( A ) Wild ‐ type and rho0 cells harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to amino ‐ acid or nitrogen starvation ( ‐ N ) medium supplemented with indicated carbon sources . Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α ‐ GFP and α ‐ Cdc11 antibodies . Quantification of ATG8 induction is shown in the lower left panel . Total GFP signals ( GFP ‐ Atg8 and free GFP ) were quantified and normalized to Cdc11 signals . Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation . Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals ( GFP ‐ Atg8 and free GFP ) after 6 h starvation . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

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

(A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Fluorescence microscopical analysis . Wild ‐ type , rho0 , and Δatg7 cells expressing prATG8 ‐ GFP ‐ ATG8 were grown as described in ( A ) and exposed to amino ‐ acid ( left panel , galactose ) or nitrogen starvation ( right panel , glucose ( ‐ N ) ) for 6 h . Vacuoles were visualized by over night FM4 ‐ 64 ( 1 μM ) staining ( red ) . Arrowhead indicates a punctate GFP ‐ Atg8 structure . Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries . Cellular localization of GFP signal was analysed in at least 150 cells ( n ⩾ 150 ) for each strain and condition . Scale bars represent 1 . 5 μm .

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

(B) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.


Autophagic response in cells with compromised respiratory chain complex III , IV , or V activity during amino ‐ acid starvation . Wild ‐ type , rho0 , atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III ( CIII : cbs1 ) , complex IV ( CIV : mss51 , pet111 , pet122 ) , or the F1Fo ‐ ATP synthase ( complex V ; CV : atp10 ) were exposed to amino ‐ acidstarvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type cells were exposed to antimycin A ( AA ) or oligomycin ( O ) during the amino ‐ acid starvation period . All strains expressed prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) . Samples were analysed as described in Figure 1A . The means and s . d . of five ( n = 5 ) independent experiments are indicated .

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

Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.


( A , B ) Wild ‐ type , rho0 , and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type cells were exposed to antimycin A ( AA ) or oligomycin ( O ) during the amino ‐ acid starvation period . ATP and protein from total cells ( upper panels ) or isolated mitochondria ( lower panels ) were determined at indicated time points .

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

(A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points.


( C ) Wild ‐ type and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with acetate or galactose for 3 h . Wild ‐ type cells were exposed to antimycin A ( AA ) , oligomycin ( O ) , or CCCP ( 50 μM ) during the amino ‐ acid starvation period when indicated . Cells were treated with the mitochondrial membrane potential ‐ dependent dye DiOC6 ( 3 ) and examined by fluorescence microscopy . Average pixel intensities for at least 10 mitochondrial tubules ( n = 10 ) in 5 representative cells were determined for each condition . Values for wild ‐ type mitochondria in the presence of acetate were defined as 100 % and relative values were calculated accordingly . The means and s . d . are shown .

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

(C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.


( A ) Wild ‐ type , rho0 , Δnpr2 , and Δnpr2 rho0 cells expressing prATG8 ‐ GFP ‐ ATG8 ( upper panel ) or prATG8 ‐ GFP ( lower panel ) were exposed to amino ‐ acid starvation medium supplemented with galactose in the absence or presence of rapamycin . Samples were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

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

(A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Wild ‐ type , rho0 , Δnpr2 , and Δatg7 cells expressing prNPR1 ‐ NPR1 ‐ HA were exposed to amino ‐ acid starvation medium supplemented with galactose in the absence ( upper panels ) or presence ( lower panels ) of rapamycin . The hyperphosphorylated ( Npr1 ‐ P ) and dephosphorylated ( Npr1 ) forms of Npr1 are indicated . Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α ‐ HA and α ‐ Cdc11 antibodies .

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

(B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies.


( C ) Wild ‐ type , rho0 , Δnpr2 , and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with galactose . Additionally , wild ‐ type cells were treated with rapamycin during starvation ( left panel ) . Phosphorylated ( Atg13 ‐ P ) and dephosphorylated ( Atg13 ) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α ‐ Atg13 and α ‐ Cdc11 antibodies .

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

(C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.


( A ) PKA ‐ dependent regulation of the autophagic response under amino ‐ acid starvation . Wild ‐ type , rho0 , pka , and ras2G19V ‐ expressing cells harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to amino ‐ acid starvation medium supplemented with galactose . PKA activity in pka was inhibited by addition of 1NM ‐ PP1 ( PP1 ; 1 μg / ml ) . Samples were analysed as described in Figure 1A ; autophagic flux was determined after 3 and 6 h .

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

(A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h.


( B ) In vivo activity of PKA . Wild ‐ type , rho0 , pka , and ras2G19V ‐ expressing cells harbouring 6xMYC ‐ cki12 − 200 ( S125 / 130A ) ( Cki1 ) were grown in galactose medium . When indicated , wild ‐ type cells were grown in galactose medium in the presence of antimycin A ( AA ) or oligomycin ( O ) for 6 h . PKA ‐ dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α ‐ Myc antibody ( upper panels ) . Ratio of phosphorylated ( Cki1 ‐ P ) and non ‐ phosphorylated ( Cki1 ) forms of Cki1 relative to wild ‐ type cells ( wt = 1 ) ( lower panels ) .

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

(B)In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels).


( C ) PKA inhibition restores autophagic flux , but not ATG8 induction in the presence of mitochondrial dysfunction . Wild ‐ type , rho0 , pka , and pka rho0 cells expressing prATG8 ‐ GFP ‐ ATG8 were treated as described in ( A ) . Samples were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated in ( A - C ) .

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

(C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild‐type, rho0, pka, and pka rho0 cells expressing prATG8‐GFP‐ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (A-C).


( A ) ATG8 induction is independent of the Atg1 - Atg13 complex and autophagic flux . Wild ‐ type , rho0 , Δatg1 , Δatg7 , Δatg9 , and Δatg11 cells harbouring prATG8 ‐ GFP ‐ ATG8 were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

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

(A)ATG8 induction is independent of the Atg1-Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function . Wild ‐ type and rho0 cells harbouring prATG8 ‐ GFP ‐ ATG8 and prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed to amino ‐ acid starvation medium supplemented with galactose for 3 h . Wild ‐ type cells were treated with antimycin A ( AA 30 ′ ) after 2 . 5 h of starvation for 30 min or with oligomycin ( O ) for 3 h of starvation . Arrowheads indicate the position of GFP ‐ Atg8 puncta . Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries . Scale bar represents 1 . 5 μm .

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

(B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm.


( C ) Steady ‐ state levels of Atg1 ‐ and Atg13 ‐ mCherry during amino ‐ acid starvation . Wild ‐ type , rho0 , and Δatg7 cells expressing prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed and treated as described in ( B ) and analysed by whole cell extraction and western blot analysis using α ‐ dsRed and α ‐ Cdc11 antibodies .

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

(C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.


C , Growth curves for E . coli 113 / 3 in defined media and in the presence or absence of B12 ( and o , r espectively ) . When challenged with 40 nM isolated R - domain ( ) , cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A ( ▫ ) a pproached that of the no colicin control , consistent with OmpF threading causing the disengagement of ColE9 from BtuB . Error bars represent the standard deviation across three biological replicates .

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

C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, respectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) approached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.


ColE9 * denotes ColE9 H551A , an active site mutation that inactivates the toxin . A , Widefield fluorescence microscopy images of E . coli JM83 cells labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) and Hoechst DNA stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment with or without prior treatment with CCCP . Each panel shows the same cell in DIC ( grey ) , Hoechst DNA stain ( blue ) and ColE9 * K469CAF647 fluorescence ( red ) . Overlays of Hoechst and ColE9 * K469CAF647 fluorescence are also shown . ColE9 * K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin . Trypsin treatment in the absence of CCCP yields some cell - associated ColE9 * K469CAF647 fluorescence that likely represents internalised molecules ( see text ) . Scale bar , 1 µm .

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

ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.


C , Box and whisker plots showing mean pixel intensities for ColE9 * K469CAF647 fluorescence per cell measured for the indicated cells and condition used , whiskers represent minimum and maximum mean pixel intensity , box shows 1st and 3rd quartile with the median shown as a line . From left - to - right : E . coli JM83 cells ; E . coli btuB deletion strain showing loss of all ColE9 * K469CAF647 cell - associated fluorescence ; E . coli JM83 cells in the presence of CCCP ; E . coli JM83 cells following trypsin treatment showing significant ColE9 * K469CAF647 fluorescence remains associated with cells indicative of import ; E . coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9 * K469CAF647 fluorescence . n , number of cells used , typically from 2 - 4 biological replicates . * * * * , indicates a t value < 0 . 05 in a student T - test as a statistically significant result , ns indicates no significant difference as determined by student T - test .

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C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.


B , Overnight plate assays comparing the cytotoxic activity of wild - type ColE9 with ColE9 K469C labelled with AF488 , AF568 or AF647 against a lawn of E . coli with either OmpF ( JW2203 ) or OmpC ( JW0912 ) in the outer membrane ( shown as + OmpF and + OmpC , respectively ) . Each plate was spotted with a serial dilution of the colicin ( 10 µM - 2 . 4 pM , 4 - fold dilution per spot ) . Wild - type ColE9 was more active against E . coli cells with OmpF in the outer membrane than OmpC . Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC .

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B, Overnight plate assays comparing the cytotoxic activity of wild-type ColE9 with ColE9 K469C labelled with AF488, AF568 or AF647 against a lawn of E. coli with either OmpF (JW2203) or OmpC (JW0912) in the outer membrane (shown as +OmpF and +OmpC, respectively). Each plate was spotted with a serial dilution of the colicin (10 µM - 2.4 pM, 4-fold dilution per spot). Wild-type ColE9 was more active against E. coli cells with OmpF in the outer membrane than OmpC. Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC.


C , Widefield fluorescence microscopy images of E . coli JW2203 ( + OmpF ) and JW0912 ( + OmpC ) cells labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) and Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment . Each panel shows the same cell in DIC ( grey ) , Hoechst DNA stain ( blue ) and fluorescence of ColE9 * K469CAF647 ( red ) . Overlays of Hoechst and ColE9 * K469CAF647 fluorescence are also shown . Data show that following trypsin - treatment , significant ColE9 * K469CAF647 fluorescence remains associated with ompF - expressing E . coli whereas little or no fluorescence remains associated with ompC - expressing E . coli . Scale bar , 1 µm . D , Box and whisker plots of E . coli JW2203 and E . coli JW0912 cells labelled with ColE9 * K469CAF647 with and without trypsin treatment : whiskers represent minimum and maximum mean pixel intensity , box shows 1st and 3rd quartile with the median shown as a line . Microscopy data were collected as in c . The mean pixel intensity of ColE9 * K469CAF647 per cell was measured for each cell condition . n , number of cells , typically from 3 or 4 biological replicates . Data show that ColE9 * K469CAF647 translocates across the OM through OmpF and that this is impeded when E . coli has OmpC in the outer membrane .

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C, Widefield fluorescence microscopy images of E. coli JW2203 (+OmpF) and JW0912 (+OmpC) cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst stain (20 µM) for 30 min at 37 °C with or without trypsin treatment. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and fluorescence of ColE9* K469CAF647 (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. Data show that following trypsin-treatment, significant ColE9* K469CAF647 fluorescence remains associated with ompF-expressing E. coli whereas little or no fluorescence remains associated with ompC-expressing E. coli. Scale bar, 1 µm. D, Box and whisker plots of E. coli JW2203 and E. coli JW0912 cells labelled with ColE9* K469CAF647 with and without trypsin treatment: whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. Microscopy data were collected as in c. The mean pixel intensity of ColE9* K469CAF647 per cell was measured for each cell condition. n, number of cells, typically from 3 or 4 biological replicates. Data show that ColE9* K469CAF647 translocates across the OM through OmpF and that this is impeded when E. coli has OmpC in the outer membrane.


C . TIRF microscopy revealed time courses of the CAR - GFP microcluster formation as CAR T cells spread on the CD19 and ICAM - 1 - coated SLBs .

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C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs.


D . Enrichment of phosphor - tyrosine ( pY ) at CAR microclusters . CAR T cells were fixed 20 min after being plated on the SLB , followed by staining with an anti - pan phosphor - tyrosine antibody . No CD19 was included in the control experiment ( only ICAM - 1 ) . Scale bar : 2 μm .

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D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.


T cells expressing GFP - CAR were plated on CD19 and ICAM - 1 coated SLBs . TIRF microscopy was implemented to visualize CAR localization . A . CAR microclusters underwent centripetal movement to form cSMAC ( upper panel ) or remained separated ( lower panel ) . B . Quantification of the categories regarding the formation of CAR cSMAC . Unstable cSMACs are defined by a cSMAC duration below 12 min . Data are shown as mean ± SD . n = 3 independent experiments . 37 , 38 , and 47 cells were quantified in individual experiments . C . Quantification of time from cell landing to the start of retrograde flow . Data are shown as mean ± SD . n = 33 ( stable ) , 31 ( unstable ) , 10 ( kinapse ) , and 41 ( no cSMAC ) cells . Results are pooled from three independent experiments .

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T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization. A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel). B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments. C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments.


D . Localization of CAR and CD45 in the immunological synapse . CAR T cells were fixed 20 min after being plated on the SLB , followed by staining with an anti - CD45 antibody . E . Line scan of CAR and CD45 across the long axis of the immunological synapse . Shown are the normalized fluorescence intensity .

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D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody. E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.


A . WT or LAT - deficient Jurkat cells ( Jcam2 . 5 ) expressing CAR - GFP were stimulated on SLB - coated with OKT3 ( anti - TCR antibody ) or CD19 ( CAR antigen ) ; in both conditions , the lipid bilayers were also coated with ICAM - 1 to facilitate cell attachment . TIRF microscopy revealed clustering of Ax647 - labeled streptavidin - Biotin - OKT3 or CD19 , which serves as a probe for TCR or CAR respectively . Scale bar , 2 µm . B . Quantification of clustering as normalized variance . n = 100 cells . Shown are the mean ± SD . Statistical test : unpaired two - tailed t - test . * * * * p < 0 . 0001 . * * 0 . 005 < p < 0 . 05 .

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A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.


B . WT or LAT - deficient ( Jcam2 . 5 ) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM - 1 . TIRF microscopy revealed clustering of CAR and Ax647 - labeled streptavidin - Biotin - CD19 . Scale bar , 2 µm . C . Quantification of clustering level of Ax647 - labeled streptavidin - Biotin - CD19 . Shown are means ± SD . Clustering is quantified as normalized variance . N = 50 cells for CAR 3rd WT and 1st WT , 63 cells for CAR 3rd lat - , and 51 cells for CAR 1st lat - . * * : 0 . 0005 < p < 0 . 005 . Statistical test : unpaired two - tailed t - test .

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B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm. C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . A . Gads was recruited to CAR microclusters in both the wild - type and LAT - deficient ( Jcam2 . 5 ) cells . n = 33 or 34 cells . Shown are the means ± SE . n . s . , p = 0 . 58 by unpaired two - tailed t - test . As a control , Gads is not recruited to LAT - deficient cells activated by OKT3 .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . B . Phosphorylated SLP76 , recognized by an anti - SLP76 pY145 antibody , was enriched in CAR microclusters in both the wild - type and LAT - deficient cells . In contrast , phosphorylated SLP76 was not enriched in TCR microclusters in LAT - deficient cells . n = 50 cells . Shown are the means ± SE . n . s . , p = 0 . 95 by unpaired two - tailed t - test .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . C . Actin remodeling was induced in LAT - deficient cells by CD19 - , but not OKT3 - coated SLB . No ICAM - 1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA - 1 / ICAM - 1 pathway . Actin network was revealed by F - tractin - mScarlet . n = 6 cells . Shown are the means ± SD . n . s . , p = 0 . 68 , and 0 . 98 by unpaired two - tailed t - test .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.


A . Cell - cell conjugation assay . Wild - type or Lat - null ( J . LAT ) Jurkat T cells expressing CAR - GFP were mixed with Raji cells expressing mCherry - CAAX at a 1 : 1 ratio for 1 hr before being fixed . Three independent experiments were performed and over 90 cells were scored in each experiment . Scale bar : 5 µm .

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A. Cell-cell conjugation assay. Wild-type or Lat-null (J.LAT) Jurkat T cells expressing CAR-GFP were mixed with Raji cells expressing mCherry-CAAX at a 1:1 ratio for 1 hr before being fixed. Three independent experiments were performed and over 90 cells were scored in each experiment. Scale bar: 5 µm.


B . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio for 24 hrs and IL - 2 from the supernatant were measured by ELISA . C . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio and fixed at 24hr , stained with an anti - CD69 antibody , and analyzed by FACS . CD69 positive cells were scored within the CAR - GFP + population .

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B. CAR T cells were incubated with Raji B cells at a 1:1 ratio for 24 hrs and IL-2 from the supernatant were measured by ELISA. C. CAR T cells were incubated with Raji B cells at a 1:1 ratio and fixed at 24hr, stained with an anti-CD69 antibody, and analyzed by FACS. CD69 positive cells were scored within the CAR-GFP+ population.


A . Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR . Shown are the means ± SD . n = 4 independent experiments .

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A. Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR. Shown are the means ± SD. n = 4 independent experiments.


B . CAR T cells were stimulated on SLBs coated with biotin - OKT3 ( left ) or biotin - CD19 ( right ) supplemented with ICAM - 1 . TIRF microscopy revealed clustering of Alexa647 - labeled streptavidin - Biotin - OKT3 or CD19 . Scale bar , 5 µm . C . Quantification of clustering of OKT3 or CD19 as normalized variance . n > 200 cells from two independent donors were scored for each condition . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 48 .

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B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm. C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48.


D . Immunofluorescence analysis revealed membrane - localized phosphorylated SLP76 . Cells were plated on supported lipid bilayer coated with ICAM - 1 only ( control ) or ICAM - 1 supplemented with CD19 or OKT3 as indicated . The white dashed circle indicated cell boundary . Scale bar , 5 µm . E . Quantification of pSLP76 level on the membrane . n > 200 cells were scored , and the cells were isolated from two independent donors . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 11 .

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

D. Immunofluorescence analysis revealed membrane-localized phosphorylated SLP76. Cells were plated on supported lipid bilayer coated with ICAM-1 only (control) or ICAM-1 supplemented with CD19 or OKT3 as indicated. The white dashed circle indicated cell boundary. Scale bar, 5 µm. E. Quantification of pSLP76 level on the membrane. n > 200 cells were scored, and the cells were isolated from two independent donors. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.11.


F . T cells were incubated with Raji B cells at a 4 : 1 ratio and IL - 2 production was measured by ELISA at 24 hr . Data were pooled from three donors . * , 0 . 005 < p < 0 . 05 ; * * , 0 . 0005 < p < 0 . 005 . Unpaired two - tailed t - test was applied .

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F. T cells were incubated with Raji B cells at a 4:1 ratio and IL-2 production was measured by ELISA at 24 hr. Data were pooled from three donors. *, 0.005 < p < 0.05; **, 0.0005 < p < 0.005. Unpaired two-tailed t-test was applied.


Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of Ca2 + monitored at 280 nm absorption wavelength . Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength .

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Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.


MICU2 _ WT ( A ) , MICU2 _ K172A ( B ) were pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + . MICU2 _ EF1mut was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + ( C ) , while MICU2 _ EF2mut was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( D ) . MICU2 _ R352A was not pulled - down by GST - MICU1 in the absence of 2 mM Ca2 + ( E ) , while MICU2 _ E329A was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( F ) . MICU2 _ D185A was partially pulled - down by GST - MICU1 in the presence of Ca2 + , while they showed a strong interaction in the absence of 2 mM Ca2 + . MICU2 _ E196K was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + , while a strong interaction was observed in the absence of 2 mM Ca2 + .

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MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.


GST - MICU1 _ EF1mut failed to pull down MICU2 _ WT in the absence of 2 mM Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A & H385A failed to pull - down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . C GST - MICU1 _ E242K failed to pull - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ D231A partly pulled - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A failed to pull down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . GST - MICU1 _ H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2 + .

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GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.


ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA ( G ) and CaCl2 ( H ) .

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ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).


A . Chemical structure of Nec - 1 . The schematic diagram was drawn by S . Lee using ChemDraw Professional 15 . 0 . software .

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A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.


B , C . MTT cytotoxicity assay . ( B ) HT22 cell line was treated with various concentrations of Nec - 1 ( 0 - 200 μM ) . Aggregated Aβ ( 1 - 42 ) was used as the positive control for the induction of neural cell death . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


B , C . MTT cytotoxicity assay . ( C ) HT22 , BV2cell lines and primary astrocytes were pre - treated with Nec - 1 ( 50 μM ) 15 minutes prior to application of 10 μM of pre - aggregated Aβ ( 1 - 42 ) for indicated times . Cell proliferation was then measured by MTT assay . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


D , E . Neural cell death levels measured by staining with the Live / Dead Viability / Cytotoxicity Assay Kit ( Molecular Probes ) . Pre - treatment of cells with 50 μM Nec - 1 preceded the application of 10 μM pre - aggregated Aβ ( 1 - 42 ) by 15 minutes . The assay was performed 24 hours after Aβ ( 1 - 42 ) application . ( D ) Representation of the stained cells . Scale bar = 500 μm ( E ) Quantification of dead cells stained by EthD - 1 .

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

D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.


F . Internalization of Aβ ( 1 - 42 ) into HT22 and BV2 cell lines in the presence or the absence of Nec - 1 pre - treatment for indicated periods .

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F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.


G . Cellular expression levels of indicated proteins in HT22 , BV2 cell lines , and primary astrocyte stimulated with Aβ ( 1 - 42 ) aggregates in the presence or the absence of Nec - 1 pre - treatment for 24 hours .

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G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.


A - E . Behavioural tests of APP / PS1mice . ( B - D ) Y - maze and passive avoidance tests on 7 - month - old APP / PS1mice after Nec - 1 administration for 12 weeks . Average alternation ( % ) for each test group ( B ) and total entry number into each arm ( C ) on Y - maze . ( D ) Average latency time in seconds for each test group on passive avoidance test .

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A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.


A - E . Behavioural tests of APP / PS1mice . ( E ) Survival of Wt and APP / PS1mice after injection of Nec - 1 ( 6 . 25 mg / kg ) .

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A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).


F - I . Behavrioural tests of Aβ ( 1 - 42 ) infusion mice . ( H , I ) Y - maze tests on Aβ ( 1 - 42 ) infusion mice model injected with Nec - 1 . Average alternation ( % ) for each test group ( H ) and total entry number into each arm ( I ) on Y - maze .

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F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.


A . Co - immunoprecipitation of RIPK3 with Aβ ( 1 - 42 ) in HT22 , BV2 cell lines , and primary astrocytes in response to Aβ ( 1 - 42 ) aggregates . IP , immunoprecipitation ; IB , immunoblot .

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A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.


B . A close - up view of the interactions between Nec - 1 and surrounding residues in RIPK1 . Nec - 1 is shown in green . The hydrophobic residues around Nec - 1 in RIPK1 kinase domain are shown in sticks . Hydrogen bonds in this and all other figures are represented by yellow dashed lines . Docking score of Nec - 1 in RIPK1 was - 9 . 550 .

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B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.


C . Surface treated view of 12 - mer Aβ fibrils ( 2LMO , VDW radius = 0 . 55 Å ) having hydrophobic pockets plausible for Nec - 1 binding .

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C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.


D . Docked structure of 12 - mer Aβ fibrils ( 2LMO ) with 4 molecules of Nec - 1 . Docking scores for four top - ranked Glide docking poses of Nec - 1 in Aβ plaques ; - 8 . 338 , - 4 . 755 , - 8 . 493 , and - 6 . 754 in clockwise order starting from the top - left corner .

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D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.


E . Surface plasmon resonance sensorgrams of Nec - 1 targeting Aβ ( 1 - 42 ) aggregates ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


4 - month - old APP / PS1 ( n = 13 , male ) and age - matched wild - type ( Wt , n = 14 , male ) mice were intravenously injected with Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for 12 weeks . After the completion of behavioural tests , the brains of each group of mice were subjected to analysis . A . ThS - stained insoluble Aβplaques in whole brain and the hippocampal region of each group . Scale bars = 1 mM ( upper ) , 200 μM ( lower ) . B - G . Statistics of ThS - positive Aβplaques in the brains of APP / PS1mice . Total numbers and areas of ThS - positive Aβplaques in the whole brains ( B and C ) , cortex ( D and E ) and hippocampus ( F and G ) .

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4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).


A . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . Diffuse plaques in the brain sections were stained by anti - Aβ antibody ( 6E10 clone , green colour ) and anti - GFAP antibody ( red colour ) . Hoechst 33342 ( blue colour ) was applied for nuclear counterstaining . Scale bars = 200 μM .

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

A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.


B - D . Dot blot analysis of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) and protein oligomer ( anti - amyloidogenic protein oligomer A11 ) in the cortical ( B ) and hippocampal ( C ) region of indicated groups . ( D ) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups . Relative intensity of each band was determined by densitometry of dot blots ( B and C ) using ImageJ software and normalized to vehicle - administered APP / PS1mouse group .

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

B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.


E . Sandwich ELISA of Aβ - soluble and - insoluble fractions from cortical and hippocampal lysates of APP / PS1mice .

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E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice.


F . Western blot analyses of cortical and hippocampal lysates from APP / PS1mice for APP and sAPP expressions .

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F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions.


G . ThT assays for the inhibition of Aβ aggregation . Fluorescence intensity was normalized to Aβaggregates ( 100 % , day 5 ) .

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G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5).


H . SDS - PAGE analysis of PICUP cross - linked Aβaggregates visualized by silver staining .

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H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.


A . Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . The membranes analysed in ( A ) are identical to those in Fig 5F .

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

A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.


B . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for phosphorylated tau expression levels . Anti - tau ( phospho Ser199 ) antibody ( red colour ) was applied for staining tau phosphorylation on Serine - 199 and Hoechst 33342 ( blue colour ) for nuclear counterstaining in the brain sections . Scale bars = 200 μM .

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

B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.


C . Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP / PS1mice injected with Nec - 1 ( upper ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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

C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


C . Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec - 1 ( lower ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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

C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


D , E . ThT assays for the inhibition of tau aggregation ( D ) and for disaggregation of tau ( E ) . Fluorescence intensity was normalized to tauaggregates ( 100 % , day 5 ) .

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

D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).


F . Surface plasmon resonance sensorgrams of Nec - 1 targeting tau ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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

F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h and stained as in ( A ) . aspect ratio ( O ) , branch length ( P ) , and roundness ( Q ) of mitochondria in the cells in ( N ) .

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

MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


TMRE ( 100 nM ) and MitoTracker green ( 200 nM ) staining in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE intensity in ( A ) on a per cell basis . MitoSOX ( 5 μM ) and MitoTracker Green ( 200 nM ) staining in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX intensity for ( C ) on a per cell basis .

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

TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed and imaged in pairs ( SD and HFD ) .

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

NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).


Citrate synthase staining in the arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 .

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

Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.


imaging was performed in the areas of the cortex


NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from the mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H intensity from ( B ) calculated on a per cell basis ; 16 - 25 cells from each of the 5 mice per group were quantified ( 87 - 109 total cells per treatment group ) .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).


TMRE ( 100 nM ) and MitoTracker Green staining ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) intensity for ( E ) calculated on a per cell basis ; 15 - 24 cells from each of the 5 mice in each group were quantified ( 99 - 104 total cells per treatment group ) .

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TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).


MitoSOX ( 5 μM ) and MitoTracker Green staining in eWAT resected from the mice in ( D ) treated as in ( B and E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) intensity calculated as in ( F and G ) ; 10 - 13 cells from each of the 5 mice in each group were quantified ( 57 - 58 total cells per treatment group ) .

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

MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).


Chop , Xbp1s , Atf6 , and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , and Friday for the final 3 weeks ; n = 7 - 9 .

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Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 weeks then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with the amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

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

Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Average daily distance run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , and Fridays over 4 weeks when housed with a running wheel .

O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.


Body weight change in the mice in ( O , P ) over the 24 h after the first treatment ( Q ) or after the 3 week course of treatment ( R ) ; n = 9 .

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Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Representative H & E stained liver sections from the mice shown in Fig . 6F after 4 weeks of treatment with vehicle or 120 mg / kg SH - BC - 893 . Scale bar , 10 μm .

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Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.


Blood glucose levels ( F ) or area under the curve ( AUC , ( G ) ) during an oral glucose tolerance test performed in the same mice as in ( E ) .

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

Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).


Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH - BC - 893 from the mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 weeks on HFD ) ; n = 8 . Insulin levels in the plasma collected 4 h after treatment with vehicle or SH - BC - 893 from the same mice as in Fig . 5A , B ; n = 8 .

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Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


C TIMM50S levels in primary and immortalized skin fibroblast from control ( C1 - 4 ) and mutant TIMM50 ( Pt ) . SDHA and ETHE1 are shown as loading controls .

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C TIMM50S levels in primary and immortalized skin fibroblast from control (C1-4) and mutant TIMM50 (Pt). SDHA and ETHE1 are shown as loading controls.


D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts . Data are shown as mean ± SD , n = 4 .

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D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts. Data are shown as mean ± SD, n = 4.


A Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium .

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

A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.


B Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium .

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B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.


C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC - 1 staining in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


D TMRM live staining of control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium . Scale bar corresponds to 10 μm .

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

D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm.


A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP . The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1 . Coomassie Blue ( CB ) staining was used as loading control and the radiolabeled protein ( TNT ) as reference for quantification . The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . Western blot for TIMM50 is shown as cell line control . B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 .

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

C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05.


Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Two biological replicates are shown for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red .

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

Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red.


B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Two biological replicates were obtained for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red .

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

B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red.


Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.