figf1a , Immunoblot analysis of Atg5 and LC3 . Brain homogenates were prepared from six - week - old control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice . Immunoblot analysis was performed using antibodies against Atg5 and LC3 . GSK - 3β was used as a loading control . The positions of the Atg12 - Atg5 conjugate , LC3 - I and LC3 - II ( LC3 - PE conjugate ) are indicated . Atg5 monomer was not detected in either lane ( data not shown ) . b , A representative male control ( Atg5flox / + ; nestin - Cre ) and an Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) littermate at three weeks of age . c , Left , paw placement records of eight - week - old mice . Right , stride lengths corrected for paw base widths ( stride / width ratio ) of Atg5flox / flox ; nestin - Cre and control ( Atg5flox / + ; nestin - Cre ) littermate mice . Values represent means ± s . d . of four mice . Asterisk , P 0 . 01 ( Students t - test ) . d , Abnormal limb - clasping of an Atg5flox / flox ; nestin - Cre mouse compared with a control mouse ( Atg5flox / + ; nestin - Cre ) when suspended by its tail . e , Rotarod testing of Atg5flox / + ; nestin - Cre ( open symbols ) and Atg5flox / flox ; nestin - Cre ( closed symbols ) mice . One male and one female mouse were analysed for each genotype . The time until drop from the rod ( rotating at 20 r . p . m . ) is shown .

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

figf1a, Immunoblot analysis of Atg5 and LC3. Brain homogenates were prepared from six-week-old control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice. Immunoblot analysis was performed using antibodies against Atg5 and LC3. GSK-3β was used as a loading control. The positions of the Atg12-Atg5 conjugate, LC3-I and LC3-II (LC3-PE conjugate) are indicated. Atg5 monomer was not detected in either lane (data not shown).b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age.c, Left, paw placement records of eight-week-old mice. Right, stride lengths corrected for paw base widths (stride/width ratio) of Atg5flox/flox; nestin-Cre and control (Atg5flox/+; nestin-Cre) littermate mice. Values represent means ± s.d. of four mice. Asterisk, P 0.01 (Students t-test).d, Abnormal limb-clasping of an Atg5flox/flox; nestin-Cre mouse compared with a control mouse (Atg5flox/+; nestin-Cre) when suspended by its tail.e, Rotarod testing of Atg5flox/+; nestin-Cre (open symbols) and Atg5flox/flox; nestin-Cre (closed symbols) mice. One male and one female mouse were analysed for each genotype. The time until drop from the rod (rotating at 20 r.p.m.) is shown.


figf2a , HE - stained sections of the cerebral cortex , the gracile nucleus and cerebellum from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at three months of age . Purkinje cells are indicated with arrowheads . Arrows indicate eosinophilic spheroids , which represent axon swelling . Scale bar , 10 µm . b , Immunohistochemistry using an anti - calbindin antibody on cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre mice at six weeks of age . Scale bar , 25 µm . c , Loss of Purkinje cells in Atg5flox / flox ; nestin - Cre mice . Purkinje cells were counted in comparable areas for each mouse , and three fields were counted in each area for each mouse . Data are normalized against values from control mice ( Atg5flox / + ; nestin - Cre ) . Values represent mean ± s . d . of three mice . Asterisk , P 0 . 01 ( t - test ) . d , Apoptotic death of granular cells . Cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre mice at six weeks of age were subjected to TUNEL staining . Nuclei were stained with Hoechst 33258 . Scale bar , 500 µm .

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

figf2a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age. Purkinje cells are indicated with arrowheads. Arrows indicate eosinophilic spheroids, which represent axon swelling. Scale bar, 10 µm.b, Immunohistochemistry using an anti-calbindin antibody on cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age. Scale bar, 25 µm.c, Loss of Purkinje cells in Atg5flox/flox; nestin-Cre mice. Purkinje cells were counted in comparable areas for each mouse, and three fields were counted in each area for each mouse. Data are normalized against values from control mice (Atg5flox/+; nestin-Cre). Values represent mean ± s.d. of three mice. Asterisk, P 0.01 (t-test).d, Apoptotic death of granular cells. Cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age were subjected to TUNEL staining. Nuclei were stained with Hoechst 33258. Scale bar, 500 µm.


figf3a , Immunohistochemistry of brain sections from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at six weeks of age , stained with an anti - ubiquitin antibody ( 1B3 ) . Scale bar , 10 µm . b , Ubiquitin - positive inclusions in neurons . Sections of medulla from Atg5flox / flox ; nestin - Cre mice at six weeks of age were stained with an antibody against NeuN ( a neuron - specific nuclear protein ) and ubiquitin . Nuclei were stained with Hoechst 33258 . Scale bar , 10 µm . c , Immunoelectron microscopy of ubiquitin - positive inclusion bodies . DRG neurons isolated from Atg5 - / - neonates were analysed by immunoelectron microscopy using an anti - ubiquitin antibody . Scale bars , 500 nm .

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 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

figf3a, Immunohistochemistry of brain sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at six weeks of age, stained with an anti-ubiquitin antibody (1B3). Scale bar, 10 µm.b, Ubiquitin-positive inclusions in neurons. Sections of medulla from Atg5flox/flox; nestin-Cre mice at six weeks of age were stained with an antibody against NeuN (a neuron-specific nuclear protein) and ubiquitin. Nuclei were stained with Hoechst 33258. Scale bar, 10 µm.c, Immunoelectron microscopy of ubiquitin-positive inclusion bodies. DRG neurons isolated from Atg5-/- neonates were analysed by immunoelectron microscopy using an anti-ubiquitin antibody. Scale bars, 500 nm.


figf4a , Immunohistochemistry of DRG neurons from Atg5 + / + and Atg5 - / - mice at different developmental stages ( embryonic day ( E ) 13 . 5 , E15 . 5 or postnatal day ( P ) 0 ) , stained with an antibody directed against ubiquitin . Scale bar , 25 µm . b , Accumulation of Triton - X - 100 - solublepolyubiquitinated proteins in the brains of Atg5flox / flox ; nestin - Cre mice . Brain homogenate prepared at the indicated times from control ( Atg5flox / + ; nestin - Cre ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice were separated into Triton - X - 100 - soluble ( S ) and - insoluble ( P ) fractions and analysed by immunoblotting using anti - ubiquitin antibodies . Arrowhead indicates the stacking gel . c , Immunohistochemistry of liver sections from control ( Atg5flox / + ; CAG - Cre ) and Atg5flox / flox ; CAG - Cre ( Atg5flox / flox ) mice at four months of age , using an anti - ubiquitin antibody . Scale bar , 25 µm . d , Immunohistochemistry of liver sections from six - week - old Atg5flox / flox ; Mx1 - Cre mice at the indicated time points after pIpC injection , using anti - ubiquitin antibodies . Arrowheads indicate ubiquitin - positive inclusion bodies . Scale bar , 25 µm . e , Five thousand hepatocytes were randomly selected , and the number of cells with diffuse cytosolic ubiquitin signals , with ( black ) or without ( white ) inclusion bodies , were counted .

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

figf4a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E)13.5, E15.5 or postnatal day (P)0), stained with an antibody directed against ubiquitin. Scale bar, 25 µm.b, Accumulation of Triton-X-100-solublepolyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel.c, Immunohistochemistry of liver sections from control (Atg5flox/+; CAG-Cre) and Atg5flox/flox; CAG-Cre (Atg5flox/flox) mice at four months of age, using an anti-ubiquitin antibody. Scale bar, 25 µm.d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm.e, Five thousand hepatocytes were randomly selected, and the number of cells with diffuse cytosolic ubiquitin signals, with (black) or without (white) inclusion bodies, were counted.


f1Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5 - GFP or GFP - ATG8 . ( a , b ) Control conditions : cytosolic signal associated with rare fluorescent puncta . ( c , d ) High increase of fluorescent puncta after 2 h of nutrient starvation . Note ring - like ATG5 structures in c . ( e , f ) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures . ( g , h ) Maximum intensity projections of vacuolar focal planes , allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A . Vacuolar ATG5 - GFP fluorescence is not detected ( g ) , while GFP - ATG8 signals are abundant ( h ) . An inset in g , corresponding to a cortical focal plane , shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment . Maximum intensity projections of 10 - μm - deep deconvolved stacks , of cortical ( a - f , and inset in g ) or vacuolar focal planes ( g , h ) . Scale bar , 10 μm .

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

f1Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.


f2 ( a ) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A , in nutrient starvation conditions . Autophagic bodies accumulation in the vacuole is clearly visible in wild - type seedlings , absent in the atg5 - 1 mutant and restored in atg5 - 1 seedlings expressing ATG5 - GFP . ( b ) Confocal microscopy . The ATG5 - GFP fluorescence pattern in the atg5 - 1 background is identical to that observed in wild - type seedling , including ring - like structures . Scale bar , 15 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

f2(a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.


f3 ( a ) Dual observation of ATG5 - GFP and mCh - ATG8 under nutrient starvation reveals a partial colocalization of the two reporters . Merge of the GFP ( green ) and ATG8 ( magenta ) channels of maximum intensity projections of 20 - μm - deep deconvolved stacks . Scale bar , 3 μm . ( b ) Real - time imaging of an individual autophagosomal structure , showing one deconvolved image per 20 s . The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure . The following steps , defined as the decay phase , being more dynamic , are depicted also at higher time resolution . Single - plane merged images of ATG5 - GFP ( green ) and mCh - ATG8 ( magenta ) channels , deconvolved . Scale bar , 1 μm . ( c ) Representative FRAP experiment on mCh - ATG8 during the ring formation phase . Deconvolved images . Scale bar , 1 μm . The corresponding fluorescence recovery kinetic , measured on non - deconvolved images , is shown on the right . ( d ) Representative FRAP experiment of mCh - ATG8 during the ring decay phase , presented as in c . ( e ) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation ( n = 11 ) and decay ( n = 17 ) . Whiskers comprise the 10 - 90 percentile , the box the two central quartiles . The mean recovery values are indicated by an horizontal line . Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann - Whitney statistical test ( * * * P0 . 001 ) .

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

f3(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm.(b) Real-time imaging of an individual autophagosomal structure, showing one deconvolved image per 20 s. The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure. The following steps, defined as the decay phase, being more dynamic, are depicted also at higher time resolution. Single-plane merged images of ATG5-GFP (green) and mCh-ATG8 (magenta) channels, deconvolved. Scale bar, 1 μm.(c) Representative FRAP experiment on mCh-ATG8 during the ring formation phase. Deconvolved images. Scale bar, 1 μm. The corresponding fluorescence recovery kinetic, measured on non-deconvolved images, is shown on the right.(d) Representative FRAP experiment of mCh-ATG8 during the ring decay phase, presented as in c.(e) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation (n=11) and decay (n=17). Whiskers comprise the 10-90 percentile, the box the two central quartiles. The mean recovery values are indicated by an horizontal line. Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann-Whitney statistical test (***P0.001).


f6 ( a ) Correlative light - electron microscopy of GFP - ATG8 - labelled autophagosomal structures . Correlation of a TEM micrograph of a root transverse section ( 1 ) with fluorescent imaging of GFP - ATG8 ( arrowheads , 2 ) confirms the association of ATG8 signal with autophagosome ( 3 ) . A closer view shows close contacts with ER - like membranes ( empty arrowheads , 4 ) . ( b ) Immunogold labelling of GFP - ATG8 on cryosections , employing an anti - GFP antibody ( arrowheads ) . Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed ( samples c ) or after chemical fixation ( d ) . Empty arrowheads : connections with ER - like membranes . Scale bars , 5 μm in a ( 1 - 3 ) ; 0 . 5 μm in a ( 4 ) and b ( 1 - 3 ) .

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 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

f6(a) Correlative light-electron microscopy of GFP-ATG8-labelled autophagosomal structures. Correlation of a TEM micrograph of a root transverse section (1) with fluorescent imaging of GFP-ATG8 (arrowheads, 2) confirms the association of ATG8 signal with autophagosome (3). A closer view shows close contacts with ER-like membranes (empty arrowheads, 4).(b) Immunogold labelling of GFP-ATG8 on cryosections, employing an anti-GFP antibody (arrowheads). Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed (samples c) or after chemical fixation (d). Empty arrowheads: connections with ER-like membranes. Scale bars, 5 μm in a(1-3); 0.5 μm in a(4) and b(1-3).


Figure 1 ( A and B ) THP1 - derived macrophages were treated with the TLR ligands Pam3 , lipopolysaccharide ( LPS ) , or FSL - 1 and analyzed by RNA - seq 8 h later . Circos plots show genome - wide differential expression of protein - coding genes ( A ) and non - protein - coding genes ( B ) between untreated and treated macrophages . The outermost to innermost circles show downregulated ( blue ) or upregulated ( red ) genes in cells stimulated with FSL - 1 , LPS , and Pam3 , respectively . Chromosomes are indicated by the numbers 1 - 22 and X and Y . ( F ) RT - qPCR validation of differentially expressed lncRNAs and mRNAs in THP1 - derived macrophages stimulated for 8 h with flagellin ( FLA ) , FSL - 1 , imiquimod , LPS , Pam3 , or poly ( I : C ) . Heat map shows the fold change in expression of 13 mRNAs ( left ) and lncRNAs ( right ) .

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

Figure 1(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.(F) RT-qPCR validation of differentially expressed lncRNAs and mRNAs in THP1-derived macrophages stimulated for 8 h with flagellin (FLA), FSL-1, imiquimod, LPS, Pam3, or poly(I:C). Heat map shows the fold change in expression of 13 mRNAs (left) and lncRNAs (right).


Figure 2 ( A ) Activation and suppression of nearby genes by eight cis - acting lncRNAs . RT - qPCR analysis of THP1 - derived macrophages transduced with lentiviruses expressing non - targeting control ( NC ) shRNA or shRNAs targeted to the indicated lncRNAs and protein - coding genes . Mean ± SD of n = 3 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by Student ' s t test . Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection . Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 IL - 6 ELISA quantification ( C ) of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . ELISA were performed at 10 h post - infection . Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 1α ( D ) . RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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

Figure 2(A) Activation and suppression of nearby genes by eight cis-acting lncRNAs. RT-qPCR analysis of THP1-derived macrophages transduced with lentiviruses expressing non-targeting control (NC) shRNA or shRNAs targeted to the indicated lncRNAs and protein-coding genes. Mean ± SD of n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001 by Student's t test.Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 IL-6 ELISA quantification (C) of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. ELISA were performed at 10 h post-infection.Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-1α (D). RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


Figure 3lnc - MARCKS ( B ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs . , MARCKS ( C ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs . IL - 6 ( D ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs . Overexpression of lnc - MARCKS decreases MARCKS expression RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . Lnc - MARCKS and MARCKS expression ( F ) Overexpression of lnc - MARCKS enhances IL - 6 expression . RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . IL - 6 expression ( G ) . lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect lnc - MARCKS ( H ) lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS ( I ) , lnc - MARCKS regulates IL - 6 in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect IL - 6 expressions ( J ) .

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Figure 3lnc-MARCKS (B) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs., MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F)Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H)lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I),lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).


Figure 4 ( A ) ROCKI is recruited to the MARCKS promoter in Pam3 - stimulated THP1 - derived macrophages . ChIRP analysis of THP1 - derived macrophages treated with Pam3 for 8 h . ChIRP assays were performed using a non - specific lacZ probe or probes specific for lnc - MARCKS . Specificity of ROCKI probes ( left ) , and enrichment at the MARCKS promoter ( right ) . ( B ) Mass spectrometry identifies APEX1 as a candidate ROCKI - binding protein in THP1 - derived macrophages . APEX1 MS / MS count represents the ratio between the number of APEX1 peptides and all peptides detected . Mean of n = 2 samples . ROCKI and ROCKI - AS represent ROCKI and antisense strand of ROCKI transcripts , respectively . ( C ) ROCKI inhibition of MARCKS expression is dependent on APEX1 . RT - qPCR analysis of MARCKS , ROCKI , and APEX1 expression in THP1 - derived macrophages overexpressing ROCKI and / or depleted of APEX1 . ( D ) APEX1 specially binds to ROCKI . Western blot analysis of APEX1 in lacZ , or ROCKI pull - down fractions . Vimentin , which non - specifically binds to all RNA probes , is shown as a loading control . ( E ) ROCKI associates with APEX1 . Left : RT - qPCR analysis of ROCKI present in anti - APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3 - treated THP1 - derived macrophages . Right : APEX1 western blot of the same immunoprecipitates .

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Figure 4(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right).(B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively.(C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1.(D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control.(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.


Figure 5ChIP analysis of THP1 - derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter . RT - qPCR analysis of fragments of the MARCKS promoter region ( a and b ) or a non - specific region ( nc ) co - immunoprecipitated with anti - APEX1 antibody . ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA ( NC ) or ROCKI shRNA and treated with Pam3 for 8 h . The y - axis shows the percentage of enrichment in normalized to input . An unrelated region of the ACTB2 promoter was amplified as a control . Left : Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti - APEX1 antibody immunoprecipitates from NC ( control ) or ROCKI knockdown THP1 - derived macrophages . Right : RT - qPCR analysis of ROCKI in the same immunoprecipitates . Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter . ChIP - qPCR analysis of HDAC1 was performed using an anti - HDAC1 antibody . Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter . ChIP - qPCR analysis of H3K27ac was performed using an anti - H3K27ac antibody .

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Figure 5ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody.ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control.Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates.Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed using an anti- HDAC1 antibody.Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.


Figure 6Heatmap of representative genes commonly upregulated in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages . RT - qPCR validation of inflammatory gene expression in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages stimulated with Pam3 for 8 h . E . V . , empty vector ; NC , control shRNA . MARCKS is a negative regulator of Ca2 + signaling . Fluo - 4 fluorescence of MARCKS , ROCKI , or NC ( control ) shRNA - expressing THP1 - derived macrophages before and after treatment with PMA and Pam3 . Triton X - 100 was added to obtain the maximal [ Ca2 + ] signal . Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence ( ΔF / Fmax ) .

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Figure 6Heatmap of representative genes commonly upregulated in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages.RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA.MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).


Figure 7 ( A ) QTL signals for ROCKI ( LINC01268 ) gene expression in whole blood ( top , black ) , H3K27ac promoter signal in monocytes ( middle , green ) , and H3K4me1 signal in monocytes ( bottom , orange ) in a 400 kb window around the MARCKS / ROCKI locus . The signals were strongly co - localized ( Pshared = . 97 , Pshared = . 98 ) suggesting they represent the same signal . ( C ) RT - qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M . tuberculosis - infected donors .

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Figure 7(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.(C) RT-qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M. tuberculosis-infected donors.


Figure 1 ( B ) Southern Blot analysis of targeted ES cells . Positions of wild type ( WT - 14 . 7kb ) and targeted ( loxP - 12 . 8kb ) alleles are indicated . ( C ) Genotyping analysis of wild type ( Clpp + / + ) , heterozygous ( Clpp + / - ) and homozygous ( Clpp - / - ) Clpp knockout animals . DNA fragments corresponding to the wild type ( WT - 199 bp ) , and knockout ( KO - 273 bp ) Clpploci are indicated . ( D ) Analysis of Clpp transcript levels in heart ( n = 4 ) . Bars represent mean ± standard deviation ( SD ) . Student ' s t - test was used to determine the level of statistical difference . ( E ) Western blot analysis of CLPPprotein levels in heart ( He ) , quadriceps ( Quad ) , liver ( Li ) , kidney ( Ki ) , lung ( Lu ) , brain ( Br ) , spleen ( Sp ) and pancreas ( Pa ) . TUBULIN and ACTIN are used as a loading control . The arrowhead indicates positions of CLPP , and the star indicates position of cross - reacting bands .

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Figure 1(B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated.(C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpploci are indicated.(D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student's t-test was used to determine the level of statistical difference.(E) Western blot analysis of CLPPprotein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.


Figure 2 ( C ) Respiratory chain enzyme activities . Relative enzyme activities of respiratory chain enzyme C I ( NADH coenzyme Q reductase ) , C I / III ( NADH cytochrome c reductase ) , C II ( succinate dehydrogenase ) , C II / III ( succinate cytochrome c reductase ) and C IV ( cytochrome c oxidase ) in mitochondrial extracts from wild type and Clpp knockout hearts . ( n = 5 ) . Bars present mean levels ± SD . Statistical difference was calculated by Student ' s t - test and changes are presented in the figure ( n = 5 ) . ( D ) Substrate - dependent oxygen consumption rates in isolated heartmitochondria . Complex I substrates ( pyruvate , malate , glutamate ) ; complex II substrate ( succinate ) . State 3 - ADP stimulated oxygen consumption rate ; State 4 - respiration rate after addition of oligomycin ; Uncoupled - oxygen consumption rate in the presence of FCCP . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 7 ) . ( E ) Decrease of steady - state levels of respiratory chain subunits in Clpp - knockout hearts relative to wild type ( set to 1 ) . VDAC or TOMM20 were used as loading controls . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . ( F ) Blue Native Page Analysis ( BN - PAGE ) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I ( anti - NDUFA9 ) ; C III ( anti - UQCRC1 ) and C IV ( anti - COX4A ) .

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Figure 2(C) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student's t-test and changes are presented in the figure (n=5).(D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7).(E) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=4).(F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).


Figure 3 ( A ) Quantification of mtDNA levels after Southern blot analysis . Bars present mean levels ± SD ( n = 4 ) . ( B - C ) Northern blot analysis and corresponding quantification of mitochondrial ( B ) rRNAs and mRNAs or ( C ) tRNAs . 18S rRNA is used as loading control . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) . ( D ) Western blot analysis of LRPPRC levels . VDAC was used as a loading control . ( E ) In organello transcription analysis upon 60 minutes labelling with - 32P - UTP .

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Figure 3(A) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4).(B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).(D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control.(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.


Figure 4 ( A ) De novo synthesized mitochondrial proteins were isolated after labelling with 35S - methionine for 1 hour . Positions of individual mitochondrial encoded proteins are indicated . ( B ) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin , doxycycline and chloramphenicol ( + antibiotic ) , followed by a 48 hours recovery period ( - antibiotic ) . During both periods , proteins are isolated at 12h , 24h and 48h . Antibodies against subunits of C I ( NDUFB6 ) , C III ( UQCRC1 ) , C IV ( COXI ) , MRPS35 and MRPL37 were used as indicated . ( C ) Quantitative analysis of the COXI recovery upon treatment with antibiotics ( Actinonin , DOXY - doxycycline and CAP - chloramphenicol ) as in B . The values represent ratios of recovered ( 48h , - antibiotics ) versus untreated ( 0h ) COXI levels . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) . ( Actinonin and CAP n = 3 , DOXY n = 4 ) . ( D ) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunobloting with antibodies against MRPS35 and MRPL12 . The profile of a given mt - RNA is calculated after qRT - PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis . Results of individual experiments are provided for 12S and 16S rRNA , and for mt - mRNAs , results of distribution from two independent experiments using probes against ATP6 , COXIII and ND5 were combined . Error bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 3 ) . ( E ) Relative association of 12S rRNA , 16S rRNA and mt - mRNAs with mitoribosomes ( 55S ) in Clpp knockout mitochondria comparing to wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S and 16S n = 3 ; mRNA n = 6 ) .

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 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 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 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

Figure 4(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.(C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY - doxycycline and CAP - chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonin and CAP n=3, DOXY n=4).(D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=3).(E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).


Figure 5 ( B ) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( Clpp + / + ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . The arrowhead indicates positions of EFG1 . ( C ) Expression levels of potential CLPP substrates from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( set to 1 ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . ( D ) Cycloheximide ( CHX ) chase assay to determine the stability of ERAL1 and EFG1 . Proteins are visualized by Western blot with respective antibodies . Proteins are isolated after 2 , 4 , 6 , 8 , 10 and 12 hours after beginning of chase ( 0 ) . The arrowhead indicates positions of ERAL1 . ( E ) Western blot analysis and subsequent quantification of protein levels in wild type ( C1 , C2 ) and Clpp knockout ( KO1 , KO2 ) MEFs upon Clpx siRNA ( Clpx ) relative to untreated ( Ctrl ) wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 6 ) . The arrowhead indicates positions of either CLPX or ERAL1 , and stars indicate position of cross - reacting bands .

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 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 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

Figure 5(B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1.(C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4).(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.(E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.


Figure 6 ( A ) Sucrose density gradient sedimentation profile ( " short gradients " ) of ribosomal proteins and transcripts from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunoblotting with antibodies against MRPS35 and MRPL12 . 12S rRNA was analysed by Northern blot . ( B ) Sedimentation profiles of ERAL1 , MRPS28 , EFG1 , CLPP , CLPX , EFTU and MTIF2 in heartmitochondria as in A . ( C ) Relative abundance of proteins bound to the 28S ribosomal subunit ( fractions 3 and up ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( ERAL1 n = 4 ; EFG1 , CLPX , MTIF2 n = 3 ; EFTU n = 2 ) . ( D ) Sucrose density gradient sedimentation profile ( " long gradients " as in figure 4D ) of ERAL1 , MRPS35 , MRPL37 , CLPX and EFG1 from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis ( Input ) . Only fractions 4 - 11 containing the 28S , 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions ( 1 - 3 ) .

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 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 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 6(A) Sucrose density gradient sedimentation profile ("short gradients") of ribosomal proteins and transcripts from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunoblotting with antibodies against MRPS35 and MRPL12. 12S rRNA was analysed by Northern blot.(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heartmitochondria as in A.(C) Relative abundance of proteins bound to the 28S ribosomal subunit (fractions 3 and up). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (ERAL1 n=4; EFG1, CLPX, MTIF2 n=3; EFTU n=2).(D) Sucrose density gradient sedimentation profile ("long gradients" as in figure 4D) of ERAL1, MRPS35, MRPL37, CLPX and EFG1 from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis (Input). Only fractions 4 - 11 containing the 28S, 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions (1-3).


Figure 7 ( A ) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins . Western blot analysis of input ( I ) , control ( C ) and eluate ( E ) is shown . Asterix indicates the cross - reacting band . ( B ) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit ( MRPS35 ) upon Eral1 siRNA . TOMM20 was used as a loading control . ( C ) De novo synthesis of mitochondrial proteins in wild type ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) MEFs upon Eral1 siRNA . Proteins were isolated after labeling with 35S - methionine for 1 hour from cells on control siRNA ( Ctrl ) or cells on Eral1 siRNA ( Eral1 ) . Positions of individual mitochondrial encoded proteins are indicated . Coomassie blue staining was used to ensure equal loading . ( D ) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1 . Levels relative to wild type cells treated with control siRNA ( set to 1 ) are presented . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; ATP5A1 n = 2 ; the rest n = 6 ) . Combined results from 2 - 3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment . Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp - knockout cells upon Eral1 siRNA . ( E ) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs . Cells ( 0 ) were treated with doxycycline for 48 hours ( T ) and upon doxycycline removal allowed to recover for 24 hours ( R ) . ( F ) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA . The values are calculated as ratio between recovered ( R ) to doxycycline untreated ( 0 ) protein levels in Eral1 - knockdown cells ( Eral1 ) and compared to the same results obtained in control cells ( Ctrl ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; NDUFB6 n = 3 ) . Numbers within bars represent statistical difference between control / Eral1 siRNA treated cells and numbers above bars represent difference between + / + ; Eral1 siRNA and - / - ; Eral1 siRNA .

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 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 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 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

Figure 7(A) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins. Western blot analysis of input (I), control (C) and eluate (E) is shown. Asterix indicates the cross-reacting band.(B) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit (MRPS35) upon Eral1 siRNA. TOMM20 was used as a loading control.(C) De novo synthesis of mitochondrial proteins in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) MEFs upon Eral1 siRNA. Proteins were isolated after labeling with 35S-methionine for 1 hour from cells on control siRNA (Ctrl) or cells on Eral1 siRNA (Eral1). Positions of individual mitochondrial encoded proteins are indicated. Coomassie blue staining was used to ensure equal loading.(D) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1. Levels relative to wild type cells treated with control siRNA (set to 1) are presented. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; ATP5A1 n=2; the rest n=6). Combined results from 2-3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment. Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp-knockout cells upon Eral1 siRNA.(E) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs. Cells (0) were treated with doxycycline for 48 hours (T) and upon doxycycline removal allowed to recover for 24 hours (R).(F) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA. The values are calculated as ratio between recovered (R) to doxycycline untreated (0) protein levels in Eral1-knockdown cells (Eral1) and compared to the same results obtained in control cells (Ctrl). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; NDUFB6 n=3). Numbers within bars represent statistical difference between control/Eral1 siRNA treated cells and numbers above bars represent difference between +/+; Eral1 siRNA and -/-; Eral1 siRNA.


figf1a , TG levels in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) and cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , n = 3 ) . b , TG levels in vector - infected ( VEC ) and siAtg5 cells in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM . c , TG levels in wild - type ( WT ) or Atg5 knockout mice embryonic fibroblasts ( Atg5 - / - ) ( * P 0 . 01 or * * P 0 . 0001 , n = 5 ) . d f , Cells from a and b stained with BODIPY 493 / 503 ( d ) . Nuclei are highlighted with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; d ) . Error bars , s . e . m . d f , Cells from a and b stained with oil red O ( e ) or visualized by electron microscopy ( f ) . Right : quantifications of LD number and size ( * P 0 . 01 , * * P 0 . 001 with untreated wild - type cells ; # P 0 . 01 , # # P 0 . 001 with cells in RM ; n = 4 6 ) .

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figf1a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).


figf2a , b , VEC and siAtg5 cells cultured with oleate ( OL ) or in MCDM were examined for their rates of TG synthesis ( a ) and β - oxidation ( b ) as compared to cells in regular medium alone ( * P 0 . 03 , * * P 0 . 004 with VEC cells in the same medium , n = 3 4 ) . c , d , Rates of TG decay in OL ( c ) and MCDM ( d ) ( * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 as compared to VEC cells , n = 3 7 ) . e , TG levels in wild - type cells treated with dimethyl sulphoxide vehicle ( DMSO ) , 3 - methyladenine ( 3MA ) or diethylumbelliferyl phosphate ( DEUP ) ( * P 0 . 00001 with DMSO - treated cells , # P 0 . 003 with 3MA - treated cells , n = 6 ) . Error bars , s . e . m . f , Co - localization of BODIPY 493 / 503 ( green ) with LAMP1 ( red ) or LC3 ( red ) in hepatocytes in MCDM . g , High - magnification regions of hepatocytes in MCDM alone ( none ) or treated with vinblastine and stained as labelled . Arrows indicate co - localization .

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

figf2a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37).e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM.g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.


figf3a , Electron micrographs of cultured hepatocytes . dm , double - membrane cytosolic vesicles . Arrows indicate membranes in LDs . Insets show the double membrane in nucleus ( N ) , lipid - containing vesicles ( L ) and mitochondria ( mit ) , but not in LDs . b , Mouse liver LC3 immunogold . Insets show higher magnification . Arrowheads indicate gold particles ( black ) and LC3 - labelled bilayer membranes ( white ) . c , Percentages of autophagic vacuoles ( AVs ) containing only lipid ( Lipids , * ) , other cargo ( Other ) or mixed cargo ( Mixed ) in cells treated as in b ( * P 0 . 01 , * * P 0 . 001 with cells in RM , n = 4 6 ) . Left : representative examples . Error bars , s . e . m .

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figf3a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs.b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.


figf4a , Immunoblots of cellular homogenates ( Hom ) and LDs from fed ( F ) or 24 - h starved ( S ) mice . IκB , inhibitor of the nuclear factor of kappa light polypeptide gene enhancer ; GPDH , glyceraldehyde - 3 - phosphate dehydrogenase . b , Percentages of autophagic vacuoles ( AVs ) containing only lipid , other cargo or mixed cargo calculated from samples processed as in Supplementary Fig . 16b ( * P 0 . 001 , * * P 0 . 0001 , n = 4 6 ) . ( Supplementary Figure 16 Effects of starvation on hepatic lipid droplets . a , Mouseliverelectron micrographs . Arrows : Lipid - containing double membrane vesicles . b , Electron micrographs of autophagic vacuoles ( AVs ) isolated from mice fed or starved for the indicated time periods . The percentages of double membrane vesicles containing lipid in isolated AVs from 3 - 4 mice per condition are shown ( * P < 0 . 01 as compared to fractions from fed animals ) . ( * ) indicate lipid ) c , Immunoblots of Hom , AV and lysosomes ( Lys ) . L - 1 , LAMP1 . d , Immunoblots of liver Hom and LDs from HFD - fed mice . e , Liver homogenate immunoblots . PDI , protein disulphide isomerase . f , Total hepatic TG and cholesterol content ( * P 0 . 01 , * * P 0 . 00001 , n = 817 ) . g , Hepatic TG concentration ( * P 0 . 05 , n = 3 ) . h , Percentage of cellular cholesterol in lysosomes ( * P 0 . 02 , n = 4 ) . i , Immunoblots of homogenates and isolated LDs . Error bars , s . e . m .

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figf4a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase.b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1.d, Immunoblots of liver Hom and LDs from HFD-fed mice.e, Liver homogenate immunoblots. PDI, protein disulphide isomerase.f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).g, Hepatic TG concentration (*P  0.05, n = 3).h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.


Figure 1E , F : EM images of negatively stained Dam1c alone ( E ) and in the presence of Bim1 ( F ) , at low salt conditions . The final concentrations of Dam1c and Bim1 were 0 . 3 µM and 0 . 1 µM , respectively . The scale bars are 100 nm , boxes mark representative complexes shown as magnified inserts .

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Figure 1E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.


Figure 2B : Chromatogram and SDS - PAGE of elution fractions from gel filtration . Binding of Bim1 to Dam1 ∆ SxIPc lacking the SxIP motif in the C - terminus of Duo1 was examined by analytical size exclusion chromatography . Elution fractions were analyzed by SDS - PAGE . C : Solid phase binding assay to test for binding of different Dam1 complexes to Bim1 . Recombinant GST - Bim1185 - 344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1 ∆ SxIPc . Input and pull down samples were analyzed by SDS - PAGE . D : Pull down assay to compare binding of Dam1WTc and Dam1 ∆ SxIPc to immobilized GST - Bim1185 - 344 . Soluble cell lysates from yeast strains expressing the respective Duo1 - 6xFlag alleles were incubated with GST - Bim1185 - 344 immobilized on beads . Duo1 - 6xFlag bound to GST - Bim1185 - 344 was analyzed by western blot . An anti - Pgk1 antibody was used to confirm equal protein amounts in input samples .

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Figure 2B: Chromatogram and SDS-PAGE of elution fractions from gel filtration. Binding of Bim1 to Dam1∆SxIPc lacking the SxIP motif in the C-terminus of Duo1 was examined by analytical size exclusion chromatography. Elution fractions were analyzed by SDS-PAGE.C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.D: Pull down assay to compare binding of Dam1WTc and Dam1∆SxIPc to immobilized GST-Bim1185-344. Soluble cell lysates from yeast strains expressing the respective Duo1-6xFlag alleles were incubated with GST-Bim1185-344 immobilized on beads. Duo1-6xFlag bound to GST-Bim1185-344 was analyzed by western blot. An anti-Pgk1 antibody was used to confirm equal protein amounts in input samples.


Figure 3C : Representative images from fluorescence live cell microscopy of Dad1 - GFP strains with Duo1WT - 6xFlag or Duo1 ∆ SxIP - 6xFlag . Displayed cells are in either metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . D : Quantification of signal intensities of Dad1 - GFP at metaphase and anaphase kinetochore clusters of Duo1WT - 6xFlag and Duo1 ∆ SxIP - 6xFlag strains . Bars represent the mean + / - standard deviation . Each spot represents the value measured for a single kinetochore cluster . Values were normalized to the mean value of metaphase clusters of the Duo1WT - 6xFlag strain . A one - way ANOVA test with Sidak ' s multiple comparisons test was used to calculate p values . n ≥ 76 kinetochore clusters . F : Analysis of cell cycle progression of yeast strains with different Duo1 alleles . Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot ( left ) . An antibody against Pgk1 was used to confirm equal protein amounts in samples . The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS ( right ) . Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content , respectively . An intermediate DNA content is detected for cells in S phase .

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Figure 3C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters.F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase.


Figure 4B : Representative images from live cell microscopy of Dam1WT and dam1 - 19 strains with Dad1 - GFP . Displayed cells are in metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . C : Quantification of Dad1 - GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1 - 19 strains . Bars represent the mean + / - standard deviation . Each spot represents the value measured for a single kinetochore cluster . Values were normalized to the mean value of metaphase clusters of the Dam1WT strain . p - values were obtained by a one - way ANOVA test with Sidak ' s multiple comparisons test . n ≥ 86 kinetochore clusters . E , F : GST - Bim1185 - 344 pull down assays . Binding of immobilized GST - Bim1185 - 344 to Duo1 - 6xFlag was analyzed in context of ( E ) selective inhibition of Mps1 ( mps1 - 737 ) or overexpression of Mps1 from a galactose - inducible promotor ( F ) . For inhibition of Mps1 , cells were grown at the restrictive temperature of 37 ° C . Overexpression of Mps1 was achieved by growing cells in medium containing galactose . GST - Bim1185 - 344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions . Binding of Duo1 was analyzed by western blot . Pgk1 indicates equal protein amounts in all samples . G : Live cell microscopy of a Dad1 - GFP strain under conditions of Mps1 overexpression . The localization of Dam1c was visualized by Dad1 - GFP in either an Mps1WT or Mps1 overexpression setup ( pGal - Mps1 ) . Ndc80 - mRuby2 was used as reference point for the localization of kinetochores . Images show representative metaphase cells from both strains . The area in the white dashed box is shown as magnification in the lower left corner . White arrowheads point at nuclear microtubules decorated by Dad1 - GFP . Please note that brightness and contrast of the Dad1 - GFP image in the lower row were optimized to make the nuclear microtubules clearly visible . Scale bar : 2 μm .

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Figure 4B: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters.E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1 (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing galactose. GST-Bim1185-344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by western blot. Pgk1 indicates equal protein amounts in all samples.G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules decorated by Dad1-GFP. Please note that brightness and contrast of the Dad1-GFP image in the lower row were optimized to make the nuclear microtubules clearly visible. Scale bar: 2 μm.


Figure 5A , B : 5 µM , 7 . 5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1 - Bik1 complex were analyzed by size exclusion chromatography . SDS - PAGE analysis of elution fractions is shown in B . High molecular weight species of Dam1WTc were eluted after 0 . 9 - 1 . 0 ml ( see chromatogram in A ) .

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Figure 5A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A).


Figure 6B : Pull down assay for analysis of Dam1c - Bim1 binding to Ndc80c . Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre - formed Dam1c - Bim1 complex with increasing amounts of Bim1 . Input and elution samples were analyzed by SDS - PAGE . LC and HC denote the light and heavy chains of the M2 anti - Flag antibody used for immobilization of Ndc80c on beads . Protein names in blue , red and green refer to Dam1c , Ndc80c and Bim1 , respectively . D : Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80 - FRB - GFP background . Ndc80WT or Ndc80 ∆ 490 - 510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1 ∆ SxIP allele ( at DUO1 locus ) . Cells were spotted on YEPD medium without and with 1 µg / ml Rapamycin and incubated at the indicated temperatures .

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Figure 6B: Pull down assay for analysis of Dam1c-Bim1 binding to Ndc80c. Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre-formed Dam1c-Bim1 complex with increasing amounts of Bim1. Input and elution samples were analyzed by SDS-PAGE. LC and HC denote the light and heavy chains of the M2 anti-Flag antibody used for immobilization of Ndc80c on beads. Protein names in blue, red and green refer to Dam1c, Ndc80c and Bim1, respectively.D: Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.


Figure 7B : Serial dilution assay of yeast strains with 6xFlag - tagged Duo1WT or Duo1 ∆ SxIP and Ask1WT , Ask12A or Ask12D . Cells were grown at the indicated temperatures and images were taken two days ( 25 ° C , 30 ° C and 37 ° C ) or four days ( 16 ° C ) after spotting .

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Figure 7B: Serial dilution assay of yeast strains with 6xFlag-tagged Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting.


Figure 1E - H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected ( E ) , 2 months after intraductal injection with MCF7 , arrows point to undilated ductal tips ( F ) , MM134 , dotted line highlight the subtending duct ( G ) , or SUM44 cells ( H ) , ( n > 6 ) scale bar , 1 mm . I Ex vivo luminescence images of bones , brain , lungs and liver from MM134 engrafted mice 1 month post injection ( top ) and ovaries ( bottom ) 12 months post injection . Scale bar , 1 cm . J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis . Number of mice analyzed MM134 and SUM44 for 30 - 180 : N = 5 , 10 , 180 - 270 days : N = 9 , 5 , 270 - 360 days : N = 14 , 20 , respectively . Dashed line indicates background levels .

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Figure 1E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm.I Ex vivo luminescence images of bones, brain, lungs and liver from MM134 engrafted mice 1 month post injection (top) and ovaries (bottom) 12 months post injection. Scale bar, 1 cm. J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis. Number of mice analyzed MM134 and SUM44 for 30-180: N=5, 10, 180-270 days: N=9, 5, 270-360 days: N=14, 20, respectively. Dashed line indicates background levels.


Figure 2E , F Representative micrographs of IHC for Ki67 , ER , and PR on histological sections of MM134 ( E ) and SUM44 ( F ) xenografts 5 months after intraductal injection counter stained with hematoxylin . Scale bars , 200 μm . Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for > 1000 cells counted on 3 glands . G Representative micrographs of PR - IHC on glands from 5 - month - old MM134 xenografts from hosts treated for 2 months with vehicle ( left ) or E2 ( right ) pellets . Scale bars , 200 μm . Right , bar plot showing percentage of PR + cells in MM134 and SUM44 xenografts with n > 1000 cells per condition on glands from 3 different mice . Data represent mean ± SEM unpaired , Student ' s t - test , two - tailed ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Figure 2E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001.


Figure 4D Representative RNAscope image of sections from patient ILCs ( n = 5 ) stained with anti - LOXL1 probes ( red ) and counterstained with DAPI ( blue ) . Scale bars ; 50 μm . E Representative immunofluorescence image of primary ILC ( n = 3 ) stained with anti - LOXL1 ( green ) and anti - ER ( red ) antibodies counterstained with DAPI ( blue ) . Scale bars ; 50 μm and right inset 30 μm .

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Figure 4D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1 probes (red) and counterstained with DAPI (blue). Scale bars; 50 μm.E Representative immunofluorescence image of primary ILC (n=3) stained with anti-LOXL1 (green) and anti-ER (red) antibodies counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.


Figure 5E Radiance - based tumor growth curves ( mean ± SE ) for T47D treated with vehicle ( n = 8 ) or BAPN ( n = 12 ) 8 days after intraductal injections . n . s not significant . F Radiance - based tumor growth curves for ( mean ± SE ) ILC ER + PDXs T125 and T137 treated with vehicle ( n = 6 , 7 ) or BAPN ( n = 7 , 8 ) 66 days after engraftment . * * p < 0 . 01 . G Radiance - based tumor growth curves ( mean ± SE ) for non - ILC ER + PDXs T99 and T157 treated with vehicle ( n = 7 , 7 ) or BAPN ( n = 8 , 12 ) , 8 days after intraductal injection 3 weeks after intraductal injections . n . s not significant .

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Figure 5E Radiance-based tumor growth curves (mean ± SE) for T47D treated with vehicle (n=8) or BAPN (n=12) 8 days after intraductal injections. n.s not significant.F Radiance-based tumor growth curves for (mean ± SE) ILC ER+ PDXs T125 and T137 treated with vehicle (n=6, 7) or BAPN (n=7, 8) 66 days after engraftment. ** p < 0.01.G Radiance-based tumor growth curves (mean ± SE) for non-ILC ER+ PDXs T99 and T157 treated with vehicle (n=7, 7) or BAPN (n=8, 12), 8 days after intraductal injection 3 weeks after intraductal injections. n.s not significant.


Figure 6E Representative fluorescent stereo - micrographs of glands injected with MM134 : RFP - luc2 cells ( left , top and bottom ) infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 ( bottom ) , n = 5 and 3 . Scale bar , 1000 μm . F Barplot showing the ratio of GFP / RFP signal from fluorescent stereo - micrographs of glands engrafted with MM134 cells , n = 8 . Statistical analysis by paired Student t - test , two - tailed . * p < 0 . 05 . H Representative fluorescent stereo - micrographs of glands engrafted with SUM44 : RFPluc2 cells infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 , n = 6 . Scale bar , 1000 μm . I Barplot ( mean ± SEM ) showing quantification of the ratio GFP / RFP from fluorescent stereo - micrographs of SUM44 . Statistical analysis by unpaired Student ' s t - test , two - tailed , n = 7 sh - scramble and n = 6 for sh - LOXL1 , * * * * p < 0 . 0001 . J , K Representative photomicrographs of picrosirius red stained histological sections for sh - scramble or sh - LOXL1 MM134 ( J ) and SUM44 ( K ) xenografts . Scale bars , 100 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 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 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

Figure 6E Representative fluorescent stereo-micrographs of glands injected with MM134:RFP-luc2 cells (left, top and bottom) infected with either GFP:sh-scramble (top) or GFP+ shLOXL1 (bottom), n=5 and 3. Scale bar, 1000 μm. F Barplot showing the ratio of GFP/RFP signal from fluorescent stereo-micrographs of glands engrafted with MM134 cells, n=8. Statistical analysis by paired Student t-test, two-tailed. *p < 0.05. H Representative fluorescent stereo-micrographs of glands engrafted with SUM44:RFPluc2 cells infected with either GFP:sh-scramble (top) or GFP+ shLOXL1, n=6. Scale bar, 1000 μm. I Barplot (mean ± SEM) showing quantification of the ratio GFP/RFP from fluorescent stereo-micrographs of SUM44. Statistical analysis by unpaired Student's t-test, two-tailed, n=7 sh-scramble and n=6 for sh-LOXL1, ****p < 0.0001. J, K Representative photomicrographs of picrosirius red stained histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.