Figure 5Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC - 0946 for two days . Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live ( Annexin V low ; DAPI low ) , apoptotic ( Annexin V high ; DAPI low ) and dead ( Annexin V high ; DAPI high ) cells . Quantification of several independent FACS experiments , including the experiment shown in panel ( A ) . Mean with individual data points of 2 - 4 replicates each of two independent lines per genotype . p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to corresponding DMSO control .

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

Figure 5Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells. Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.


Figure 2 ( B ) The inherent elastic response of a lipid membrane ( POPC ) was studied by enforcing indentations via the isolated SNARE C - termini in the presence or absence of a short linker peptide , or the linker plus a fluorescent protein tag ( FP ) . We define " relative indentation " as the change in C - terminus ( Nyv1 ) - C - terminus ( Vam3 ) distance that occurs when the SNARE complex performs mechanical work . It reflects the depth of the formed well upon squeezing . The slope of the obtained work profile estimates the required force ( thin black line ) . Perforation ( red arrow ) requires a force of 80 pN for the wild - type C - termini . The plateau within the work profile indicates that the membrane gives up its elastic response ( perforation transition ) . C - terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime . Furthermore , if the unstructured peptide - spacer would be alpha - helical - and thus much shorter - indentation forces would still be only slightly affected .

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

Figure 2(B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.


Figure 3 ( B ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting . ( C ) In vivo vacuole morphology was assayed by FM4 - 64 staining . Scale bar : 5 µm . ( D ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments . ( E ) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8 - EGFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Scale bar : 5 µm . ( F ) Fusion activity : vacuole fusion was assayed by measuring the percentage of co - localization of the two Pho8 - EGFP and Pho8 - mCherry signals . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments .

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 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

Figure 3(B) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.(C) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.


Figure 4 ( A ) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and / or Nyv1 . 10 µg of the organelles were analyzed by SDS - PAGE and Western blotting using antibodies to Vti1 , Vam3 and Nyv1 . ( B ) Localization in vivo . The indicated strains were grown logarithmically in synthetic complete ( SC ) medium . After 16 h at 30 ° C , when the final OD of each culture was about 1 , the cells were observed by confocal fluorescence and differential interference ( DIC ) microscopy . Scale bar : 5 µm .

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

Figure 4(A) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1.(B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.


Figure 5 ( A ) In vivo morphology . Cells expressing the indicated SNARE variants were grown in SC medium ( 16 h , 30 ° C ) , stained with FM4 - 64 and their relative morphology was assessed by fluorescence and DIC microscopy . Scale bar : 5 µm . The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments . ( B ) and ( C ) SNARE activation on isolated vacuoles . Vacuoles were isolated from a strain co - expressing NYV1 - S9 - EGFP and VAM3 - S9 - mCitrine and from an isogenic wild - type . 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP - regenerating system and recombinant , purified Sec18 / NSF ( rSec18 , 50 µg / ml ) . After 10 min of incubation at 27 ° C , the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1 . Co - immunoprecipitated proteins analyzed by SDS - PAGE and Western blotting . The histograms provide quantifications of the band intensities as the means and s . d . from three independent experiments .

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

Figure 5(A) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.(B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.


Figure 6 ( A ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting . ( B ) Vacuole morphology was assessed for the indicated cells as in Fig . 3C . Scale bar : 5 µm . ( C ) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated . Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [ 65 ] but ensures that trans - SNARE complexes can only form in one orientation , between the Q - SNARE in DKY6281 and the R - SNARE in BJ3505 . The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay . In parallel , identical samples were incubated either incubated on ice , which prevents fusion , or in the presence of 0 . 5 % Triton X - 100 , which allows fusion - independent access of the maturase Pep4 to pro - ALP and controls for the levels of these two reporter enzymes in the samples . ( D ) Using strains from C , lipid and content mixing ( left and right panel , respectively ) were performed in parallel in the presence or absence of ATP . Anti - Vam3 antibody ( 3 µg ) was added to some reactions in order to inhibit trans - SNARE pairing and fusion . Means and the s . d . are shown for three independent experiments .

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

Figure 6(A) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.(B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm.(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.


Figure 7Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( A ) Protein levels on isolated vacuoles from these strains were compared as in Fig . 3B . Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( B ) Vacuole morphology was assessed as in Fig . 3C . Scale bar : 5 µm . ( C ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments . Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( D ) Fusion activity : vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8 - mCFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments . Scale bar : 5 µm . ( E ) Vacuole fusion was assayed by measuring the percentage of co - localisation of the two Pho8 - mCFP and Pho8 - mCherry signals .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B.Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.


Figure 8 ( A ) Yeast strain expressing a plasmid with Vph1 - GFP was grown in SC - URA medium for 16 h at 30 ° C . The mobility of Vph1 - GFP in the absence ( upper panels ) or presence ( lower panel ) of CPZ ( 150 µM ) was assessed by FRAP . ( B ) The histogram shows the GFP signals in the bleached area during the FRAP procedure . ( C ) The half - time is shown . Means and s . d . are shown for 20 cells of 3 independent experiments . ( D ) Yeast cells were grown in SC medium for 16 h at 30 ° C . After staining with FM4 - 64 , cells were treated with CPZ ( 150 µM ) . Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition . Scale bar : 5 µm . ( E ) Fusion activity . Vacuoles were isolated from BJ cells carrying Nyv1 - S9 - EGFP and DKY cells deleted for Nyv1 and carrying Vam3 - S9 - mCitrine or from wild - type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine ( CPZ , 150 µM ) and anti - Vam3 ( 3 µg ) . Content mixing was assayed via alkaline phosphatase activity . Means and s . d . are shown for three independent experiments .

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 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

Figure 8(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.


Figure 1Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family . Quantitative real - time PCR of XRCC4 relative to GAPDH mRNA in affected ( II - 1 and II - 2 ) and control ( CT1 and CT2 ) fibroblasts . Each value refers to the mean of three independent experiments performed in duplicate . Error bars indicate the standard deviation . Immunoblot analysis of total lysates from fibroblasts ( fbs ) and of in vitro - synthesized proteins ( i . v . p ) . fl : in vitro - translated full - length XRCC4 protein ; ret . : reticulocyte lysate used for in vitro protein synthesis ; 225 * : in vitro - translated XRCC4 R225 * truncated protein ; Ct : lysate of control fbs ; and II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . α - XRCC4 ( Abcam , 1 : 1 , 000 ) and α - GAPDH antibodies were used . Immunoblot analysis of total lysates from fibroblasts using α - LIG4 ( GeneTex , 1 : 1 , 000 ) and α - SDHA antibodies . Ct : lysate of control fbs ; II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . The position of the molecular weight ( MW ) marker proteins is indicated .

O B-PANEL_START 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 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 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

Figure 1Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family.Quantitative real-time PCR of XRCC4 relative to GAPDH mRNA in affected (II-1 and II-2) and control (CT1 and CT2) fibroblasts. Each value refers to the mean of three independent experiments performed in duplicate. Error bars indicate the standard deviation.Immunoblot analysis of total lysates from fibroblasts (fbs) and of in vitro-synthesized proteins (i.v.p). fl: in vitro-translated full-length XRCC4 protein; ret.: reticulocyte lysate used for in vitro protein synthesis; 225*: in vitro-translated XRCC4 R225* truncated protein; Ct: lysate of control fbs; and II-1 and II-2: lysates of fbs from patient II-1 and II-2. α-XRCC4 (Abcam, 1:1,000) and α-GAPDH antibodies were used.Immunoblot analysis of total lysates from fibroblasts using α-LIG4 (GeneTex, 1:1,000) and α-SDHA antibodies. Ct: lysate of control fbs; II-1 and II-2: lysates of fbs from patient II-1 and II-2. The position of the molecular weight (MW) marker proteins is indicated.


Figure 2γ - H2AX nuclear foci in XRCC4wt and mutant fibroblasts ( XRCC4m / m1 and XRCC4m / m2 ) were determined at different times after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated control ( 1 . 3 foci / nucleus in XRCC4wt cells and 1 . 4 and 0 . 9 foci / nucleus in XRCC4m / m1 and XRCC4m / m2 cells , respectively ) , are the mean ± SD of three independent experiments . Percentages of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts ( pooled values ) remaining at the indicated time - points . Immunofluorescence micrographs of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts . Data information : * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ( XRCC4m / m vs . XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2γ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.


Figure 3A , B Percentages of γ - H2AX nuclear foci remaining in CENP - F - negative ( A ) and CENP - F - positive ( B ) cells at the indicated time - points after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated controls , are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * P < 0 . 05 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . C Immunofluorescence micrographs of γ - H2AX foci in CENP - F - positive and negative fibroblasts at 2 h after irradiation . Arrow heads indicate the CENP - F - positive cells .

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

Figure 3A, B Percentages of γ-H2AX nuclear foci remaining in CENP-F-negative (A) and CENP-F-positive (B) cells at the indicated time-points after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated controls, are the mean ± SD of three independent experiments. ***P < 0.001; *P < 0.05 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.C Immunofluorescence micrographs of γ-H2AX foci in CENP-F-positive and negative fibroblasts at 2 h after irradiation. Arrow heads indicate the CENP-F-positive cells.


Figure 4RAD51 foci in XRCC4wt and XRCC4m / m fibroblasts irradiated with 0 . 5 Gy of γ - rays . Values are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * * P < 0 . 01 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m / mfibroblasts . Arrowheads indicate the CENP - F - positive cells .

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

Figure 4RAD51 foci in XRCC4wt and XRCC4m/m fibroblasts irradiated with 0.5 Gy of γ-rays. Values are the mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m/mfibroblasts. Arrowheads indicate the CENP-F-positive cells.


Figure 5XRCC4wt and XRCC4m / m cells irradiated with 0 . 5 Gy of γ - rays were exposed to the PARP1 inhibitor 3 ′ - AB or to solvent ( DMSO ) . The γ - H2AX foci number is the mean ± SD of three independent experiments . * P < 0 . 05 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , * * * P < 0 . 001 ( XRCC4m / m + 3 ′ - AB versus XRCC4wt + 3 ′ - AB ) , two - way ANOVA , Bonferroni post hoc test .

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Figure 5XRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.


Figure 6A , B Percentages of DSBs repaired in increasing time intervals ( 0 . 5 - 2 h , 0 . 5 - 6 h , 0 . 5 - 24 h ) were calculated in XRCC4wt ( A ) and XRCC4m / m ( B ) cells from the number of γ - H2AX foci in presence or absence of a PARP - 1 inhibitor , 3 ′ - AB . * * P < 0 . 01 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , two - way ANOVA , Bonferroni post hoc test .

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Figure 6A, B Percentages of DSBs repaired in increasing time intervals (0.5-2 h, 0.5-6 h, 0.5-24 h) were calculated in XRCC4wt (A) and XRCC4m/m (B) cells from the number of γ-H2AX foci in presence or absence of a PARP-1 inhibitor, 3′-AB. **P < 0.01 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), two-way ANOVA, Bonferroni post hoc test.


Figure 1Vero and Caco - 2 cells were infected with SARS - CoV - 2 at MOIs of 0 . 2 for 8 h . Infected cells were then permeabilized and immunostained for the intracellular SARS - CoV - 2 nucleoprotein ( NP , red ) . Nuclei were stained with Hoechst ( blue ) before imaging by fluorescence confocal microscopy . Scale bars : 100 µm . Infection of Vero and Caco - 2 cells was monitored over 24 h using the same flow cytometry - based assay used for the experiment shown in panel D . Infection is given as the total fluorescence associated with the NP protein - positive cells . MFI , mean fluorescence intensity . n = 3 .

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Figure 1Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm.Infection of Vero and Caco-2 cells was monitored over 24 h using the same flow cytometry-based assay used for the experiment shown in panel D. Infection is given as the total fluorescence associated with the NP protein-positive cells. MFI, mean fluorescence intensity. n = 3.


Figure 2Cells were pretreated with the indicated concentrations of aprotinin ( A ) and SB412515 ( B ) , which are inhibitors of TMPRSS2 and cathepsin L , respectively . Infection of Calu - 3 , Caco - 2 , A549 * , and Vero cells with SARS - CoV - 2 at MOIs of 0 . 3 , 0 . 4 , 0 . 2 , and 0 . 3 , respectively , was achieved in the continuous presence of the drug . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where inhibitors had been omitted . n = 2 - 4 biological replicates . SARS - CoV - 2 particles were bound to A549 * and Vero cells ( MOIs 0 . 2 and 0 . 3 , respectively ) ( C ) or Calu - 3 and Caco - 2 cells ( 0 . 6 and 0 . 5 , respectively ) ( D ) on ice for 90 min and subsequently warmed rapidly to 37 ° C to allow infectious penetration . SB412515 ( 10 µM , C ) or aprotinin ( 30 µM , D ) was added at different times post warming to block further proteolytic activation . Infection was analyzed by flow cytometry , and data were normalized to samples where protease inhibitors had been omitted . n = 2 .

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Figure 2Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates.SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.


Figure 3Cells were pretreated with endosomal - pH interfering drugs at the indicated concentrations and subsequently infected with SARS - CoV - 2 as described in Fig . 2A and 2B in the continuous presence of NH4Cl ( A ) , chloroquine ( B ) , bafilomycin A1 ( C ) , or concanamycin B ( D ) . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to control samples that were not treated with inhibitors . n = 2 - 6 biological replicates .

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Figure 3Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates.


Figure 4A549 * cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate ( B ) , SB412515 ( C ) , and bafilomycin A1 ( D ) . Infection with SARS - CoV - 2 ( MOI ~ 0 . 2 ) was achieved in the continuous presence of the drug . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where inhibitors had been omitted . n = 5 - 6 biological replicates .

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Figure 4A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.


Figure 5The timing of the MG - 132 - sensitive step during SARS - CoV - 2 infectious entry into Calu - 3 and Caco - 2 cells was assayed as detailed in D but using 60 µM MG - 132 for Caco - 2 cells and MOIs of 0 . 5 and 0 . 6 , respectively . n = 2 . A549 * cells expressing or lacking TMPRSS2 were pretreated with MG - 132 at the indicated concentrations and subsequently infected with SARS - CoV - 2 ( MOI ~ 0 . 2 ) in the continuous presence of the drug . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to that from control samples for which MG - 132 had been omitted . n = 6 biological replicates .

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Figure 5The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.


Figure 6SARS - CoV - 2 was subjected to pretreatment with trypsin and furin for 15 min at 37 ° C prior to infection of Caco - 2 and Vero cells ( MOIs of 0 . 4 and 0 . 3 , respectively ) . Infected cells were quantified by flow cytometry as described in Fig . 1D . Data were normalized to samples not pretreated with trypsin . n = 4 - 6 biological replicates . A549 * expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS - CoV - 2 at MOIs of 0 . 1 and 0 . 2 , respectively , for 24 h and then cocultured for 5 h along with target cells , not infected but prestained with CMFDA , a cytosolic green dye . Cells were subsequently treated with trypsin or furin for 5 min at 37 ° C and left to coculture for an additional hour at 37 ° C . After fixation , nuclei were stained with Hoechst ( blue ) , and infected cells were subjected to immunofluorescence staining against SARS - CoV - 2 nucleoprotein ( magenta ) . Samples were ultimately imaged by confocal fluorescence microscopy . White stars indicate syncytia with at least four nuclei . Scale bars : 100 µm . Images of microscope fields obtained in ( E ) were quantified [ A549 * : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 80 , and n ( furin ) = 60 ; A549 * TMPRSS2 : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 79 , and n ( furin ) = 60 ; Vero : n ( no virus ) = 40 , n ( no protease ) = 112 , n ( trypsin ) = 114 , and n ( furin ) = 60 ] . The fusion index is given as f = 1 - [ ( number of cells in a field after fusion ) / ( number of nuclei ) ] .

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Figure 6SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm. Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].


Figure 7SARS - CoV - 2 particles ( MOI of 0 . 3 ) were first subjected to trypsin treatment for 15 min at 37 ° C and then exposed to buffers at the indicated pH for 10 min at 37 ° C , and vice versa . Calu - 3 and Vero cells were then infected at a MOI of 0 . 3 and analyzed by flow cytometry as described in Fig . 1D . n = 4 - 8 biological replicates .

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Figure 7SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.


Figure 8SARS - CoV - 2 was first treated with trypsin or thermolysin and then allowed to infect A549 * and Vero cells ( MOIs ~ 0 . 2 and 0 . 3 ) in the continuous presence of SB412515 ( cathepsin L inhibitor ) . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where SB412515 had been omitted . n = 4 biological replicates .

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Figure 8SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.


Figure 1 ( A ) Live - cell nanoscopy of HeLa cells . COX8A - SNAP was labeled with SNAP - cell SiR and imaged with STED nanoscopy . ( B ) 3D architecture of cristae in HeLa cells visualized by FIB - SEM . The OM and the IBM are together shown as a transparent surface . Cristae of two mitochondria are depicted in light and dark blue . A twisted crista is shown in grey . ( C ) Live - cell recording as in ( A ) . In addition , mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy . Arrows mark sites where nucleoids but no lamellar cristae are present . ( D ) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA . Arrows mark sites where nucleoids are present . ( E ) Dual - color STED nanoscopy of cells labeled for COX8A - SNAP and immunolabeled for Mic60 . Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae .

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Figure 1(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy.(B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey.(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.(E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.


Figure 2 ( A ) Composition of MICOS in WT cells and MICOS mutants . Mic60 specific antibodies were used to pull - down Mic60 and interacting proteins from insolated mitochondria . * * Unspecific band , due to the cross reaction of the anti - Mic25 antibody with Mic19 . ( B ) Oxygen consumption rate ( OCR ) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer . Basal and maximal OCR are shown relative to the WT . Error bars : SEM , n = 6 . ( C ) Mitochondrial networks of WT and MICOS mutant cells . Cells were immunolabeled for TOM20 and visualized by confocal microscopy . The inset shows a magnification of the respective overview image . Scale bar : 10 µm . ( D ) Quantification of the mitochondrial networks as shown in ( C ) . The evaluation was performed manually in a blinded approach based on pre - defined morphology criteria . Average and SD of 3 independent biological replicates are shown ( > 170 cells per sample and replicate ) .

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Figure 2(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.(B) Oxygen consumption rate (OCR) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer. Basal and maximal OCR are shown relative to the WT. Error bars: SEM, n=6.(C) Mitochondrial networks of WT and MICOS mutant cells. Cells were immunolabeled for TOM20 and visualized by confocal microscopy. The inset shows a magnification of the respective overview image. Scale bar: 10 µm. (D) Quantification of the mitochondrial networks as shown in (C). The evaluation was performed manually in a blinded approach based on pre-defined morphology criteria. Average and SD of 3 independent biological replicates are shown (>170 cells per sample and replicate).


Figure 3 ( A ) Representative TEM recordings of WT cells and MICOS - KO mutants , as indicated . ( B ) Quantification of the overall cristae morphology on TEM recordings . Data information : n : number of mitochondrial sections ( C ) Quantification of detailed morphology of CMs in MICOS mutants ( left ) . TEM recordings were assigned to 8 classes ( right ) . Data information : n : number of mitochondrial sections ( D ) CJ frequency on TEM recordings . The number of CJs was manually determined and normalized to the length of the OM . Samples were compared to WT by a One - way ANOVA test . Error bars : SEM . Data information : n : number of CJs ( E ) Diameter of CJs . CJs were manually measured on TEM recordings . Red line : Average . Error bars : SD . Samples were compared to WT by a One - way ANOVA test . Data information : n : number of mitochondrial sections ( F ) Representative ET reconstructions of mitochondria from a WT cell ( left ) , a Mic10 - KO cell ( center ) and a Mic60 - KO cell ( right ) . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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Figure 3(A) Representative TEM recordings of WT cells and MICOS-KO mutants, as indicated.(B) Quantification of the overall cristae morphology on TEM recordings. Data information: n: number of mitochondrial sections(C) Quantification of detailed morphology of CMs in MICOS mutants (left). TEM recordings were assigned to 8 classes (right). Data information: n: number of mitochondrial sections(D) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the length of the OM. Samples were compared to WT by a One-way ANOVA test. Error bars: SEM. Data information: n: number of CJs(E) Diameter of CJs. CJs were manually measured on TEM recordings. Red line: Average. Error bars: SD. Samples were compared to WT by a One-way ANOVA test. Data information: n: number of mitochondrial sections(F) Representative ET reconstructions of mitochondria from a WT cell (left), a Mic10-KO cell (center) and a Mic60-KO cell (right). The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Figure 4 ( A Nanoscopy of mitochondria of WT and Mic10 - KO cells , as indicated . ( A ) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy . ( B ) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy . Image data were deconvolved . Depicted are cross - sections of a mitochondrion ( side view ) . ( C ) 3D MINFLUX nanoscopy . Mic10 - KO cells were immunolabeled for Mic60 using a directly labeled antibody . Colors encode depth information . ( D ) 3D SIM of living WT and Mic10 - KO cells . Cells were labeled with Mitotracker Green . Images show maximum intensity projections . ( E ) FIB - SEM of Mic10 - KO cells . Shown is a representative reconstructed mitochondrion . Dashed box : Orthoslice view on the smaller mitochondrion . The OM and IBM are displayed together in clear grey , the CM in is shown in blue . ( F ) 2D STED nanoscopy of mitochondria . Mic19 - KO cells were transfected with a plasmid for Mic10 - FLAG expression under the control of a tetracycline - inducible promoter . Left panel : Mitochondria from noninduced cell . Right : Mitochondria from induced cell expressing Mic10 - FLAG .

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Figure 4(A Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy.(B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view).(C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information.(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.(E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue.(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.


Figure 5 ( A ) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h . ( B ) Protein levels of MICOS proteins after Mic60 induction . Cell lysates were analyzed by western blotting at the indicated time points . ( C ) Recovery of mitochondrial networks upon Mic60 re - expression . Cells were induced with doxycycline hyclate for 72 h , immunolabeled for TOM20 and visualized by confocal microscopy . ( D - G ) TEM of Mic60 - TO cells before and after induction . ( D ) TEM recordings of Mic60 - TO cells at the indicated time points . ( E ) Quantification of the cristae morphology . ( F ) CJ frequency . The number of CJs on TEM recordings was normalized to the length of the OM . At least 90 mitochondrial sections were analyzed for each sample . ( G ) Quantification of the cristae morphology of Mic60 - TO cells . Only mitochondria exhibiting at least one CJ were analyzed . Data information : n : Number of mitochondrial sections . Scale bars : 20 µm ( A ) , 10 µm ( C ) , 500 nm ( D ) .

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

Figure 5(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h.(B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points.(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.(D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed. Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).


Figure 6 ( A , B ) Live - cell nanoscopy of Mic10 - TO cells . Cells expressing COX8A - SNAP were labeled with SNAP - cell SiR and recorded with STED nanoscopy . ( A ) Cristae architecture in noninduced cells . ( B ) Cristae architecture after Mic10 re - expression ( 24 h induction with doxycycline ) . STED image data were deconvolved . ( C ) STED nanoscopy of fixed Mic10 - TO cells . Cells were induced for 24 h and immunolabeled for Mic60 and Mic10 - FLAG . Upper row : Cell with weak Mic10 expression level . Lower row : Cell with strong Mic10 expression level . ( D - G ) TEM of Mic10 - TO cells . ( D ) Cells induced for the indicated period of time were recorded with TEM . Arrow indicates an intermediately shaped crista . ( E ) Quantification of the cristae morphology . n : Number of mitochondrial sections . ( F ) CJ frequency in Mic10 - TO cells . Number of CJs was normalized to the length of the OM . The same sections were analyzed as in ( E ) . ( G ) Diameter of CJs estimated on TEM recordings . Data information : n : Number of analyzed mitochondrial sections ( E ) or CJs ( G ) . .

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

Figure 6(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.(C) STED nanoscopy of fixed Mic10-TO cells. Cells were induced for 24 h and immunolabeled for Mic60 and Mic10-FLAG. Upper row: Cell with weak Mic10 expression level. Lower row: Cell with strong Mic10 expression level.(D-G) TEM of Mic10-TO cells. (D) Cells induced for the indicated period of time were recorded with TEM. Arrow indicates an intermediately shaped crista. (E) Quantification of the cristae morphology. n: Number of mitochondrial sections. (F) CJ frequency in Mic10-TO cells. Number of CJs was normalized to the length of the OM. The same sections were analyzed as in (E). (G) Diameter of CJs estimated on TEM recordings. Data information: n: Number of analyzed mitochondrial sections (E) or CJs (G)..


Figure 7 ( A ) FIB - SEM of Mic10 - TO cells . Cells were induced for 0 h , 16 h and 24 h . The representative reconstructions show mitochondria from noninduced , rescued and intermediate cells , as indicated . The OM is shown together with the IBM in clear grey , the CM in blue . ( B ) ET of Mic10 - TO cells . Mic10 expression was induced for 0 h and 16 h . Shown are reconstructions of representative noninduced and almost completely rescued mitochondria . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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 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

Figure 7(A) FIB-SEM of Mic10-TO cells. Cells were induced for 0 h, 16 h and 24 h. The representative reconstructions show mitochondria from noninduced, rescued and intermediate cells, as indicated. The OM is shown together with the IBM in clear grey, the CM in blue.(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Figure 8Knockdown ( KD ) of OPA1 in HeLa cells . WT and Mic60 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 5 d . ( A ) Cell lysates were analyzed by western blotting . ( B ) TEM recordings from OPA1 depleted WT and Mic60 - KO cells . Arrows point to a septum . ( C , D ) Quantification of the cristae morphology of OPA1 deficient WT ( C ) and Mic60 - KO ( D ) cells . The number of CJs was normalized to the length of the OM ( C ) . The number of septated mitochondria in Mic60 - KO cells was estimated ( D ) . ( E , F ) ET of mitochondria from OPA1 - KD cells . WT cells were transfected with a scrambled control ( E ) or siRNA pools against OPA1 ( F ) for 48 h . Reconstructions of representative mitochondria are displayed . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting. (B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Figure 9WT and Mic10 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 72 h . ( A , B ) STED nanoscopy of OPA1 depleted WT ( A ) and Mic10 - KO ( B ) cells . Cells were immunolabeled for Mic60 and ATPB . ( C ) TEM of OPA1 depleted Mic10 - KO cells . Left : Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1 - KD cell . The CMs are colored in green and the OM and IBM together in magenta . Right : Quantification of mitochondria containing CJs or septa junctions . Sections that contained CJ - like structures were analyzed . Of these , the number of sections exhibiting septa or cristae structures ( other ) was quantified . ( D Depletion of ATP5ME in HeLa cells . Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d . TEM recording ( left ) and quantification of the number of cristae per mitochondrial section ( right ) . ( E ) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED . The arrows point to crossing points of the opposite Mic60 distribution bands .

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

Figure 9WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB.(C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified.(D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.


Figure 1 . ( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit . ( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole . ( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit . ( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole .

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

Figure 1.(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.


Figure 2 . ( A ) GFP or Rpl25 - GFP were expressed in cells transformed ( + ) or not transformed ( − ) with a plasmid encoding 6His - ubiquitin . ( top ) Expression levels of GFP and Rpl25 - GFP in whole cell lysates were verified with anti - GFP blotting . ( middle ) Purified 6His - ubiquitin conjugates were examined by anti - GFP blotting . ( bottom ) 6His - ubiquitin expression and efficiency of purification was controlled using an anti - 6His antibody . White lines indicate that intervening lanes have been spliced out . ( B ) Analysis of ubiquitylated forms of Rpl25 - GFP in wild - type and the indicated mutant cells as in A . ( C ) Expression of Rpl25 - GFP and its modified form were analyzed by anti - GFP blot of extracts from wild - type and mutant cells starved for the indicated periods . Ub , ubiquitin ; WT , wild type .

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

Figure 2.(A) GFP or Rpl25-GFP were expressed in cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin. (top) Expression levels of GFP and Rpl25-GFP in whole cell lysates were verified with anti-GFP blotting. (middle) Purified 6His-ubiquitin conjugates were examined by anti-GFP blotting. (bottom) 6His-ubiquitin expression and efficiency of purification was controlled using an anti-6His antibody. White lines indicate that intervening lanes have been spliced out.(B) Analysis of ubiquitylated forms of Rpl25-GFP in wild-type and the indicated mutant cells as in A.(C) Expression of Rpl25-GFP and its modified form were analyzed by anti-GFP blot of extracts from wild-type and mutant cells starved for the indicated periods. Ub, ubiquitin; WT, wild type.


Figure 3 . ( A ) Indicated strains were transformed with plasmids encoding either Rpl25 - GFP ( Rpl25 ) or rpl25 K74 , 75R - GFP ( rpl25KR ) before the deletion of a genomic copy of RPL25 . ( top ) Comparable expression levels of Rpl25 - GFP or rpl25 K74 , 75R - GFP in whole cell lysates were confirmed with anti - GFP blotting . Purified 6His - ubiquitin - conjugated forms of Rpl25 - GFP or rpl25 K74 , 75R - GFP ( middle ) and 6His - ubiquitin expression ( bottom ) were analyzed as in Fig . 2 . Ub , ubiquitin . ( B ) Wild - type and ubp3Δ cells expressing Rpl25 - GFP or rpl25 K74 , 75R - GFP were starved for the indicated period of time . ( C ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts , and the ratio between cleaved GFP and full - length protein was quantified for all time points in four independent experiments . a . u . , arbitrary units . ( D ) Localization of GFP - tagged proteins . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole ( E ) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti - Rpl3 ( gift from V . Albanese , Institut Jacques Monod , Paris , France ) or anti - Mex67 ( as a control ) antibodies . Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0 . Significance of the differences observed for the ribophagy efficiency was evaluated using Student ' s t test . * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 . White lanes indicate that intervening lanes have been spliced out . The errors bars correspond to standard deviations .

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Figure 3.(A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin-conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin.(B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole(E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student's t test. *, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.


Figure 4 . ( A ) Wild - type cells expressing Bre5 - GFP , Ltn1 - Flag , or ltn1 - ΔRing - Flag were starved for the indicated periods of time . ( B ) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132 . Expression of tagged proteins was analyzed by anti - GFP or anti - Flag blotting of whole cell extracts . Five times less extracts were analyzed for ltn1 - ΔRing - Flag cells . A nonspecific band served as a loading control . White lanes indicate that intervening lanes have been spliced out . WT , wild type .

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Figure 4.(A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time.(B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.


Figure 1A APB Northern blot using a tRNAHis probe . m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points . Both queuosinylation and methylation levels could be restored by the addition of queuine . The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate , producing a single faster - migrating band ( ox ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) . B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) . C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa , whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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Figure 1A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


Figure 2B Corresponding queuosinylation and methylation levels , as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) . C Representative metaplot of SF cells . 25 - 32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero . In - frame 27 - 30 mer ribosome footprints showing the correct periodicity . D Representative metagene plot of 27 - 30 mer ribosome footprints in S and SF culture conditions for two biological replicates ( rep1 and rep2 ) . Data information : SF ( serum free medium ) , q ( queuine )

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Figure 2B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity.D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)


Figure 3A Heatmap showing codon occupancy according to the color scale . Green bars indicate Q - decoded codons . The Asp - GAC codon translated by Q - and m5C38 - tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange . Lack of Q reduces ribosome translation speed at all Q dependent codons and at near - cognate decoded codons . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) . B Translational speed of C ending codons relative to U ending codons . Error bars : ± SE of the permutated ratios of C - ending codon occupancy relative to U for the indicated conditions ; ( n = 4 ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) . C Heatmap displaying increase or decrease of codon frequencies ( ‰ ) in SILAC down - regulated proteins relative to the average frequency of that codon in unchanged proteins . D Codons translated by Q - tRNA are depleted in down - regulated proteins and increased in up - regulated proteins in both forward and reverse SILAC .

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Figure 3A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).B Translational speed of C ending codons relative to U ending codons. Error bars: ±SE of the permutated ratios of C-ending codon occupancy relative to U for the indicated conditions; (n=4). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).C Heatmap displaying increase or decrease of codon frequencies (‰) in SILAC down-regulated proteins relative to the average frequency of that codon in unchanged proteins.D Codons translated by Q-tRNA are depleted in down-regulated proteins and increased in up-regulated proteins in both forward and reverse SILAC.


Figure 4A The volcano plot shows differentially translated transcripts against adjusted p - values for SF compared to SF + q . Red dots indicate padj < 0 . 1 in multiple tests . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q . As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified ; * p < 0 . 05 ( t - test ) ; n = 5 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , RO ( ribosome occupancy ) . D Proliferation analysis of HeLa cells grown under the indicated culture conditions . Population doubling levels of three biological replicates were calculated for each time point . Error bars : ± SD ; n = 3 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

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Figure 4A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine)C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy).D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)


Figure 5A Representative fluorescence confocal images of live HeLa cells expressing Fluc - EGFP and FlucR188Q - EGFP maintained in SF compared to SF + q . Scale bars , 10 μm . Data information : SF ( serum free medium ) , q ( queuine ) B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell . The number of cells showing at least one aggregate is indicated . 10 cells were imaged for Fluc - EGFP and 20 cells for FlucR188Q - EGFP from 3 independent transfections ; * p < 0 . 05 ( t - test ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , AU ( arbitrary units ) . C Specific luminescence activity of EGFP - tagged sensor proteins maintained in SF compared to SF + q . Luci ferase activity was normalized to the luciferase protein content in the sample . Error bars : ± SD ; * p < 0 . 05 ( t - test ) ; n = 8 . Data information : SF ( serum free medium ) , q ( queuine )

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Figure 5A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine)B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units).C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luciferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)


Figure 6A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion . Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks . Scale bar 250 nm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) . B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q . Anti - KDEL ( ER stress marker ) ; anti - eIF2 - pS51 ( ER stress - induced inhibitor of translation ) ; anti - ATF4 ( transcription factor that activates ER stress - responsive genes ) ; anti - Hps70 ( chaperone ) . Nuclei were stained with Hoechst . Scale bar 10 µm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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 O O O O O O O O O O O O O O O O O O O O O O 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 6A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine).B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).


Figure 7A APB Northern blot using a tRNAHis probe . m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue . Both queuosinylation and methylation levels could be restored by the addition of q to the feed . The results are shown for three biological replicates . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days . B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q - free synthetic diet . Addition of q to the q - free diet significantly rescued the translation rate . n = 3 . Asterisks indicate statistically significant ( p < 0 . 05 , t - test ) differences . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days . C Box plot showing the body weight of mice fed a q - free synthetic diet for 60 days , center lines show the medians ; box limits indicate the 25th and 75th percentiles , whiskers extend 1 . 5 times the interquartile range ; n = 3 . Addition of q to the q - free diet significantly rescued body weights . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days . D Dimethyl - labeling analysis of - q versus + q liver tissue . Top 10 % of deregulated proteins are indicated in red and blue . UPR effectors : HSPA5 / BiP , HSP90b1 and CALR are in orange . Data information : - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days . F Liver sections from indicated mice were stained with anti - KDEL antibody . Arrowheads indicate ER stressed hepatocytes . Scale bar 25 µm . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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

Figure 7A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange. Data information: -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


Figure 1B . UTP6 in U - 2 OS cells exemplify proteins localized to whole nucleoli ( HPA025936 ) . C . Fibrillar center localization shown by a UBTF IF staining in U - 2 OS cells ( HPA006385 ) . E . Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP - tagged HeLa cells ( magenta ) , also supported by IF antibody staining using HPA040741 ( green ) . F . The multilocalizing ribosomal protein RPL13 detected in the nucleoli , cytosol and ER in MCF - 7 cells ( HPA051702 ) .

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Figure 1B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936).C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385).E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green).F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).