Figure 1Schematic of single ‐ plane acquisition FRAP on endogenously tagged Swi6 ‐ EGFP . Representative still images of a FRAP experiment are shown . See also Movie EV1 . Green sphere : yeast nucleus containing Swi6 ‐ EGFP ; green dots : heterochromatic Swi6 ‐ EGFP ; red circle : bleached area . Normalized average intensities ( black dots ) of FRAP on Swi6 ‐ EGFP heterochromatic loci ( n = 31 ) fitted to a one ‐ component model ( blue line ) . The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half ‐ recovery time ( t 1 / 2 ) . Fluorescence t 1 / 2 values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box . Normalized fluorescence recovery values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box . FLIP experiment showing a bleached ( FRAP , red circle ) and unbleached , adjacent locus ( FLIP , blue circle ) in the same nucleus . The graph displays average relative fluorescence of FLIP ( blue ) , FRAP ( red ) , and control loci ( no bleach , green ) in other non ‐ bleached cells . Schematic of a line ‐ scan FRAP experiment . Fluorescence intensities are measured along a line of a non ‐ bleached nucleus ( Ctrl nucleus ) and a bleached nucleus ( FRAP nucleus ) in Swi6 ‐ EGFP clr4 ∆ cells . Red box : bleached region . Right : raw data example . Average relative intensities of a line ‐ scan FRAP experiment on Swi6 ‐ EGFP in heterochromatin ( blue line ) , euchromatin ( green line ) , and clr4 ∆ ( red line ) . The curves have a gliding average of 40 .

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Figure 1Schematic of single‐plane acquisition FRAP on endogenously tagged Swi6‐EGFP. Representative still images of a FRAP experiment are shown. See also Movie EV1. Green sphere: yeast nucleus containing Swi6‐EGFP; green dots: heterochromatic Swi6‐EGFP; red circle: bleached area.Normalized average intensities (black dots) of FRAP on Swi6‐EGFP heterochromatic loci (n = 31) fitted to a one‐component model (blue line). The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half‐recovery time (t 1/2).Fluorescence t 1/2 values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.Normalized fluorescence recovery values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.Schematic of a line‐scan FRAP experiment. Fluorescence intensities are measured along a line of a non‐bleached nucleus (Ctrl nucleus) and a bleached nucleus (FRAP nucleus) in Swi6‐EGFP clr4∆ cells. Red box: bleached region. Right: raw data example.Average relative intensities of a line‐scan FRAP experiment on Swi6‐EGFP in heterochromatin (blue line), euchromatin (green line), and clr4∆ (red line). The curves have a gliding average of 40.


Figure 2A , BAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red ) . C , DAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type ( brown ) or cid14 ∆ ( dark green ) cells expressing Swi6 ‐ EGFP . E , FAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type cells expressing heterochromatic NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 KR25A ‐ EGFP ( red ) , or cid14 ∆ cells expressing NLS ‐ Swi6 ‐ EGFP ( green ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( yellow ) . Data information : In ( B ) , ( D ) and ( F ) , the box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Figure 2A, BAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP (blue) or NLS‐Swi6‐KR25A‐EGFP (red).C, DAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type (brown) or cid14∆ (dark green) cells expressing Swi6‐EGFP.E, FAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type cells expressing heterochromatic NLS‐Swi6‐EGFP (blue) or NLS‐Swi6 KR25A‐EGFP (red), or cid14∆ cells expressing NLS‐Swi6‐EGFP (green) or NLS‐Swi6‐KR25A‐EGFP (yellow).Data information: In (B), (D) and (F), the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Figure 3Schematic of a chromosome indicating Swi6 ‐ EGFP heterochromatic domains ( green ) . Cnp1 ‐ mCherry and Taz1 ‐ mCherry ( red ) mark centromeres and telomeres , respectively . Representative images demonstrating Swi6 ‐ EGFP co ‐ localization with centromeres ( Cnp1 ‐ mCherry ) and telomeres ( Taz1 ‐ mCherry ) are shown . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6 ‐ EGFP . Fluorescence t 1 / 2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS ‐ Swi6 ‐ EGFP ( black dots ) or the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red dots ) from G1 / S cell cycle arrest . Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest . Swi6 is dispersed in M phase due to H3S10 phosphorylation , and M phase was therefore not included in the FRAP analysis . Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 . A total of 40 , 66 , and 104 individual cells residing in the G1 , S , and G2 phases of the cell cycle were analyzed , respectively . Representative bright ‐ field images of cells in G1 , S , or G2 are shown . Scale bars = 2 μm . Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS ‐ Swi6 ‐ EGFP and NLS ‐ Swi6 ‐ KR25A ‐ EGFP that reside in the G2 of the cell cycle . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP .

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Figure 3Schematic of a chromosome indicating Swi6‐EGFP heterochromatic domains (green). Cnp1‐mCherry and Taz1‐mCherry (red) mark centromeres and telomeres, respectively. Representative images demonstrating Swi6‐EGFP co‐localization with centromeres (Cnp1‐mCherry) and telomeres (Taz1‐mCherry) are shown.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6‐EGFP.Fluorescence t 1/2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS‐Swi6‐EGFP (black dots) or the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP (red dots) from G1/S cell cycle arrest. Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest. Swi6 is dispersed in M phase due to H3S10 phosphorylation, and M phase was therefore not included in the FRAP analysis.Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6. A total of 40, 66, and 104 individual cells residing in the G1, S, and G2 phases of the cell cycle were analyzed, respectively. Representative bright‐field images of cells in G1, S, or G2 are shown. Scale bars = 2 μm.Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS‐Swi6‐EGFP and NLS‐Swi6‐KR25A‐EGFP that reside in the G2 of the cell cycle.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.


Figure 4H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on centromere 1 . Centromeric repeat elements ( dg / dh ) are indicated . Inverted repeats ( IRC1 ) constitute heterochromatin boundaries in wild ‐ type cells ( Cam et al , 2005 ) . The y ‐ axes represent log2 ChIP ‐ seq enrichments in 200 ‐ bp genomically tiled windows calculated over clr4Δ cells . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) . Positions of genomic elements on the plus and minus strands are indicated . Blue , protein ‐ coding genes ; white , repeats ; green , long terminal repeat ( LTR ) . H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on the telomere of the right arm of chromosome 1 . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) . Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis . Expression data for swi6 + and swi6 ∆ cells were taken from a previous publication ( Woolcock et al , 2012 ) .

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Figure 4H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on centromere 1. Centromeric repeat elements (dg/dh) are indicated. Inverted repeats (IRC1) constitute heterochromatin boundaries in wild‐type cells (Cam et al, 2005). The y‐axes represent log2 ChIP‐seq enrichments in 200‐bp genomically tiled windows calculated over clr4Δ cells. Two independent biological replicates for each genotype were performed (replicates 1 and 2). Positions of genomic elements on the plus and minus strands are indicated. Blue, protein‐coding genes; white, repeats; green, long terminal repeat (LTR).H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on the telomere of the right arm of chromosome 1. Two independent biological replicates for each genotype were performed (replicates 1 and 2).Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis. Expression data for swi6+ and swi6∆ cells were taken from a previous publication (Woolcock et al, 2012).


Figure 5C - EH3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , and swi6 ‐ L315E ( CSD dimerization mutant ) ( C ) , swi6 ‐ W104A ( H3K9me2 binding mutant ) ( D ) , and epe1 ∆ ( E ) cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three ( C and E ) or four ( D ) independent experiments . P ‐ values were generated by the Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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Figure 5C-EH3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, and swi6‐L315E (CSD dimerization mutant) (C), swi6‐W104A (H3K9me2 binding mutant) (D), and epe1∆ (E) cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three (C and E) or four (D) independent experiments. P‐values were generated by the Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


Figure 6sRNA reads mapping to centromere 1 in swi6 + ( green ) and swi6 ∆ ( blue ) cells . The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6 . Centromeric repeat elements and the central core are indicated . Counts were normalized to the library size . Asterisks denote tRNA fragments . Note that tRNA genes flank IRC3 ( Fig EV4 ) but not IRC1R . H3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , tas3 L479E , and tas3 L479E swi6 ∆ cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three independent experiments . P ‐ values were generated by Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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Figure 6sRNA reads mapping to centromere 1 in swi6+ (green) and swi6∆ (blue) cells. The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6. Centromeric repeat elements and the central core are indicated. Counts were normalized to the library size. Asterisks denote tRNA fragments. Note that tRNA genes flank IRC3 (Fig EV4) but not IRC1R.H3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, tas3 L479E, and tas3 L479E swi6∆ cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three independent experiments. P‐values were generated by Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


figf1a , Northern blot analysis of lamp - 2 expression . Total RNA was hybridized using a lamp - 2 cDNA2 probe and a murine glyceraldehyde - 3 - phosphate dehydrogenase probe . b , Western blot analysis of LAMP - 2 expression . In control liver and kidney extracts the glycosylated LAMP - 2 molecules were detected . In LAMP - 2 - / - tissues no LAMP - 2 product was found . c , Increased mortality in LAMP - 2 deficient outbred ( C57B6 / Jx129SV ) mice . Forty - two control ( closed circles ) and LAMP - 2 - deficient mice ( open circles ) were monitored over 300 days . d , Loss of weight in early dying ( n = 7 males ) and surviving LAMP - 2 - deficient mice ( n = 11 males ) in comparison with control mice ( n = 12 males ) monitored for 60 days after birth .

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figf1a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe.b, Western blot analysis of LAMP-2 expression. In control liver and kidney extracts the glycosylated LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2 product was found.c, Increased mortality in LAMP-2 deficient outbred (C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient mice (open circles) were monitored over 300 days.d, Loss of weight in early dying (n = 7 males) and surviving LAMP-2-deficient mice (n = 11 males) in comparison with control mice ( n = 12 males) monitored for 60 days after birth.


figf2a , Ultrastructure of autophagic vacuoles ( arrowheads ) in pancreatic acinar gland cells from a 6 . 5 - month - old LAMP - 2 - deficient mouse . b , Autophagic vacuoles containing glycogen ( g ) , cytosol and cellular constituents in a hepatocyte of an 8 - day - old LAMP - 2 - / - mouse . m , mitochrondria ; bc , bile canaliculus . c , Accumulation of numerous early autophagic vacuoles in cultured LAMP - 2 - / - hepatocytes . Arrow , large late autophagic vacuole containing partially degraded material . Insert , magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents . Scale bars : a , 3 . 5 µm ; b , 0 . 2 µm ; c , 1 . 3 µm ; insert , 0 . 9 µm .

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figf2a, Ultrastructure of autophagic vacuoles (arrowheads) in pancreatic acinar gland cells from a 6.5-month-old LAMP-2-deficient mouse.b, Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria; bc, bile canaliculus. c, Accumulation of numerous early autophagic vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late autophagic vacuole containing partially degraded material. Insert, magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents. Scale bars: a, 3.5 µm; b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.


figf3a , Vacuole in a fibre of the soleus muscle . b , Ultrastructure of a vacuole in a cardiomyocyte . Scale bars : a , 1 µm ; b , 1 . 5 µm . c , Typical original twitch recordings of developed force of heart muscles from control ( thin line ) and LAMP - 2 - deficient ( thick line ) mice . d , Average values of developed force . Preparations of LAMP - 2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz ; P 0 . 01 .

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figf3a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm; b, 1.5 µm.c, Typical original twitch recordings of developed force of heart muscles from control (thin line) and LAMP-2-deficient (thick line) mice. d, Average values of developed force. Preparations of LAMP-2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.


figf4a , Volume density of early ( Avi ) and late ( Avd ) autophagic vacuoles from four independent cultures . Examples of Avi and Avd are shown in b and c , respectively . Bars represent 0 . 4 µm . d , Labelling density of cathepsin - D as estimated in two independent experiments . e , Labelling density of LAMP - 1 . f , Degradation of long - lived proteins followed over 24 h in amino acid and fetal - calf - serum - containing medium . g , Effect of amino - acid ( AA ) deprivation and 3 - methyladenine ( MA ) on protein degradation during 4 h of chase . The error bars in a , d , e and g represent s . e . m .

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figf4a, Volume density of early (Avi) and late (Avd) autophagic vacuoles from four independent cultures. Examples of Avi and Avd are shown in b and c, respectively. Bars represent 0.4 µm.d, Labelling density of cathepsin-D as estimated in two independent experiments. e, Labelling density of LAMP-1.f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.


Figure 1Localization of R - LPS ( green ) and S - LPS ( red ) in exponential phase bacteria . Scale bars : 2 μm . Distribution of R - LPS and S - LPS in exponential phase culture analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) . Numbers in each corner corresponds to % of relative frequencies .

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Figure 1Localization of R-LPS (green) and S-LPS (red) in exponential phase bacteria. Scale bars: 2 μm. Distribution of R-LPS and S-LPS in exponential phase culture analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Numbers in each corner corresponds to % of relative frequencies.


Figure 2Roughness measurements on R - LPS labeled ( green ) WT and Δgmd cells . AFM images of whole bacteria and of the separated areas ( coloured squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The arithmetic roughness Ra is indicated below each area . Scale bars : 1 μm . Quantitative roughness measurement of R - LPS labeled WT and Δgmd cells . The areas with the lower roughness ( more regular surfaces ) of Δgmd cells were assigned as areas 1 . mAb : monoclonal antibody . Ab : antibody . nWT = 13 bacteria . nΔgmd = 7 bacteria . Differences were statistically analyzed by t - test . * * P < 0 . 01 . ns : not significant . Detection of forces between AFM tip functionalized with mAb against R - LPS and unlabeled WT cells . Height image and the corresponding adhesion image are shown on top and zooms of depicted areas ( black squares , 0 . 4 x 0 . 4 μm2 ) below . The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area . Scale bar : 1 μm . Correlation between roughness and detection of R - LPS on unlabeled WT cells . The areas with the lower roughness ( more regular surfaces ) were assigned as areas 1 . The boxplot represents the mean values ( squares ) , the median ( horizontal line ) , the 25 % and 75 % quartiles ( box limits ) and the standard deviations ( whiskers ) . n = 9 bacteria . Error bars : s . d . Statistical differences were analyzed by t - test . * * P < 0 . 01 . * P < 0 . 05 .

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Figure 2Roughness measurements on R-LPS labeled (green) WT and Δgmd cells. AFM images of whole bacteria and of the separated areas (coloured squares in first images, 0.4 x 0.4 μm2) are shown. The arithmetic roughness Ra is indicated below each area. Scale bars: 1 μm. Quantitative roughness measurement of R-LPS labeled WT and Δgmd cells. The areas with the lower roughness (more regular surfaces) of Δgmd cells were assigned as areas 1. mAb: monoclonal antibody. Ab: antibody. nWT = 13 bacteria. nΔgmd = 7 bacteria. Differences were statistically analyzed by t-test. **P < 0.01. ns: not significant.Detection of forces between AFM tip functionalized with mAb against R-LPS and unlabeled WT cells. Height image and the corresponding adhesion image are shown on top and zooms of depicted areas (black squares, 0.4 x 0.4 μm2) below. The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area. Scale bar: 1 μm. Correlation between roughness and detection of R-LPS on unlabeled WT cells. The areas with the lower roughness (more regular surfaces) were assigned as areas 1. The boxplot represents the mean values (squares), the median (horizontal line), the 25% and 75% quartiles (box limits) and the standard deviations (whiskers). n = 9 bacteria. Error bars: s.d. Statistical differences were analyzed by t-test. **P < 0.01. *P < 0.05.


Figure 3Co - localization of Omp2b ( red ) and R - LPS ( green ) in exponential phase bacteria . Scale bars : 2 μm . Co - detection of Omp2b ( red ) and R - LPS by AFM . R - LPS was detected using AFM tips functionalized with mAb against R - LPS . AFM images of whole bacteria and of the separated areas ( red squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The percentages of adhesion Padh are indicated below each area . Scale bars : 1 μm . Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area . Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure . nWT = 13 bacteria , nΔgmd = 13 bacteria . Statistical analysis with Mann - Whitney U test showed * * P < 0 . 01 .

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Figure 3Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm.Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm.Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area. Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure. nWT = 13 bacteria, nΔgmd = 13 bacteria. Statistical analysis with Mann-Whitney U test showed **P < 0.01.


Figure 4Localization of initially labeled Omp25 ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . As a control , Omp25 localization after pulse - chase labeling with TRSE is shown at the bottom panel . Scale bars : 2 μm . Localization of initially labeled Omp2b ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . Scale bars : 2 μm . Co - localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels , respectively . Error bars correspond to s . d . from independent experiments . Differences were statistically analyzed by t - test . * * * P < 0 . 001 . nOmp25 = 279 bacteria , 85666 pixels ( 5 biological replicates ) . nOmp2b = 252 bacteria , 74630 pixels ( 3 biological replicates ) .

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Figure 4Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm.Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm.Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).


Figure 5Short pulse labeling of exponential phase bacteria with the fluorescent D - amino acid HADA . Merge is showing old pole marker PdhS - mCherry ( red ) and PG insertion sites ( white ) . Scale bars : 2 μm . Demographic representation of HADA labeling . Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS - mCherry fluorescence signal . NFI : normalized fluorescence intensity . n = 393 bacteria ( 3 biological replicates ) .

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Figure 5Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm.Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates).


Figure 6Localization of S - LPS ( green ) in the eFluor - labeled ( magenta ) inducible rough strain Δgmd plac - gmd possessing the old pole marker PdhS - mCherry ( red ) . The strains Δgmd and Δgmd plac - gmd - IPTG were used as negative controls . Scale bars : 2 μm . Demograph represents fluorescence profile of S - LPS labeling from ( A ) . Cells were oriented with the old pole at the bottom of the graph by PdhS - mCherry and sorted by cell length . NFI : normalized fluorescence intensity . n = 468 bacteria ( 3 biological replicates ) . Short pulse labeling of LPS ( 2 h ) by Kdo - N3 ( green ) on eFluor - labeled ( magenta ) bacteria expressing the old pole marker PdhS - mCherry ( red ) . Scale bars : 2 μm . Demographs represent fluorescence intensities of eFluor ( OM , left ) and Kdo - N3 ( LPS , right ) labeling . Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS - mCherry localization . NFI : normalized fluorescence intensity . n = 566 bacteria ( 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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 6Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm.Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates).Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm.Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).


Figure 7Distribution of R - LPS and S - LPS in Δgmd plac - gmd after 3 , 6 , 9 and 24 h post - induction analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) . Quantification of S - LPS labeling from ( A ) as relative frequencies . Error bars correspond to s . d . from 3 independent experiments . n0h = 77 bacteria , n3h = 254 bacteria , n6h = 616 bacteria , n9h = 937 bacteria , n24h = 1059 bacteria .

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Figure 7Distribution of R-LPS and S-LPS in Δgmd plac-gmd after 3, 6, 9 and 24 h post-induction analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events).Quantification of S-LPS labeling from (A) as relative frequencies. Error bars correspond to s.d. from 3 independent experiments. n0h = 77 bacteria, n3h = 254 bacteria, n6h = 616 bacteria, n9h = 937 bacteria, n24h = 1059 bacteria.


Figure 4 ( A ) Binding assay for Hsc70 binding in the presence of WT DNAJB1 : control of DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 / Apg2 , control of DNAJB1 / Hsc70 / Apg2 . The ' * ' highlights the condition where an increase of Hsc70 in the bound fraction is observed . P , pellet ; S , supernatant . The amount of Hsc70 recruited to the fibres is shown in the pellet fractions . ( B ) Binding assay for Hsc70 binding in the presence of the truncated ΔJ - DNAJB1 , lacking the J domain . The gel layout is similar to the previous one with ΔJ - DNAJB1 replacing WT DNAJB1 . Both Apg2 and J domain are required for enhancement of Hsc70 binding . ( C ) Histogram of Hsc70 bound fraction in each sample ( N = 3 independent experiments , WT : WT DNAJB1 , ΔJ : ΔJ - DNAJB1 mutant , - Apg2 : condition with fibres + DNAJB1 / Hsc70 , + Apg2 : condition with fibres + DNAJB1 / Hsc70 / Apg2 ) . A 2 - way ANOVA was performed with Tukey ' s multiple comparisons test ( * * : P = 0 . 003 ; * * * * : P < 0 . 0001 ) . Mean Hsc70 binding values with SD are shown . ( D ) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2 . Scale bar , 1 µm . ( E , F ) Quantitation of fluorescence intensity of Hsc70 ( E ) and αSyn ( F ) in the two conditions ( + / - Apg2 , N = 3 independent experiments , n = 65 fibres ) . Mean Hsc70 and αSyn fluorescence intensities with SD are shown . ( G ) Quantitation of differences in Hsc70 binding between the two ends of the fibres , normalised to the local αSyn intensity , in the absence and presence of Apg2 ( N = 3 independent experiments , n = 54 fibres ) . Mean Hsc70 fluorescence intensities with SD are shown . Data information : ( E - G ) For each plot , data were tested for normality by performing a Shapiro - Wilk test . As the data did not display a normal distribution , they were analysed using a two - tailed Mann - Whitney test ( * * * * : P < 0 . 0001 , ns : not significant ) .

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Figure 4(A) Binding assay for Hsc70 binding in the presence of WT DNAJB1: control of DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70/Apg2, control of DNAJB1/Hsc70/Apg2. The '*' highlights the condition where an increase of Hsc70 in the bound fraction is observed. P, pellet; S, supernatant. The amount of Hsc70 recruited to the fibres is shown in the pellet fractions. (B) Binding assay for Hsc70 binding in the presence of the truncated ΔJ-DNAJB1, lacking the J domain. The gel layout is similar to the previous one with ΔJ-DNAJB1 replacing WT DNAJB1. Both Apg2 and J domain are required for enhancement of Hsc70 binding. (C) Histogram of Hsc70 bound fraction in each sample (N = 3 independent experiments, WT: WT DNAJB1, ΔJ: ΔJ-DNAJB1 mutant, -Apg2: condition with fibres + DNAJB1/Hsc70, +Apg2: condition with fibres + DNAJB1/Hsc70/Apg2). A 2-way ANOVA was performed with Tukey's multiple comparisons test (**: P=0.003; ****: P<0.0001). Mean Hsc70 binding values with SD are shown.(D) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2. Scale bar, 1 µm. (E, F) Quantitation of fluorescence intensity of Hsc70 (E) and αSyn (F) in the two conditions (+/- Apg2, N = 3 independent experiments, n = 65 fibres). Mean Hsc70 and αSyn fluorescence intensities with SD are shown. (G) Quantitation of differences in Hsc70 binding between the two ends of the fibres, normalised to the local αSyn intensity, in the absence and presence of Apg2 (N = 3 independent experiments, n = 54 fibres). Mean Hsc70 fluorescence intensities with SD are shown. Data information: (E-G) For each plot, data were tested for normality by performing a Shapiro-Wilk test. As the data did not display a normal distribution, they were analysed using a two-tailed Mann-Whitney test (****: P<0.0001, ns: not significant).


Figure 5Binding assay with different dilutions of Apg2 incubated with DNAJB1 / Hsc70 and ( A ) with αSyn fibres or ( B ) without αSyn fibres ( control ) . 5 mM ATP was present in the buffer throughout .

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Figure 5Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5 mM ATP was present in the buffer throughout.


Figure 2A - L ) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F - domains for 15 minutes and analyzed by immunoblotting for pAKT ( S473 ) , pERK1 / 2 ( T202 / Y204 ) , and pFAK ( Y397 ) phosphorylation . A , E , I ) pAKT , B , F , J ) pERK1 / 2 , and C , G , K ) pFAK phosphorylation levels are quantified and normalized to Icos - cage signaling levels . Appendix Figure S1B shows the detection of two bands expected for pERK1 / 2 . Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F - domain : D ) helical bundles , H ) trimeric and tetrameric homo - oligomers and tetrahedral nanocages , L ) Icosahedral nanocages and trimeric subunit . M - P ) Dose - response curves , EC50 , and EMAX for pAKT and pERK1 / 2 activation . The curves , EC50 and EMAX values are calculated using Prism , GraphPad . N , P ) Darker red color indicate lower EC50 and higher EMAX values .

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Figure 2A-L) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F-domains for 15 minutes and analyzed by immunoblotting for pAKT (S473), pERK1/2 (T202/Y204), and pFAK (Y397) phosphorylation. A, E, I) pAKT, B, F, J) pERK1/2, and C, G, K) pFAK phosphorylation levels are quantified and normalized to Icos-cage signaling levels. Appendix Figure S1B shows the detection of two bands expected for pERK1/2. Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F-domain: D) helical bundles, H) trimeric and tetrameric homo-oligomers and tetrahedral nanocages, L) Icosahedral nanocages and trimeric subunit.M-P) Dose-response curves, EC50, and EMAX for pAKT and pERK1/2 activation. The curves, EC50 and EMAX values are calculated using Prism, GraphPad. N, P) Darker red color indicate lower EC50 and higher EMAX values.


Figure 3C ) Comparison of cell migration induced by Ang1 , Ang2 , and designed F - domain scaffolds after 12 hours of treatment . The change in wound area is normalized to vehicle control . All experiments have at least 3 biological replicates . ( * ) is the comparison between F - domain scaffolds vs Ang1 , and ( # ) is the comparison between F - domain scaffolds vs Vehicle . E ) Representative increase in vascular stability by Icos1 . The number of nodes ( blue ) , meshes ( red ) , and tubes ( green ) were quantified using angiogenesis analyzer plugin in ImageJ . Scale bar is100 µm .

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Figure 3C) Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 hours of treatment. The change in wound area is normalized to vehicle control. All experiments have at least 3 biological replicates. (*) is the comparison between F-domain scaffolds vs Ang1, and (#) is the comparison between F-domain scaffolds vs Vehicle.E) Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) were quantified using angiogenesis analyzer plugin in ImageJ. Scale bar is100 µm.


Figure 4A - C ) Representative images of FOXO1 localization upon scaffolds administration . Scale bars are 10 µm D ) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain , 100 cells were counted per sample . E ) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters . Dark gray bars : Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane . K , L ) Representatives immunoblot gel ( K ) and quantifications ( L ) for α5 integrin knockdown experiments using 20 nM of siRNA .

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Figure 4A-C) Representative images of FOXO1 localization upon scaffolds administration. Scale bars are 10 µm D) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain, 100 cells were counted per sample. E) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters. Dark gray bars: Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane.K, L) Representatives immunoblot gel (K) and quantifications (L) for α5 integrin knockdown experiments using 20 nM of siRNA.


Figure 5C , D ) CCI - TBI brain sections were stained for reactive astrocytes ( GFAP , blue ) , microglia ( Iba1 , green ) and serum albumin ( magenta ) to highlight tissue margins ( dotted white line ) and quantify secondary damage as a function of lesion area ( E ; * p = 0 . 04 , n = 4 , the mean ± SEM is plotted ) . Bar , 1 mm . E ) Cumulative blood extravasation into brain tissue ( white outline ) was imaged by albumin immunofluorescence to establish an unbiased intensity - threshold mask ( red ) and quantify the area of blood serum extravasation ( mm2 ) in Icos1ø vs . Icos1 treated animals ( * * p = 0 . 01 , n = 4 , the mean ± SEM is plotted ) . Scale bar is 500 µm . F - H ) Brain tissues stained by GFAP - ( magenta ) and CD31 - immunofluorescence ( green ) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter ( G ; * * p = 0 . 003 , n = 4 , the boxes show 25th to 75th percentiles and whiskers represent the min and max values . ) and quantify the number of CD31 - vessels ( H ; * * p = 0 . 007 , n = 4 , the mean ± SEM is plotted ) at the site of injury in Icos1ø and Icos1 treated animals 4 days post - injury . Scale bars in G and H is 100 µm . I ) Activation of pAKT ( Magenta ) was examined in Icos1ø and Icos1 treated - animals within or around vascular ( CD31 + , green ) and pericytes ( NG2 + cells , yellow ) of the BBB . High - resolution confocal images ( insets ) reveal the prevalence of activated pAKT ( magenta ) co - stain ( cyan arrows ) in pericytes ( yellow ) of the BBB along vasculature ( green , or dotted outline ) versus reactive neuroglia ( NG2 + pAKT - ) in the parenchyma ( red arrows ) in Icos1ø vs . Icos1 treated animals . Scale bar is 10 µm .

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

Figure 5C, D) CCI-TBI brain sections were stained for reactive astrocytes (GFAP, blue), microglia (Iba1, green) and serum albumin (magenta) to highlight tissue margins (dotted white line) and quantify secondary damage as a function of lesion area (E; *p = 0.04, n = 4, the mean ± SEM is plotted ). Bar, 1 mm. E) Cumulative blood extravasation into brain tissue (white outline) was imaged by albumin immunofluorescence to establish an unbiased intensity-threshold mask (red) and quantify the area of blood serum extravasation (mm2) in Icos1ø vs. Icos1 treated animals (**p = 0.01, n = 4, the mean ± SEM is plotted). Scale bar is 500 µm. F-H) Brain tissues stained by GFAP- (magenta) and CD31-immunofluorescence (green) were imaged by confocal microscopy to measure angiogenesis as a function of vessel diameter (G; **p = 0.003, n = 4, the boxes show 25th to 75th percentiles and whiskers represent the min and max values.) and quantify the number of CD31-vessels (H; **p = 0.007, n = 4, the mean ± SEM is plotted) at the site of injury in Icos1ø and Icos1 treated animals 4 days post-injury. Scale bars in G and H is 100 µm.I) Activation of pAKT (Magenta) was examined in Icos1ø and Icos1 treated-animals within or around vascular (CD31+, green) and pericytes (NG2+ cells, yellow) of the BBB. High-resolution confocal images (insets) reveal the prevalence of activated pAKT (magenta) co-stain (cyan arrows) in pericytes (yellow) of the BBB along vasculature (green, or dotted outline) versus reactive neuroglia (NG2+pAKT-) in the parenchyma (red arrows) in Icos1ø vs. Icos1 treated animals. Scale bar is 10 µm.


Figure 1A Age , gender and BMI information of the Ashkenazi Jewish Centenarian cohort . Table showing the age , gender and BMI information in control and centenarian groups . Pie chart showing the percentage of male / female individuals in control and centenarian groups . Right panel showing the age and BMI distribution of all individuals recruited in targeted capture - seq . Data were presented as mean and SD , Students t - test , two tailed , * * * p < 0 . 001 . B , C Histograms of normalized allele counts in individuals 75 + years old compared to all age groups ( N = 125 , 748 ) across ( B ) all chromosome 19 SNPs and small insertions / deletions , and ( C ) missense SNPs and indels only . Dotted vertical lines show centSIRT6 . E Michaelis - Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD + . n = 3 technical replicates ( TR ) ; error bars = SD .

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 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

Figure 1A Age, gender and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture-seq. Data were presented as mean and SD, Students t-test, two tailed, *** p<0.001.B, C Histograms of normalized allele counts in individuals 75+ years old compared to all age groups (N=125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.E Michaelis-Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD+. n=3 technical replicates (TR); error bars=SD.


Figure 2A , B Deacetylase activity on H3K9 ( A ) and H3K18 ( B ) residues shows reduced activity in centSIRT6 allele . Designer histones were incubated with purified SIRT6 and 5 mM NAD + , then resolved by SDS - PAGE and analyzed by immunoblotting with acetyl - specific histone antibodies . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisks indicate p < 0 . 05 compared to WT , color corresponds to allele . H3K9ac , H3K18ac and H3 are blots , SIRT6 loading Coomassie stain HeLa histone preps were also probedC Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels . Relative fraction represents the portion of the peptide encompassing H3K9 - 17 compared to the summed total of all of the peptide quantitation values for the same region . n = 3 biological replicates ( BR ) ; error bars = SD . Students t - test , two tailedD Whole cell histone H3 acetylation levels in cumate - inducible SIRT6 human fibroblasts , assessed by Western blot . Cu , cumate ; PQ , paraquat . Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot , and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest , with PQ - treated cells receiving PQ 24hrs after initial cumate induction . E - H Relative abundance of H3K9ac at SIRT6 - dependent NF - κB target genes . Anti - H3K9ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 BR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition . Red asterisk indicated p < 0 . 05 compared to + Cu WT . I , J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity . Anti - H3K18ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 TR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition .

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

Figure 2A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probedC Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailedD Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.E-H Relative abundance of H3K9ac at SIRT6-dependent NF-κB target genes. Anti-H3K9ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu WT.I, J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti-H3K18ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition.


Figure 3A Self - ribosylation of SIRT6 using biotin - labeled NAD + . Recombinant SIRT6 was incubated with NAD + conjugated with a biotin residue and then run on an SDS - PAGE . Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over 0 hr control . Red asterisks indicated significance over time - matched WT SIRT6 . B Self - ribosylation of SIRT6 with titration of NAD + . mADPr - specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins . Plot was fit the Michaelis - Menten best - fit with GraphPad software . Km NAD was 142 + 29 for centSIRT6 and 106 + 45 for wild type SIRT6 and maximal signal was ~ 2x greater for centSIRT6 compared to the wild type SIRT6 . Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed . n = 3 TR ; error bars = SD . C Activation of PARP1 by SIRT6 variants . SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly - ADPr antibody . Poly - ADPr activity of PARP1 results in a wide range of product size . Activity was assessed by quantifying poly - ADPr signal in whole lanes for each sample . n = 3 TR ; error bars = SD . Two - way ANOVA . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over HPRT control . Red asterisks indicated significance over wild type SIRT6 .

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 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

Figure 3A Self-ribosylation of SIRT6 using biotin-labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.B Self-ribosylation of SIRT6 with titration of NAD+. mADPr-specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ~2x greater for centSIRT6 compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n=3 TR; error bars=SD.C Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicated significance over wild type SIRT6.


Figure 4A qRT - PCR analysis of LINE1 expression in cumate - inducible SIRT6 fibroblasts . Primers assessed both 5 ' ( ORF1 ) and 3 ' ( ORF2 ) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons . Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes . Asterisk indicate p < 0 . 05 . n = 3 TR ; error bars = SD . Students t - test , two tailed . B LINE1 EGFP retrotransposition assay . Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicated significance over SIRT6 KO control . Black asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA . C In vitro KAP1 ribosylation by SIRT6 . Asterisk indicate p < 0 . 05 . Red asterisk indicate significance over 0 hr , black asterisk indicate significance over compared to WT SIRT6 . n = 3 BR ; error bars = SD . Students t - test , two tailed . D , E Stimulation of NHEJ and HR by SIRT6 variants . Reporter cell lines were co - transfected with SIRT6 - expressing plasmid , I - Sce1 plasmid , and DsRed transfection control . After 72 hr recovery , reactivation of the GFP reporter was measured by flow cytometry . Stimulation of NHEJ or HR was calculated as radio of GFP + / DsRed + positive cells . Blots demonstrate equivalent SIRT6 abundance in transfected cells . Asterisk indicate p < 0 . 05 Black asterisk indicate significance over control , red asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVAF Basal γH2AX foci in cumate - inducible SIRT6 fibroblasts . Foci were scored in at least 80 cells per condition . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed . G DNA repair kinetics in cumate - inducible SIRT6 fibroblasts . Cells were grown on slides and irradiated with 2 Gy gamma radiation , followed by immunostaining for γH2AX . Irradiation was conducted when the cells were at 75 % confluency on slides . Cells were fixed and foci scored at t = 0 . 5 hr , 2 hr , 4 hr , 6 hr , and 24 hr post - irradiation . Foci were scored in at least 80 cells per genotype per time point . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed . H , I Oxidative stress resistance . Cumate - inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours . Resistance was determined by apoptosis staining 48 hours after exposure . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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

Figure 4A qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.B LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicated significance over SIRT6 KO control. Black asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.C In vitro KAP1 ribosylation by SIRT6. Asterisk indicate p<0.05. Red asterisk indicate significance over 0 hr, black asterisk indicate significance over compared to WT SIRT6. n=3 BR; error bars=SD. Students t-test, two tailed.D, E Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Sce1 plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisk indicate p<0.05 Black asterisk indicate significance over control, red asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVAF Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


Figure 5A Number of adherent cells after transfection with SIRT6 variants . Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours . HCA2 are normal human foreskin fibroblasts . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed . B , C Apoptosis staining of cancer cell lines 48 hours after transfection . Cells were stained with Annexin V / PI and analyzed by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicates significance over control , black asterisk indicates significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

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

Figure 5A Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours. HCA2 are normal human foreskin fibroblasts. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.B, C Apoptosis staining of cancer cell lines 48 hours after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


Figure 6B IP experiments on lysates from cumate - induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6 , LMNA , and mADPr . SIRT6 expression was induced 48 hours prior to IP . n = 3 BR . One representative set of IPs is shown . C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6 . Quantification of the IP experiment shown in ( B ) . SIRT6 IP followed by Western blot with antibodies to LMNA . D LMNA shows enhanced interaction with centSIRT6 compared to the wild type . Quantification of the IP experiment shown in ( B ) . LMNA IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6 . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 . 05E centSIRT6 shows enhanced mADPr . Quantification of the IP experiment shown in ( B ) . SIRT6 IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6 . Quantification of the IP experiment shown in ( B ) . IP with mADPr antibody using extract from cumate - induced SIRT6 fibroblasts , followed by Western blot with antibodies to LMNA . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 .

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

Figure 6B IP experiments on lysates from cumate-induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs is shown.C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA. D LMNA shows enhanced interaction with centSIRT6 compared to the wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05E centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


Figure 4 ( H ) - ( J ) mRNA expression of ( H ) U2AF1 , ( I ) SMN2 - FL , or ( J ) SMN2 ∆ 7 in SMA patient - derived fibroblasts ( red bars ) 72 hours post - transfection with SMN2 SSO , scrambled siRNA , or U2AF1 siRNA at the indicated dose . Unaffected , SMA carrier fibroblasts ( blue bars ) treated with scrambled siRNA . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 biological replicates . Three technical replicates were performed during qPCR for each biological replicate and averaged . Scrambled siRNA condition for each experiment was set to 1 . ( K ) Representative Western blot . ( L ) - ( M ) Quantification of protein expression of ( L ) U2AF1 or ( M ) SMN normalized to GAPDH ( n = 3 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 biological replicates .

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Figure 4(H)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA. Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.(K) Representative Western blot. (L)-(M) Quantification of protein expression of (L) U2AF1 or (M) SMN normalized to GAPDH (n=3). Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates.


figf1a , Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy ( TEM ) . Shown are spacious vacuoles ( SLAPs ) containing multiple bacteria . Magnification , × 5 , 200 . Region 1 and 2 ( white boxes ) are enlarged in the lower panels . b , SCID mice were infected as in a and liver sections stained for LAMP1 ( green ) , bacteria ( red ) and DNA ( blue ) . Shown is a LAMP1 + SLAP . c , The percentage of bacteria in LAMP1 + compartments was quantified and characterized as either spacious or tight - fitting . Mean ± s . e . m . for three mice examined . The image in a ( from ref . 2 ) and the tissue sections were provided by E . Unanue .

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figf1a, Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy (TEM). Shown are spacious vacuoles (SLAPs) containing multiple bacteria. Magnification, ×5,200. Region 1 and 2 (white boxes) are enlarged in the lower panels.b, SCID mice were infected as in a and liver sections stained for LAMP1 (green), bacteria (red) and DNA (blue). Shown is a LAMP1+ SLAP.c, The percentage of bacteria in LAMP1+ compartments was quantified and characterized as either spacious or tight-fitting. Mean ± s.e.m. for three mice examined. The image in a (from ref. 2) and the tissue sections were provided by E. Unanue.


figf2a , RAW 264 . 7macrophages infected for 4 or 8 h were analysed by TEM . Shown are spacious vacuoles ( SLAPs ) containing multiple bacteria . The arrow indicates septum of dividing bacteria . Scale bars , 0 . 5 µm . b , The arrow indicates a LAMP1 + SLAP colocalizing with green fluorescent protein ( GFP ) - LC3 in cells infected for 4 h . The percentage of GFP - LC3 + ( c ) d , The arrowhead indicates a LAMP1 + SLAP devoid of cathepsin D in cells infected for 4 h . Scale bars , 5 µm . The percentage of GFP - LC3 + ( c ) or cathepsin D + ( e ) f , GFP - LC3 - transfected macrophages were infected with wild - type or D actA bacteria . Where indicated , chloramphenicol ( CM ) was added to the media at 3 h post infection . The number of bacteria per SLAP was quantified . Brackets indicate significant differences , and corresponding P values are shown . g , Macrophages were infected with wild - type bacteria for 7 h , pulsed with BrdU for 1 h , and stained for LAMP1 ( red ) , bacteria ( blue ) and BrdU ( green ) . Magnified images and the arrow indicate SLAPs containing actively replicating bacteria ( BrdU + ) . h , The percentage of BrdU + bacteria in SLAPs , compared to bacteria and PFA - killed bacteria , was quantified as in c .

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figf2a, RAW 264.7macrophages infected for 4 or 8 h were analysed by TEM. Shown are spacious vacuoles (SLAPs) containing multiple bacteria. The arrow indicates septum of dividing bacteria. Scale bars, 0.5 µm.b, The arrow indicates a LAMP1+ SLAP colocalizing with green fluorescent protein (GFP)-LC3 in cells infected for 4 h.The percentage of GFP-LC3+ (c)d, The arrowhead indicates a LAMP1+ SLAP devoid of cathepsin D in cells infected for 4 h. Scale bars, 5 µm.The percentage of GFP-LC3+ (c) or cathepsin D+ (e)f, GFP-LC3-transfected macrophages were infected with wild-type or D actA bacteria. Where indicated, chloramphenicol (CM) was added to the media at 3 h post infection. The number of bacteria per SLAP was quantified. Brackets indicate significant differences, and corresponding P values are shown.g, Macrophages were infected with wild-type bacteria for 7 h, pulsed with BrdU for 1 h, and stained for LAMP1 (red), bacteria (blue) and BrdU (green). Magnified images and the arrow indicate SLAPs containing actively replicating bacteria (BrdU+). h, The percentage of BrdU+ bacteria in SLAPs, compared to bacteria and PFA-killed bacteria, was quantified as in c.


figf3a , GFP - LC3 - transfected macrophages were infected for 4 h , and the percentage of infected cells exhibiting SLAPs was quantified . Mean ± s . e . m . for three independent experiments . P values for strains with significant differences from wild - type levels are shown . b , The arrow indicates a GFP - LC3 + SLAP with internal LLO expression in cells infected for 4 h . Scale bar , 5 µm . c , Arrowheads indicate GFP - LC3 + SLAPs devoid of Lysotracker Red in cells infected for 4 h . e , The arrow indicates a LAMP1 + SLAP colocalizing with v - ATPase staining in cells infected for 4 h . Scale bar , 5 µm . f , The percentage of v - ATPase + SLAPs was quantified as in Fig . 2c . Mean ± s . e . m . for three independent experiments . P values for conditions significantly different from PFA - killed bacteria are shown .

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figf3a, GFP-LC3-transfected macrophages were infected for 4 h, and the percentage of infected cells exhibiting SLAPs was quantified. Mean ± s.e.m. for three independent experiments. P values for strains with significant differences from wild-type levels are shown.b, The arrow indicates a GFP-LC3+ SLAP with internal LLO expression in cells infected for 4 h. Scale bar, 5 µm.c, Arrowheads indicate GFP-LC3+ SLAPs devoid of Lysotracker Red in cells infected for 4 h.e, The arrow indicates a LAMP1+ SLAP colocalizing with v-ATPase staining in cells infected for 4 h. Scale bar, 5 µm. f, The percentage of v-ATPase+ SLAPs was quantified as in Fig. 2c. Mean ± s.e.m. for three independent experiments. P values for conditions significantly different from PFA-killed bacteria are shown.


figf4a , Arrows indicate LAMP1 - wild - type bacteria with actin ' comet tails ' in cells infected for 6 h . The boxed region is magnified in the bottom left panels . The merged image for this region is shown at the bottom right . Scale bar , 5 µm . b , Macrophages were infected with IPTG - induced iLLO bacteria . Arrowheads indicate actin - iLLO bacteria within LAMP1 + vacuoles . c , Macrophages were infected with wild - type ( triangles ) or IPTG - induced iLLO ( circles ) bacteria , and the percentage of LAMP1 + bacteria was quantified . Mean ± s . e . m . for three independent experiments . d , Macrophages were infected as in c with or without IPTG induction . Intracellular bacterial replication was determined using a gentamicin - protection assay . Shown is fold replication compared to 2 h post infection . Clear triangles , wild type ; filled triangles , wild type + IPTG ; clear circles , iLLO ; filled circles , iLLO + IPTG . Mean ± s . e . m . or range for three ( wild type , wild type + IPTG , iLLO + IPTG ) or two ( iLLO - IPTG ) independent experiments , respectively . e , Wild - type or Atg5 - / - MEFs were infected with iLLO bacteria , and intracellular bacterial replication was determined as in d . Mean ± s . e . m . for three independent experiments . Clear circles , wild - type MEFs ; filled circles , wild - type MEFs + IPTG ; clear squares , Atg5 - / - MEFs ; filled squares , Atg5 - / - MEFs + IPTG . f , Wild - type ( circles ) or Atg5 - / - ( squares ) MEFs were infected as in e with IPTG induction . The percentage of LAMP1 + bacteria was quantified as in c . Mean ± s . e . m . for three independent experiments .

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figf4a, Arrows indicate LAMP1- wild-type bacteria with actin 'comet tails' in cells infected for 6 h. The boxed region is magnified in the bottom left panels. The merged image for this region is shown at the bottom right. Scale bar, 5 µm.b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles.c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments.d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively.e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG.f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g


Figure 1 ( A ) Isolation of FTCD from rat liver extract . The p47 ( 1 - 170 ) fragment , which does not contain the p97 - binding site , was immobilized on beads and incubated with rat liver extract . Bound proteins were fractionated by SDS - PAGE , and stained with CBB . ( B ) FTCD binds directly to p47 . GST - tagged p47 ( 0 . 54 μg ) or GST - tagged p37 ( 0 . 45 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , and then isolated on glutathione beads . The blots were probed with antibodies to FTCD and GST . ( C ) FTCD and p47 form a complex in cells . HepG2 cells were solubilized and incubated with anti - p47 antibodies . The immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to p47 and FTCD . ( D ) FTCD , p47 and p97 form a complex in cells . HepG2 cells were solubilized and incubated with anti - FTCD antibodies . The immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to FTCD , VCIP135 , p97 , p47 and syntaxin5 . ( E ) p47 binds to the CD domain of FTCD . GST - tagged p47 ( 0 . 50 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 63 μg ) . The blots were probed with antibodies to His - tag and GST . ( F ) p47 ( E86K , E88K ) does not bind to FTCD . GST - tagged p47wt / mutant ( 0 . 50 μg ) was incubated with the indicated His - tagged proteins ( p97 , 1 . 0 μg ; FTCD , 0 . 75 μg ) . The blots were probed with antibodies to p97 , FTCD and GST . ( G ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , does not bind to p47 . GST - tagged p47 ( 0 . 35 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . The blots were probed with antibodies to His - tag and GST . ( H ) p47 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were first incubated with either GST - tagged p47wt , p47 ( E86K , E88K ) or p47 ( F253S ) . The isolated membranes were solubilized , and then GST - tagged p47wt / mutants and their binding proteins were recovered with glutathione - beads . The bound proteins were assayed by Western blotting with antibodies to FTCD and GST .

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Figure 1(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD.(D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5.(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.(F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST.(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.


Figure 2 ( A ) FTCD directly binds to p97 . GST - tagged p97 ( 2 . 1 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , and then isolated on glutathione beads . The blots were probed with antibodies to FTCD and GST . ( B ) Nucleotide dependency of the binding of FTCD with p97 . FTCD ( 0 . 80 μg ) and GST - tagged p97 ( 2 . 1 μg ) were incubated in the presence of the indicated nucleotide ( 1 mM ) . The blots were probed with antibodies to FTCD and GST . ( C ) FTCD binds to the C - terminal region of p97 . GST - tagged p97 fragments were incubated with either FTCD or p47 . The blots were probed with antibodies to FTCD and p47 . ( D ) p97 also binds to the CD domain of FTCD . GST - tagged p97 ( 2 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 62 μg ) . The blots were probed with antibodies to His - tag and GST . ( E ) p97 ( V722K , E724K ) shows very low binding affinity to FTCD . GST - tagged p97wt / mutant ( 2 . 0 μg ) was incubated with the indicated His - tagged proteins ( FTCD , 2 . 0 μg ; p47 , 0 . 5 μg ) . The blots were probed with antibodies to p47 , FTCD and GST . ( F ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , shows a very low binding affinity to p97 . GST - tagged p97 ( 1 . 4 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . The blots were probed with antibodies to His - tag and GST . ( G ) p97 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were incubated with either GST - tagged p97wt or p97 ( V722k , E724K ) . The isolated membranes were solubilized , and then GST - tagged p97wt / mutants and their binding proteins were recovered with glutathione - beads . The bound proteins were assayed by Western blotting with antibodies to FTCD and GST .

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Figure 2(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(B) Nucleotide dependency of the binding of FTCD with p97. FTCD (0.80 μg) and GST-tagged p97 (2.1 μg) were incubated in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to FTCD and GST.(C) FTCD binds to the C-terminal region of p97. GST-tagged p97 fragments were incubated with either FTCD or p47. The blots were probed with antibodies to FTCD and p47.(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.(E) p97(V722K, E724K) shows very low binding affinity to FTCD. GST-tagged p97wt/mutant ( 2.0 μg) was incubated with the indicated His-tagged proteins (FTCD, 2.0 μg; p47, 0.5 μg). The blots were probed with antibodies to p47, FTCD and GST.(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.