rather than the MAPAs ( white arrowheads ) , colocalizes with γ - Tubulin . Scale bar = 10 µm . B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( C ) . D , E . HeLa cells were transfected with Ctrl , TOM20 , TOM22 or TOM70 shRNA vectors including nuclear localized GFP ( N - GFP ) expressing elements , then treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( E ) . F , G . HeLa cells were transfected with GFP or GFP - Bcl - xL , then treated with MG132 and immunostained with anti - p62 antibodies . Scale bar = 10 µm . Quantification is show in ( G ) . H , I . HeLa cells were stably transfected with Ctrl or LONP1 shRNA , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( I ) .

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Figure 4HeLa cells were transfected with GFP-p62, treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti-Ub and anti-γ-Tubulin antibodies. Note that the aggresome (blue arrowhead), rather than the MAPAs (white arrowheads), colocalizes with γ-Tubulin. Scale bar=10 µm.B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm.B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (C).D, E. HeLa cells were transfected with Ctrl, TOM20, TOM22 or TOM70 shRNA vectors including nuclear localized GFP (N-GFP) expressing elements, then treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (E).F, G. HeLa cells were transfected with GFP or GFP-Bcl-xL, then treated with MG132 and immunostained with anti-p62 antibodies. Scale bar=10 µm. Quantification is show in (G).H, I. HeLa cells were stably transfected with Ctrl or LONP1 shRNA, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (I).


Figure 5csCLEM analysis of cells transfected with GFP - p62 and treated with MG132 . Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy ( LM ) and transmission electron microscopy ( TEM ) . The blue arrowhead shows a MAPA observed under LM and TEM . TEM analysis of HeLa cells treated with DMSO or MG132 . Note the layered structures ( red arrowheads ) in the mitochondrial matrix ( ii , iii ) , budding from mitochondria ( iv ) , attached to mitochondria ( v - vi ) , away from mitochondria ( vii ) or inside the MAPAs ( viii , ix ) in MG132 - treated cells . Scale bar = 500 nm . M , mitochondrion . N , nucleus . Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells treated with MG132 as indicated . Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells transfected with Ub - R - GFP and treated with MG132 as indicated .

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Figure 5csCLEM analysis of cells transfected with GFP-p62 and treated with MG132. Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy (LM) and transmission electron microscopy (TEM). The blue arrowhead shows a MAPA observed under LM and TEM.TEM analysis of HeLa cells treated with DMSO or MG132. Note the layered structures (red arrowheads) in the mitochondrial matrix (ii, iii), budding from mitochondria (iv), attached to mitochondria (v-vi), away from mitochondria (vii) or inside the MAPAs (viii, ix) in MG132-treated cells. Scale bar=500 nm. M, mitochondrion. N, nucleus.Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells treated with MG132 as indicated.Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells transfected with Ub-R-GFP and treated with MG132 as indicated.


Figure 6HeLa cells were co - transfected with Mito - OM - cherry and Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 antibody . Note the colocalization of a p62 punctum with a structure that is positive for Mito - OM - cherry but not for Mito - MTX - GFP ( white arrowheads ) in MG132 - treated cells . Scale bar = 10 µm . HeLa cells were transfected with Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 and anti - TIM23 antibodies . Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito - MTX - GFP ( white arrowhead ) in MG132 - treated cells . Scale bar = 10 µm . HeLa cells were co - transfected with Mito - MTX - GFP and OTC - Myc or OTC - Δ - Myc , treated with DMSO or MG132 and immunostained with anti - Myc and anti - p62 antibodies . Note that the p62 - labled MAPAs were colocalized with OTC - ∆ ( red arrowheads ) , but not with OTC ( white arrowheads ) in the MG132 treated cells . Scale bar = 10 µm . TEM analysis of HeLa cells transfected with indicated vectors , treated with MG132 and stained with DAB . Blue arrowheads show MAPAs , red arrowheads show electron - dense Apex signals . Scale bar = 500 nm . M , mitochondrion .

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Figure 6HeLa cells were co-transfected with Mito-OM-cherry and Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 antibody. Note the colocalization of a p62 punctum with a structure that is positive for Mito-OM-cherry but not for Mito-MTX-GFP (white arrowheads) in MG132-treated cells. Scale bar=10 µm.HeLa cells were transfected with Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 and anti-TIM23 antibodies. Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito-MTX-GFP (white arrowhead) in MG132-treated cells. Scale bar=10 µm.HeLa cells were co-transfected with Mito-MTX-GFP and OTC-Myc or OTC-Δ-Myc, treated with DMSO or MG132 and immunostained with anti-Myc and anti-p62 antibodies. Note that the p62-labled MAPAs were colocalized with OTC-∆ (red arrowheads), but not with OTC (white arrowheads) in the MG132 treated cells. Scale bar=10 µm.TEM analysis of HeLa cells transfected with indicated vectors, treated with MG132 and stained with DAB. Blue arrowheads show MAPAs, red arrowheads show electron-dense Apex signals. Scale bar=500 nm. M, mitochondrion.


Figure 7Time - lapse analysis of live HeLa cells transfected with OTC - Δ - GFP and Mito - MTX - DsRed and treated with MG132 . White arrowheads show a OTC - Δ - GFP - positive Mito - MTX - DsRed - negative structure that eventually segregated from the mitochondrial network . Immunoblot analysis of the MG132 - induced deposition of FUNDC1 , TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA . C , D . Immunoblot analysis of the MG132 - induced deposition of FUNDC1 in cells transfected with ctrl shRNA , FIS1 shRNA or FIS1 shRNA together with shRNA - resistant Flag - FIS1 . Quantification is shown in ( D ) . E , F . HeLa cells were stably transfected with Ctrl or FIS1 shRNA , treated with MG132 and immunostained with indicated antibodies . Scale bar = 10 µm . Quantification is shown in ( F ) . G , H . Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA , then transiently co - transfected with Ub - R - GFP and Flag or Flag - FIS1 and treated with DMSO or MG132 . Quantification is shown in ( H ) .

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Figure 7Time-lapse analysis of live HeLa cells transfected with OTC-Δ-GFP and Mito-MTX-DsRed and treated with MG132. White arrowheads show a OTC-Δ-GFP-positive Mito-MTX-DsRed-negative structure that eventually segregated from the mitochondrial network.Immunoblot analysis of the MG132-induced deposition of FUNDC1, TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA.C, D. Immunoblot analysis of the MG132-induced deposition of FUNDC1 in cells transfected with ctrl shRNA, FIS1 shRNA or FIS1 shRNA together with shRNA-resistant Flag-FIS1. Quantification is shown in (D).E, F. HeLa cells were stably transfected with Ctrl or FIS1 shRNA, treated with MG132 and immunostained with indicated antibodies. Scale bar=10 µm. Quantification is shown in (F).G, H. Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA, then transiently co-transfected with Ub-R-GFP and Flag or Flag-FIS1 and treated with DMSO or MG132. Quantification is shown in (H).


Figure 8HeLa cells were transfected with GFP - LC3 , treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm . Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated . Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated . Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated . Quantification of the results from panels ( D ) and ( E ) . 3 replicates were analyzed using Student ' s 2 - tailed t - test and are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . HeLa cells were stably transfected with the indicated vectors , transiently transfected with GFP - LC3 , then treated with 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm . TEM analysis of F1 shRNA / F1 - Myc or F1 shRNA / ΔLIR - Myc cells treated with MG132 for 8 hours . Yellow arrowheads show the MAPAs . Blue arrows show the places where membrane is absent . White arrowheads show the layered aggregates budding from mitochondria . Scale bar = 500 nm ; M , mitochondrion .

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Figure 8HeLa cells were transfected with GFP-LC3, treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated.Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated.Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated.   Quantification of the results from panels (D) and (E). 3 replicates were analyzed using Student's 2-tailed t-test and are presented as the means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.   HeLa cells were stably transfected with the indicated vectors, transiently transfected with GFP-LC3, then treated with 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.TEM analysis of F1 shRNA/F1-Myc or F1 shRNA/ΔLIR-Myc cells treated with MG132 for 8 hours. Yellow arrowheads show the MAPAs. Blue arrows show the places where membrane is absent. White arrowheads show the layered aggregates budding from mitochondria. Scale bar=500 nm; M, mitochondrion.


Figure 9A , B . HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours , then washed with fresh medium three times and cultured for another 3 days . Cells were then fixed and subjected to SA - β - gal staining . The senescent cells show SA - β - gal - positive staining ( Blue ) . Quantification is shown in ( B ) . C , D . Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours , then washed and cultured for the indicated times . Quantification is shown in ( D ) . E , Quantification of SA - β - gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days . F . HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days . Immunoblot analysis of the cells is shown in ( F ) . Quantification of SA - β - gal staining of HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells . H . HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( H ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells . Quantification of MG132 - induced SA - β - gal staining in HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 . J . HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( J ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 . HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Cells were then subjected to SA - β - gal staining . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable . L . HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Immunoblot analysis of the cells is shown in ( L ) . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable . Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . , N . HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( N ) . Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . P . HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( P ) .

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

Figure 9A, B. HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours, then washed with fresh medium three times and cultured for another 3 days. Cells were then fixed and subjected to SA-β-gal staining. The senescent cells show SA-β-gal-positive staining (Blue). Quantification is shown in (B).C, D. Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours, then washed and cultured for the indicated times. Quantification is shown in (D).E, Quantification of SA-β-gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days.F. HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days. Immunoblot analysis of the cells is shown in (F).Quantification of SA-β-gal staining of HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.H. HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (H). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.Quantification of MG132-induced SA-β-gal staining in HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.J. HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (J). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Cells were then subjected to SA-β-gal staining. The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.L. HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Immunoblot analysis of the cells is shown in (L). The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days., N. HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (N).Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.P. HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (P).


Figure 1Single - cell expression data in pseudotime of central regulators of naïve pluripotency , epiblast marker genes , glycolytic regulators , and genes with key functions in oxidative metabolism .

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Figure 1Single-cell expression data in pseudotime of central regulators of naïve pluripotency, epiblast marker genes, glycolytic regulators, and genes with key functions in oxidative metabolism.