E Western blot analysis indicates that GCα - 3Ty is present in three distinct isoforms . A PKG - 3Ty western blot also shows three immune reactive isoforms . Catalase or actin were used as loading controls and ΔKu80 as the parental strain .

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

E Western blot analysis indicates that GCα-3Ty is present in three distinct isoforms. A PKG-3Ty western blot also shows three immune reactive isoforms. Catalase or actin were used as loading controls and ΔKu80 as the parental strain.


F PKG - 3Ty parasites were solubilised in either PBS or 1 % Triton X - 100 ( TX100 ) and split into soluble ( S ) and pellet ( P ) fractions . Total lysate is also shown .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.


G , H The signal of Myc - ACβ - iKD followed by IFA ( G ) after 48 hours ± ATc . Data information : Scale bars = 2 μm .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

G, H The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm.


G , H The signal of Myc - ACβ - iKD followed by western blot ( H ) after 48 hours ± ATc .

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

G, H The signal of Myc-ACβ-iKD followed by western blot (H) after 48 hours ± ATc.


I Invasion of ACα - KO tachyzoites or Myc - Acβ - iKD after 48 hours ± ATc is not affected . However , addition of ATc for 48 hours reduces the invasion efficiency of ACα - KO / Myc - Acβ - iKD ( data is from three independent biological replicates ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates).


J As for destabilisation of PKAc1 - iKD or stabilisation of DDmyc - PKArG321E - Ty , ATc - KO / Myc - Acβ - iKD tachyzoites treated with lytic cycle for 33 or 40 hours , exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non - treated parasites invade and initiate a new lytic cycle . Data information : Scale bars = 2 μm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ATc -KO/ Myc-Acβ-iKD tachyzoites treated with lytic cycle for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm.


B - E ( B , D ) Representative microscopy images of the mitochondrial network morphology in neurons of the amygdala ( B ) and the CA1 region of the hippocampus ( D ) in mitoCFP + / P301L - mice and mitoCFP + / P301L + mice . MitoCFP signal is displayed in gray on the images . Gray scale bars : 5 µm . ( C , E ) Mitochondrial length in neurons of the amygdala ( C ) and in Ca1 ( E ) of mitoCFP + / P301L - and mitoCFP + / P301L + mice . Data information : Values represent the mean ± SEM of n = 11 MitoCFP + / P301L - mice and n = 13 MitoCFP + / P301L + mice . Each gray open circle represents the mean of 6 technical replicates ( data from 6 images ) per animal . * P < 0 . 05 , * * P < 0 . 01 , Student unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E (B, D) Representative microscopy images of the mitochondrial network morphology in neurons of the amygdala (B) and the CA1 region of the hippocampus (D) in mitoCFP+/P301L- mice and mitoCFP+/P301L+ mice. MitoCFP signal is displayed in gray on the images. Gray scale bars: 5 µm. (C, E) Mitochondrial length in neurons of the amygdala (C) and in Ca1 (E) of mitoCFP+/P301L- and mitoCFP+/P301L+ mice. Data information: Values represent the mean ±SEM of n=11 MitoCFP+/P301L- mice and n= 13 MitoCFP+/P301L+ mice. Each gray open circle represents the mean of 6 technical replicates (data from 6 images) per animal. * P<0.05, ** P<0.01, Student unpaired t-test.


F - I ( F , H ) Representative microscopy images of the endoplasmic reticulum stained with calnexin ( in red ) and mitochondria ( mitoCFP ) in neurons of the amygdala ( F ) and Ca1 ( H ) in mitoCFP + / P301L - and mitoCFP + / P301L + mice . ( G , I ) Colocalization analysis between calnexin and mitoCFP in neurons of the amygdala ( G ) and Ca1 ( I ) of mitoCFP + / P301L - and mitoCFP + / P301L + mice . The Manders ' overlap coefficients M2 is represented on the graphs , as it indicates the proportion of overlaps between the mitoCFP signal ( channel 2 ) and calnexin signal ( channel 1 ) relative to the total pixel intensity of channel 2 ( i . e . only in neurons , as mitoCFP is expressed only in these cells ) . Data information : Values represent the mean ± SEM of n = 11 MitoCFP + / P301L - mice and n = 13 MitoCFP + / P301L + mice . Each gray open circle represents the mean of 6 technical replicates ( data from 6 images ) per animal . * P < 0 . 05 , * * P < 0 . 01 , Student unpaired t - test .

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

F-I (F, H) Representative microscopy images of the endoplasmic reticulum stained with calnexin (in red) and mitochondria (mitoCFP) in neurons of the amygdala (F) and Ca1 (H) in mitoCFP+/P301L- and mitoCFP+/P301L+ mice. (G, I) Colocalization analysis between calnexin and mitoCFP in neurons of the amygdala (G) and Ca1 (I) of mitoCFP+/P301L- and mitoCFP+/P301L+ mice. The Manders' overlap coefficients M2 is represented on the graphs, as it indicates the proportion of overlaps between the mitoCFP signal (channel 2) and calnexin signal (channel 1) relative to the total pixel intensity of channel 2 (i.e. only in neurons, as mitoCFP is expressed only in these cells). Data information: Values represent the mean ±SEM of n=11 MitoCFP+/P301L- mice and n= 13 MitoCFP+/P301L+ mice. Each gray open circle represents the mean of 6 technical replicates (data from 6 images) per animal. * P<0.05, ** P<0.01, Student unpaired t-test.


A Representative microscopy images ( z - projections ) of the mitochondrial network stained with TOMM20 in SH - SY5Y cells stably expressing a green fluorescent protein ( GFP ) - tagged wild - type form of tau protein ( wtTau - GFP ) , a GFP - tagged P301L mutant form of tau protein ( P301L - GFP ) or cell expression the GFP only ( control cells = WT - GFP ) . GFP signal is displayed in gray on the images , TOMM20 signal is in red , and the nuclei are delimited by white dotted lines . B - C Mitochondrial length ( B ) and percentage of mitochondria in the perinuclear area ( C ) in WT - GFP , wtTau - GFP and P301L - GFP expressing cells . On average 1 , 000 - 2 , 500 mitochondrial organelles were analyzed per group ( n = 20 - 25 images per group , 3 independent experiments ) . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm B - C * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . TOMM20 : translocase of the outer mitochondrial membrane complex subunit

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative microscopy images (z-projections) of the mitochondrial network stained with TOMM20 in SH-SY5Y cells stably expressing a green fluorescent protein (GFP)-tagged wild-type form of tau protein (wtTau-GFP), a GFP-tagged P301L mutant form of tau protein (P301L-GFP) or cell expression the GFP only (control cells = WT-GFP). GFP signal is displayed in gray on the images, TOMM20 signal is in red, and the nuclei are delimited by white dotted lines. B-C Mitochondrial length (B) and percentage of mitochondria in the perinuclear area (C) in WT-GFP, wtTau-GFP and P301L-GFP expressing cells. On average 1,000-2,500 mitochondrial organelles were analyzed per group (n = 20-25 images per group, 3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm B-C * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. TOMM20: translocase of the outer mitochondrial membrane complex subunit 20


Visualization of the IP3R - VDAC ( D ) , IP3R - GRP75 ( E ) interactions by in situ proximity ligation assay ( PLA ) WT - GFP , wtTau - GFP and P301L - GFP expressing cells . Interactions between the two targeted proteins are depicted in red ( 63X magnification ) and cell surfaces are delimited by a white line . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm GRP75 : glucose - regulated protein 75 IP3R : inositol trisphosphate receptor VDAC : voltage - dependent anion channel .

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

Visualization of the IP3R-VDAC (D), IP3R-GRP75 (E) interactions by in situ proximity ligation assay (PLA) WT-GFP, wtTau-GFP and P301L-GFP expressing cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. Data information: Data are presented as mean ±SEM. White scale bars: 20 µm GRP75: glucose-regulated protein 75 IP3R: inositol trisphosphate receptor VDAC: voltage-dependent anion channel.


G - I Quantitative analysis of the PLA signal represented as number of contact points between IP3R VDAC ( G ) , IP3R - GRP75 ( H ) and VAPB - PTPIP51 ( I ) per cell in percentage of the WT - GFP cells . In total , 60 - 75 cells were analyzed per group ( 5 independent experiments ) . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm G - I * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . GRP75 : glucose - regulated protein 75 PTPIP51 : protein tyrosine phosphatase - interacting protein 51 VDAC : voltage - dependent anion channel .

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

G-I Quantitative analysis of the PLA signal represented as number of contact points between IP3RVDAC (G), IP3R-GRP75 (H) and VAPB-PTPIP51 (I) per cell in percentage of the WT-GFP cells. In total, 60-75 cells were analyzed per group (5 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm G-I * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. GRP75: glucose-regulated protein 75 PTPIP51: protein tyrosine phosphatase-interacting protein 51 VDAC: voltage-dependent anion channel.


K - L Mitochondrial length ( K ) and percentage of mitochondria in the perinuclear area ( L ) in WT cells and Tau KO cells . On average 1 , 000 - 2 , 500 mitochondrial organelles were analyzed per group ( n = 20 - 25 images per group , 3 independent experiments ) . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm * P < 0 . 05 , * * * P < 0 . 001 ; Student unpaired t - test .

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

K-L Mitochondrial length (K) and percentage of mitochondria in the perinuclear area (L) in WT cells and Tau KO cells. On average 1,000-2,500 mitochondrial organelles were analyzed per group (n = 20-25 images per group, 3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test.


M - O Quantitative analysis of the PLA signal represented as number of contact points between IP3R - VDAC ( M ) , IP3R - GRP75 ( N ) and VAPB - PTPIP51 ( O ) per cell in percentage of the WT cells . In total , 60 - 75 cells were analyzed per group ( 3 independent experiments ) . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm * P < 0 . 05 , * * * P < 0 . 001 ; Student unpaired t - test . IP3R : inositol trisphosphate receptor ; PTPIP51 : protein tyrosine phosphatase - interacting protein 51 VAPB : vesicle - associated membrane protein - associated protein B ; VDAC : voltage - dependent anion channel .

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

M-O Quantitative analysis of the PLA signal represented as number of contact points between IP3R-VDAC (M), IP3R-GRP75 (N) and VAPB-PTPIP51 (O) per cell in percentage of the WT cells. In total, 60-75 cells were analyzed per group (3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test. IP3R: inositol trisphosphate receptor; PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B; VDAC: voltage-dependent anion channel.


P - R Visualization of the IP3R - VDAC ( P ) , IP3R - GRP75 ( Q ) and VAPB - PTPIP51 ( R ) interactions by in situ PLA in WT and Tau KO cells . Interactions between the two targeted proteins are depicted in red ( 63X magnification ) and cell surfaces are delimited by a white line . Data information : Data are presented as mean ± SEM . White scale bars : 20 µm * P < 0 . 05 , * * * P < 0 . 001 ; Student unpaired t - test . GRP75 : glucose - regulated protein 75 ; IP3R : inositol trisphosphate receptor ; PTPIP51 : protein tyrosine phosphatase - interacting protein 51 VAPB : vesicle - associated membrane protein - associated protein B VDAC : voltage - dependent anion channel .

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

P-R Visualization of the IP3R-VDAC (P), IP3R-GRP75 (Q) and VAPB-PTPIP51 (R) interactions by in situ PLA in WT and Tau KO cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test. GRP75: glucose-regulated protein 75; IP3R: inositol trisphosphate receptor; PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B VDAC: voltage-dependent anion channel.


G Quantification of total cholesterol levels in cell lysates from WT , P301L and Tau KO cells . Data information : ( G Data are presented as mean ± SEM of n = 12 - 18 replicates per group ( 4 independent experiments ) in percentage of the WT cells . * P < 0 . 05 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Quantification of total cholesterol levels in cell lysates from WT, P301L and Tau KO cells. Data information: (G Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test.


K - N Mitochondrial cholesterol levels ( K , L ) and pregnenolone levels ( M , N ) in WT , P301L and Tau KO cells ( K , M ) and in brain preparations from WT and pR5 mice ( L , N ) . Data information : K , M ) Data are presented as mean ± SEM of n = 12 - 18 replicates per group ( 4 independent experiments ) in percentage of the WT cells . * P < 0 . 05 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . L , N ) Data are presented as mean ± SEM of n = 6 WT and n = 5 pR5 mice with 2 technical replicates per animal . * P < 0 . 05 , * * P < 0 . 01 , Student unpaired t - test .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-N Mitochondrial cholesterol levels (K, L) and pregnenolone levels (M, N) in WT, P301L and Tau KO cells (K, M) and in brain preparations from WT and pR5 mice (L, N). Data information: K, M) Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test. L, N) Data are presented as mean ±SEM of n = 6 WT and n = 5 pR5 mice with 2 technical replicates per animal. * P<0.05, ** P<0.01, Student unpaired t-test.


O - Q Quantification of total cholesterol levels ( O ) , mitochondrial cholesterol levels ( P ) and pregnenolone levels ( Q ) in patient - derived induced pluripotent stem cells ( iPSC ) bearing a P301L tau mutation ( iPSC - P301L ) and the corresponding isogenic wild - type cells ( iPSC - WT ) . ( O - Q ) Data are presented as mean ± SEM of n = 27 replicates / group ( O ) , n = 9 replicates / group ( P ) and n = 18 replicates / group ( Q ) . * P < 0 . 05 , * * * P < 0 . 001 , Student unpaired t - test .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O-Q Quantification of total cholesterol levels (O), mitochondrial cholesterol levels (P) and pregnenolone levels (Q) in patient-derived induced pluripotent stem cells (iPSC) bearing a P301L tau mutation (iPSC-P301L) and the corresponding isogenic wild-type cells (iPSC-WT). (O-Q) Data are presented as mean ±SEM of n = 27 replicates/group (O), n = 9 replicates/group (P) and n = 18 replicates/group (Q). * P<0.05, *** P<0.001, Student unpaired t-test.


( A Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green and ( A ) TOMM20 ( mitochondria ) , in red with xy - and xz - axis orthogonal views framing the corresponding image . ( B - C Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( B ) TOMM20 M2 representing the proportion of ( C ) TOMM20 overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10 µm TOMM20 : translocase of the outer mitochondrial membrane complex subunit 20 .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green and (A) TOMM20 (mitochondria), in red with xy- and xz-axis orthogonal views framing the corresponding image. (B-C Manders' coefficients M1 representing the proportion of Filipin III overlapping with (B) TOMM20 M2 representing the proportion of (C) TOMM20 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm TOMM20: translocase of the outer mitochondrial membrane complex subunit 20.


D , Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green ( D ) ER - RFP in red with xy - and xz - axis orthogonal views framing the corresponding image . E - F Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( E ) ER - RFP M2 representing the proportion of ( F ) ER - RFP overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10 µm ER - RFP : endoplasmic reticulum red fluorescent

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (D) ER-RFP in red with xy- and xz-axis orthogonal views framing the corresponding image. E-F Manders' coefficients M1 representing the proportion of Filipin III overlapping with (E) ER-RFP M2 representing the proportion of (F) ER-RFP overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm ER-RFP: endoplasmic reticulum red fluorescent protein


G , Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green ( G ) GM130 ( Golgi apparatus ) in red with xy - and xz - axis orthogonal views framing the corresponding image . H - I , Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( H ) GM130 M2 representing the proportion of ( I ) GM130 overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10 µm GM130 : Golgi matrix protein

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (G) GM130 (Golgi apparatus) in red with xy- and xz-axis orthogonal views framing the corresponding image. H-I, Manders' coefficients M1 representing the proportion of Filipin III overlapping with (H) GM130 M2 representing the proportion of (I) GM130 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm GM130: Golgi matrix protein 130


J , Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green , ( J ) EEA1 ( endosomes ) in red with xy - and xz - axis orthogonal views framing the corresponding image . Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( K ) EEA1 M2 representing the proportion of ( L ) EEA1 overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10 µm EEA1 . early endosome antigen

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green , (J) EEA1 (endosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Manders' coefficients M1 representing the proportion of Filipin III overlapping with (K) EEA1 M2 representing the proportion of (L) EEA1 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm EEA1. early endosome antigen 1


M , Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green ( M ) LipidSpot ( lipid droplets ) in red with xy - and xz - axis orthogonal views framing the corresponding image . N - O ( N ) LipidSpot M2 representing the proportion of ( O ) LipidSpot overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (M) LipidSpot (lipid droplets) in red with xy- and xz-axis orthogonal views framing the corresponding image. N-O (N) LipidSpot M2 representing the proportion of (O) LipidSpot overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm


P ) Representative microscopy images ( z - projections ) of co - staining with Filipin III ( cholesterol ) in green ( P ) M6PR ( lysosomes ) in red with xy - and xz - axis orthogonal views framing the corresponding image . Q - R ) Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( Q ) M6PR , and M2 representing the proportion of ( R ) M6PR overlapping with Filipin III in wild - type ( WT ) versus P301L cells . Data information : Data are presented as mean ± SEM ( n = 40 - 90 images per group , 3 - 4 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; Student unpaired t - test . White scale bars : 10

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P) Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (P) M6PR (lysosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Q-R) Manders' coefficients M1 representing the proportion of Filipin III overlapping with (Q) M6PR, and M2 representing the proportion of (R) M6PR overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm


A Visualization of the VAPB - PTPIP51 interactions by in situ proximity ligation assays ( PLAs ) in wild - type ( WT ) cells and P301L cells transfected with the control ( Ctrl ) siRNA , as well as in MFN2 siRNA transfected cells . Interactions between the two targeted proteins are depicted in red ( 63X magnification ) and cell surfaces are delimited by a white line . White scale bars : 20 µm B Quantitative analysis of the PLA signal represented as number of contact points between VAPB - PTPIP51 per cell in percentage of the WT cells . In total , 60 cells were analyzed per group ( 4 independent experiments ) . Data information : Data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . PTPIP51 : protein tyrosine phosphatase - interacting protein 51 VAPB : vesicle - associated membrane protein - associated protein B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Visualization of the VAPB-PTPIP51 interactions by in situ proximity ligation assays (PLAs) in wild-type (WT) cells and P301L cells transfected with the control (Ctrl) siRNA, as well as in MFN2 siRNA transfected cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. White scale bars: 20 µm B Quantitative analysis of the PLA signal represented as number of contact points between VAPB-PTPIP51 per cell in percentage of the WT cells. In total, 60 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ± SEM. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B.


C - D Representative microscopy images ( z - projections ) of co - staining with Filipin III in green and ( C ) TOMM20 ( mitochondria ) or ( D ) ER - RFP ( endoplasmic reticulum ) in red with xy - and xz - axis orthogonal views framing the corresponding image . White scale bars : 10 µm Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . ER - RFP : endoplasmic reticulum red fluorescent protein TOMM20 : translocase of the outer mitochondrial membrane complex subunit

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Representative microscopy images (z-projections) of co-staining with Filipin III in green and (C) TOMM20 (mitochondria) or (D) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20


E - H Manders ' coefficient M1 representing the proportion of Filipin III overlapping with ( E ) TOMM20 or ( G ) ER - RFP , and M2 representing the proportion of ( F ) TOMM20 or ( H ) ER - RFP , overlapping with Filipin III . In total , 20 - 60 cells were analyzed per group ( 4 independent experiments ) . Data information : E - J ) Data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . ER - RFP : endoplasmic reticulum red fluorescent protein ; TOMM20 : translocase of the outer mitochondrial membrane complex subunit 20 ;

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

E-H Manders' coefficient M1 representing the proportion of Filipin III overlapping with (E) TOMM20 or (G) ER-RFP, and M2 representing the proportion of (F) TOMM20 or (H) ER-RFP, overlapping with Filipin III. In total, 20-60 cells were analyzed per group (4 independent experiments). Data information: E-J) Data are presented as mean ± SEM. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein; TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;


A - D ( A , C ) Representative microscopy images ( z - projections ) of tau phosphorylation using ( A ) AT8 and ( C ) AT180 antibodies ( in red ) in GFP - tagged P301L cells ( GFP signal in gray ) treated for 24 h with CHIR99021 ( CHIR ) ( right ) and untreated cells ( left ) . White scale bars : 25 µm . ( B , D ) Quantification of ( B ) AT8 and ( D ) AT180 staining fluorescence intensity . Data are presented as mean ± SEM of n = 50 - 60 cells per group ( 4 independent experiments ) . * * * P < 0 . 001 ; Student unpaired t - test . Data information : Data are presented as mean ± SEM of n = 50 replicates per group ( 5 independent experiments ) in percentage of the WT cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D (A, C) Representative microscopy images (z-projections) of tau phosphorylation using (A) AT8 and (C) AT180 antibodies (in red) in GFP-tagged P301L cells (GFP signal in gray) treated for 24 h with CHIR99021 (CHIR) (right) and untreated cells (left). White scale bars: 25 µm. (B, D) Quantification of (B) AT8 and (D) AT180 staining fluorescence intensity. Data are presented as mean ±SEM of n = 50-60 cells per group (4 independent experiments). *** P<0.001; Student unpaired t-test. Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells.


E Visualization of the VAPB - PTPIP51 interactions by in situ proximity ligation assay ( PLA ) in untreated wild - type ( WT ) cells and P301L cells as well as in CHIR - treated cells . Interactions between the two targeted proteins are depicted in red ( 63X magnification ) and cell surfaces are delimited by a white line . White scale bars : 20 µm F Quantitative analysis of the PLA signal represented as number of contact points between VAPB - PTPIP51 per cell in percentage of the WT cells . In total , 60 cells were analyzed per group ( 4 independent experiments ) . Data information : Data are presented as mean ± SEM of n = 50 replicates per group ( 5 independent experiments ) in percentage of the WT cells . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . PTPIP51 : protein tyrosine phosphatase - interacting protein 51 VAPB : vesicle - associated membrane protein - associated protein B .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Visualization of the VAPB-PTPIP51 interactions by in situ proximity ligation assay (PLA) in untreated wild-type (WT) cells and P301L cells as well as in CHIR-treated cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. White scale bars: 20 µm F Quantitative analysis of the PLA signal represented as number of contact points between VAPB-PTPIP51 per cell in percentage of the WT cells. In total, 60 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B.


G - H Representative microscopy images ( z - projections ) of co - staining with Filipin III in green and ( G ) TOMM20 ( mitochondria ) or ( H ) ER - RFP ( endoplasmic reticulum ) in red with xy - and xz - axis orthogonal views framing the corresponding image . White scale bars : 10 µm Data information : Data are presented as mean ± SEM of n = 50 replicates per group ( 5 independent experiments ) in percentage of the WT cells . ER - RFP : endoplasmic reticulum red fluorescent protein TOMM20 : translocase of the outer mitochondrial membrane complex subunit

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

G-H Representative microscopy images (z-projections) of co-staining with Filipin III in green and (G) TOMM20 (mitochondria) or (H) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20


I - L Manders ' coefficients M1 representing the proportion of Filipin III overlapping with ( I ) TOMM20 or ( K ) ER - RFP , and M2 representing the proportion of ( J ) TOMM20 or ( L ) ER - RFP , overlapping with Filipin III in untreated WT cells and P301L cells as well as in CHIR - treated cells . In total , 35 - 75 cells were analyzed per group ( 4 independent experiments ) . Data information : Data are presented as mean ± SEM of n = 50 replicates per group ( 5 independent experiments ) in percentage of the WT cells . P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; One - way ANOVA + Tukey ' s post hoc test . ER - RFP : endoplasmic reticulum red fluorescent protein TOMM20 : translocase of the outer mitochondrial membrane complex subunit 20 ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-L Manders' coefficients M1 representing the proportion of Filipin III overlapping with (I) TOMM20 or (K) ER-RFP, and M2 representing the proportion of (J) TOMM20 or (L) ER-RFP, overlapping with Filipin III in untreated WT cells and P301L cells as well as in CHIR-treated cells. In total, 35-75 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;


C ) . Western blot of LMF1 - partners after DSP crosslinking and affinity tag purification using LMF1 ' s C - terminal His tag . Tetracycline induces LMF1 expression . Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners . LMF1 complexes are labeled with asterisks and an arrow points to LMF1

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O 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). Western blot of LMF1-partners after DSP crosslinking and affinity tag purification using LMF1's C-terminal His tag. Tetracycline induces LMF1 expression. Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners. LMF1 complexes are labeled with asterisks and an arrow points to LMF1


D ) . Western blot of LMF1 - partners after pBPA photo - crosslinking and affinity tag purification . The DMSO panel does not include pBPA and serves as a negative control . Fractions were eluted with increasing concentrations of imidazole . An arrow points to LMF1

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

D). Western blot of LMF1-partners after pBPA photo-crosslinking and affinity tag purification. The DMSO panel does not include pBPA and serves as a negative control. Fractions were eluted with increasing concentrations of imidazole. An arrow points to LMF1


E ) . SYPRO orange protein stain of DSP crosslinked samples . Samples without tetracycline but with DSP and DTT ( negative control ) , and with tetracycline , DSP and DTT were sent for LC MS / MS . LMF1 complexes are labeled with asterisks and an arrow points to LMF1

O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

E). SYPRO orange protein stain of DSP crosslinked samples. Samples without tetracycline but with DSP and DTT (negative control), and with tetracycline, DSP and DTT were sent for LC MS/MS. LMF1 complexes are labeled with asterisks and an arrow points to LMF1


F ) . SYPRO orange stained gel of pBPA samples . An arrow points to LMF1

O O O O O O O O B-CONTROLLED_VAR O O O O O O O

F). SYPRO orange stained gel of pBPA samples. An arrow points to LMF1


A - B ) . HEK293 cells stably expressing LPL - V5 or PL - V5 were transfected with scrambled negative control siRNA ( NC ) or siRNA against LMF1 - interacting partners . Media fractions were probed for the lipase V5 tags , and lysate fractions were probed for V5 and the LMF1 interacting - partners . GAPDH is a loading

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B). HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with scrambled negative control siRNA (NC) or siRNA against LMF1-interacting partners. Media fractions were probed for the lipase V5 tags, and lysate fractions were probed for V5 and the LMF1 interacting-partners. GAPDH is a loading control


siRNA knockdowns were performed in triplicate , and lipase secretion was quantified relative to NC siRNA . Error bars indicate the standard error . Quantification of the percent of each protein remaining from three independent experiments . Error bars indicate the standard error .

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

siRNA knockdowns were performed in triplicate, and lipase secretion was quantified relative to NC siRNA. Error bars indicate the standard error. Quantification of the percent of each protein remaining from three independent experiments. Error bars indicate the standard error.


E ) HEK293 cells stably expressing LPL - V5 or PL - V5 were transfected with NC siRNA or siRNA against TRX . Lipase and TRX levels were tested as for Fig 1A

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

E) HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with NC siRNA or siRNA against TRX. Lipase and TRX levels were tested as for Fig 1A


F ) Txnip overexpression in HEK293 cells stably expressing LPL - V5 or PL - V5 hinders LPL , but not PL , secretion

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

F) Txnip overexpression in HEK293 cells stably expressing LPL-V5 or PL-V5 hinders LPL, but not PL, secretion


A ) . A Western blot against the C - terminal V5 tags of both lipases shows that LPL , but not PL , forms intermolecular disulfide bonds in cells . Lipase monomers are marked with a single arrow , and aggregates are marked with double and triple arrows . The LPL aggregates are not present when samples are treated with DTT . There is no PL in the pellet fraction because PL does not aggregate

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). A Western blot against the C-terminal V5 tags of both lipases shows that LPL, but not PL, forms intermolecular disulfide bonds in cells. Lipase monomers are marked with a single arrow, and aggregates are marked with double and triple arrows. The LPL aggregates are not present when samples are treated with DTT. There is no PL in the pellet fraction because PL does not aggregate


B ) . A Western blot of a non - reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 ( HEK293 ∆ LMF1 ) , normal HEK293 cells or cells with additional LMF1 ( + LMF1 ) . LPL monomers and aggregates are marked as above

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). A Western blot of a non-reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 (HEK293∆LMF1), normal HEK293 cells or cells with additional LMF1 (+LMF1). LPL monomers and aggregates are marked as above


C ) . Co - translational LPL folding was carried out in the presence of SP HEK293 or HEK293 ∆ LMF1 cells . At the indicated time points , AMS was added to reactions to differentiate reduced ( R ) and oxidized ( O ) LPL . The position of the stacker layer is indicated

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Co-translational LPL folding was carried out in the presence of SP HEK293 or HEK293∆LMF1 cells. At the indicated time points, AMS was added to reactions to differentiate reduced (R) and oxidized (O) LPL. The position of the stacker layer is indicated


The amount of reduced / total LPL intensity was quantified . Each point is the average of three independent experiments , and error bars represent the standard error . Significance was determined by a 2 - tailed student ' s t - test .

The amount of reduced/total LPL intensity was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test.


the amount of oxidized / total LPL ( E ) and the intensity of the stacker / total LPL ( F ) was quantified . Each point is the average of three independent experiments , and error bars represent the standard error . Significance was determined by a 2 - tailed student ' s t -

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

the amount of oxidized/total LPL (E) and the intensity of the stacker/total LPL (F) was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test


G and H ) . Data from ( D - F ) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293 ∆ LMF1 cells . Each point is the average of three independent experiments , and error bars represent the standard deviation

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

G and H). Data from (D-F) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293∆LMF1 cells. Each point is the average of three independent experiments, and error bars represent the standard deviation


Differential sensitivity of HEK293 cells containing and lacking LMF1 ( A ) to drugs that perturb cellular redox homeostasis . Cells were treated with BSO , DTT , or tunicamycin as described in materials and methods . Treated cell and untreated cells were counted , and the fraction of surviving cells was calculated . Three wells were counted and averaged for each data point , and three independent trials were carried out per condition . Significance was determined by a 2 - tailed student ' s t - test .

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-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.


Differential sensitivity of cld / cld and wt / cld MEFs ( B ) to drugs that perturb cellular redox homeostasis . Cells were treated with BSO , DTT , or tunicamycin as described in materials and methods . Treated cell and untreated cells were counted , and the fraction of surviving cells was calculated . Three wells were counted and averaged for each data point , and three independent trials were carried out per condition . Significance was determined by a 2 - tailed student ' s t - test .

O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differential sensitivity of cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.


C ) . The redox sensor ERroGFP - S4 shows that HEK293 ∆ LMF1 cells have a more oxidized ER than HEK293 and HEK293 + LMF1 cells both untreated and when stressed by expression of LPL - mCherry . A two - tailed student ' s t - test was used to calculate the significance of the difference in the ratio of oxidized / reduced GFP fluorescence intensity between cell types . Each data point is one of three spots measured per cell , 17 cells were measured per condition . Significance was determined by a 2 - tailed student ' s t - test

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C). The redox sensor ERroGFP-S4 shows that HEK293∆LMF1 cells have a more oxidized ER than HEK293 and HEK293+LMF1 cells both untreated and when stressed by expression of LPL-mCherry. A two-tailed student's t-test was used to calculate the significance of the difference in the ratio of oxidized/reduced GFP fluorescence intensity between cell types. Each data point is one of three spots measured per cell, 17 cells were measured per condition. Significance was determined by a 2-tailed student's t-test


) . Secretion of LPL - V5 from HEK293 ∆ LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations . Lipase levels in the lysate and pellet , and LMF1 levels in the pellet ( α - HIS ) , are also probed . GAPDH serves as a loading control .

). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.


α - His Western blot to detect the redox state of LMF1 . In the first two lanes samples were untreated and resolved in reducing or non - reducing loading dye . The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non - reducing SDS - PAGE .

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α-His Western blot to detect the redox state of LMF1. In the first two lanes samples were untreated and resolved in reducing or non-reducing loading dye. The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non-reducing SDS-PAGE.


α - His Western blot to detect acid trapped partners of LMF1 . Samples were TCA precipitated , treated with NEM , and LMF1 was purified via its C - terminal His tag . Samples were resolved by reducing or nonreducing SDS - PAGE . The lanes marked - LMF1 are HEK293 ∆ LMF1 cells included as a negative control .

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α-His Western blot to detect acid trapped partners of LMF1. Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control.


Samples were TCA precipitated , treated with NEM , and LMF1 was purified via its C - terminal His tag . Samples were resolved by reducing or nonreducing SDS - PAGE . The lanes marked - LMF1 are HEK293 ∆ LMF1 cells included as a negative control . Samples were probed with an antibody for TRX .

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Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control. Samples were probed with an antibody for TRX.


Bottom , center and top slices from a z - stack of a cld / wt or cld / cld MEF cell . YPet - tagged fibronectin is in green and mCherry - Sec61β , an ER marker , is in red . Note fibronectin assembly into the extracellular matrix in cld / wt cells . cld / wt cells had significantly higher amounts of fibronectin outside of the ER than cld / cld cells by a two - tailed student ' s t - test .

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Bottom, center and top slices from a z-stack of a cld/wt or cld/cld MEF cell. YPet-tagged fibronectin is in green and mCherry-Sec61β, an ER marker, is in red. Note fibronectin assembly into the extracellular matrix in cld/wt cells. cld/wt cells had significantly higher amounts of fibronectin outside of the ER than cld/cld cells by a two-tailed student's t-test.


Bottom , center and top slices show that LDLR is retained in the ER in cells HEK293 ∆ LMF1 cells . LDLR - GFP is in green and mCherry - Sec61β is in red . Note the clumping of LDLR in the ER in HEK293 ∆ LMF1 cells . The ratio of LDLR within the ER / total LDLR was significantly higher for HEK293 ∆ LMF1 cells than HEK293 or HEK293 + LMF1 cells by a 2 - tailed student ' s t - test .

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Bottom, center and top slices show that LDLR is retained in the ER in cells HEK293∆LMF1 cells. LDLR-GFP is in green and mCherry-Sec61β is in red. Note the clumping of LDLR in the ER in HEK293∆LMF1 cells. The ratio of LDLR within the ER/total LDLR was significantly higher for HEK293∆LMF1 cells than HEK293 or HEK293+LMF1 cells by a 2-tailed student's t-test.


( A ) Representative double - plotted actograms showing wheel - running activity of wild type ( WT ) and CRY - deficient ( CRY Knockout ; CKO ) mice during constant light ( yellow shading ) and thereafter in constant darkness . Note the 48 hour x - axis for WT versus 32 hour for CKO .

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(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 hour x-axis for WT versus 32 hour for CKO.


( B ) Mean period and amplitude ( ± SEM ) of mouse behavioural data ( n = 4 ) .

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(B) Mean period and amplitude (±SEM) of mouse behavioural data (n=4).


( C ) Longitudinal bioluminescence recordings of organotypic SCN slices from WT ( black ) and CKO ( red ) PER2 : : LUC mice ( RLU ; relative light units ) .

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(C) Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units).


( D ) Mean period and amplitude ( ± SEM ) of rhythmic SCN bioluminescence traces .

(D) Mean period and amplitude (±SEM) of rhythmic SCN bioluminescence traces.


( E ) Circadian PER2 : : LUC expression in immortalised WT and CKO adult lung fibroblasts . Left panel shows two raw traces of a representative longitudinal bioluminescence recording , right panel shows same data detrended with a 24 - hour moving average to remove differences in baseline expression .

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(E) Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, right panel shows same data detrended with a 24-hour moving average to remove differences in baseline expression.


( F ) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments ( n ≥ 3 per experiment , individual values ± SEM shown ) . P - values were calculated by an unpaired t test with Welch correction . Standard deviations differ significantly between WT and CKO ( F test : p < 0 . 0001 ) .

(F) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n≥3 per experiment, individual values ±SEM shown). P-values were calculated by an unpaired t test with Welch correction. Standard deviations differ significantly between WT and CKO (F test: p <0.0001).


( A ) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range ( n = 4 , solid lines : mean , dashed lines : ± SEM ) . Temperature was changed from 37 ° C to 32 ° C halfway through the experiment , as depicted by red / blue shading . Arrows represent medium changes . Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change . ( B ) Quantification of period from recordings presented in ( A ) . Both WT and CKO oscillations are temperature compensated with respective Q10s of 1 . 05 and 0 . 95 ( n = 3 , mean ± SEM ) . P - values were determined by two - tailed t - test . ( C ) Bioluminescence of WT and CKO PER2 : : LUC cells during temperature entrainment ( 12h 32 ° C ( blue ) - 12h 37 ° C ( red ) ) ( n = 3 , solid lines : mean , dashed lines : ± SEM ) .

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(A) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range (n=4, solid lines: mean, dashed lines: ±SEM). Temperature was changed from 37°C to 32°C halfway through the experiment, as depicted by red/blue shading. Arrows represent medium changes. Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change. (B) Quantification of period from recordings presented in (A). Both WT and CKO oscillations are temperature compensated with respective Q10s of 1.05 and 0.95 (n=3, mean ±SEM). P-values were determined by two-tailed t-test. (C) Bioluminescence of WT and CKO PER2::LUC cells during temperature entrainment (12h 32°C (blue) - 12h 37°C (red)) (n=3, solid lines: mean, dashed lines: ±SEM).


( A ) Mean number of PER2 : : LUC molecules per cell at the estimated peak of PER2 expression for each cell line ( mean of three experiments , n = 3 each ) .

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(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each).


( B ) PER2 : : LUC binding to BMAL1 in WT and CKO cells . Cells were harvested at the peak of PER2 expression , BMAL1 was immunoprecipitated , and PER2 : : LUC binding was measured by bioluminescence measurements ( n = 3 , mean ± SD ) .

(B) PER2::LUC binding to BMAL1 in WT and CKO cells. Cells were harvested at the peak of PER2 expression, BMAL1 was immunoprecipitated, and PER2::LUC binding was measured by bioluminescence measurements (n=3, mean ±SD).


( C ) Per2 mRNA levels in WT ( left ) and CKO ( right ) cells were determined by qPCR over one circadian cycle ( bottom ) , while PER2 : : LUC bioluminescence ( top , min - max normalised ) was recorded from parallel cultures . Per2 mRNA reported relative to Rns18s ( bottom ) , n = 3 , ± SEM ; PER2 : : LUC ( top ) presented as mean ( solid ) ± SEM ( dashed ) , n = 3 . The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line ( p = 0 . 0412 , extra sum - of - square F - test ) whereas CKO data was not ( ns ) .

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(C) Per2 mRNA levels in WT (left) and CKO (right) cells were determined by qPCR over one circadian cycle (bottom), while PER2::LUC bioluminescence (top, min-max normalised) was recorded from parallel cultures. Per2 mRNA reported relative to Rns18s (bottom), n=3, ±SEM; PER2::LUC (top) presented as mean (solid) ±SEM (dashed), n=3. The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line (p=0.0412, extra sum-of-square F-test) whereas CKO data was not (ns).


( D ) Detrended Per2 and Nr1d1 promoter activity in WT , CKO and quadruple Cry1 / 2 - Per1 / 2 knockout ( CPKO ) mouse embryonic fibroblasts ( MEFs ) recorded at 37 ° C , n = 3 , mean ( solid ) ± SEM ( dashed ) . Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line ( p < 0 . 0001 , extra sum - of - squares F - test ) ( right hand graphs , solid lines ; error bars , SEM ) .

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(D) Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 37°C, n=3, mean (solid) ±SEM (dashed). Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line (p<0.0001, extra sum-of-squares F-test) (right hand graphs, solid lines; error bars, SEM).


( A ) Actual PER2 : : LUC levels ( dark symbols ( 3 - hour moving average , n = 3 ± SEM , 4 outliers removed ) ) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours , compared with parallel longitudinal co - recordings from cells in the presence of 0 . 1 mM luciferin ( light lines ( n = 6 , mean ± SEM ) ) .

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(A) Actual PER2::LUC levels (dark symbols (3-hour moving average, n=3 ±SEM, 4 outliers removed)) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours, compared with parallel longitudinal co-recordings from cells in the presence of 0.1 mM luciferin (light lines (n=6, mean ±SEM)).


( B ) PER2 : : LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 ( 10 µM , applied at the arrow ) ( n = 3 , mean ± SEM ) . ( C ) Quantification of relative PER2 : : LUC induction upon proteasome inhibition , n = 3 , mean ( solid ) ± SEM ( dashed ) .

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(B) PER2::LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 (10 µM, applied at the arrow) (n=3, mean ±SEM). (C) Quantification of relative PER2::LUC induction upon proteasome inhibition, n=3, mean (solid) ±SEM (dashed).


( D ) Phase - dependent PER2 : : LUC half - life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one - phase exponential decay curve ( n = 3 , mean ± SEM ) . Left image depicts the timing of cycloheximide ( CHX , 10 µM ) pulses ( labelled I ( PER2 levels going up ) and II ( PER2 levels going down ) ) , plotted on PER2 : : LUC bioluminescence traces of control cells ( dark colours ) . A representative trace of CHX - treated cells at time point I is shown in light colours . Right image shows quantifications , p - values were calculated by unpaired t test .

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(D) Phase-dependent PER2::LUC half-life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one-phase exponential decay curve (n=3, mean ±SEM). Left image depicts the timing of cycloheximide (CHX, 10 µM) pulses (labelled I (PER2 levels going up) and II (PER2 levels going down)), plotted on PER2::LUC bioluminescence traces of control cells (dark colours). A representative trace of CHX-treated cells at time point I is shown in light colours. Right image shows quantifications, p-values were calculated by unpaired t test.


( A ) Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CK1δ / ε inhibitor PF670462 ( 0 . 3 µM ; PF ) .

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(A) Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 ( 5 µM ; CHIR ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 (5 µM; CHIR).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CRY inhibitor KL001 ( 1 µM ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CRY inhibitor KL001 (1 µM).


A representative nuclease assay with MR , MBP - NBS1 - his ( denoted MBP - NBS1 ) , MRN and pCtIP on 5 ' - end labeled 70 - bp dsDNA with all ends blocked with streptavidin . Samples were separated on 15 % denaturing polyacrylamide gel . Quantitation of nuclease assays such as in ( A ) . Averages shown ; n ≥ 3 ; error bars , SEM .

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A representative nuclease assay with MR, MBP-NBS1-his (denoted MBP-NBS1), MRN and pCtIP on 5'-end labeled 70-bp dsDNA with all ends blocked with streptavidin. Samples were separated on 15 % denaturing polyacrylamide gel. Quantitation of nuclease assays such as in (A). Averages shown; n ≥ 3; error bars, SEM.


The effect of mutations in FHA and / or BRCT domains of NBS1 on the nuclease of MR and pCtIP . Quantitation of experiments such as shown in Fig EV2D . Averages shown ; n ≥ 4 , error bars , SEM . Statistical significance denotations represent the analysis between wild type full - length NBS1 ( in black ) and the corresponding concentrations of the NBS1 variants ; ns ( p > 0 . 05 , not significant ) , * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , two - tailed t - test .

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The effect of mutations in FHA and/or BRCT domains of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2D. Averages shown; n ≥ 4, error bars, SEM. Statistical significance denotations represent the analysis between wild type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (p > 0.05, not significant), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), two-tailed t-test.


Analysis of pCtIP binding to NBS1 variants . Anti CtIP antibody was immobilized on Protein G agarose , bound to pCtIP , and tested for interactions with the indicated NBS1 variants ( see cartoon at the top ) . Western blot of eluates ( right part ) was performed with anti - CtIP and anti - NBS1 antibodies . Left part ( input ) indicates that the anti - NBS1 antibody recognizes the NBS1 constructs to a similar level .

Analysis of pCtIP binding to NBS1 variants. Anti CtIP antibody was immobilized on Protein G agarose, bound to pCtIP, and tested for interactions with the indicated NBS1 variants (see cartoon at the top). Western blot of eluates (right part) was performed with anti-CtIP and anti-NBS1 antibodies. Left part (input) indicates that the anti-NBS1 antibody recognizes the NBS1 constructs to a similar level.


Analysis of phosphorylated CtIP ( pCtIP , mock - treated ) or λ phosphatase treated CtIP ( λCtIP ) interactions with NBS1 . The CtIP variants were immobilized on protein G agarose , and incubated with NBS1 ( see cartoon on the left ) . The western blot was performed with anti CtIP and anti NBS1 antibodies .

Analysis of phosphorylated CtIP (pCtIP, mock-treated) or λ phosphatase treated CtIP (λCtIP) interactions with NBS1. The CtIP variants were immobilized on protein G agarose, and incubated with NBS1 (see cartoon on the left). The western blot was performed with anti CtIP and anti NBS1 antibodies.


The effect of the MRE11 interaction region ( MIR ) of NBS1 on the nuclease of MR and pCtIP . Quantitation of experiments such as shown in Fig EV2E . Averages shown , n ≥ 3 , error bars , SEM . The quantitation of wild type NBS1 ( NBS1 WT , in black ) is the same as in panel ( B ) and is shown again for reference . Statistical significance denotations represent the analysis between wild type full - length NBS1 ( in black ) and the corresponding concentrations of the NBS1 variants ; ns ( p > 0 . 05 , not significant ) , * * * ( p < 0 . 001 ) , two - tailed t - test .

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The effect of the MRE11 interaction region (MIR) of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2E. Averages shown, n ≥ 3, error bars, SEM. The quantitation of wild type NBS1 (NBS1 WT, in black) is the same as in panel (B) and is shown again for reference. Statistical significance denotations represent the analysis between wild type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (p > 0.05, not significant), *** (p < 0.001), two-tailed t-test.


Nuclease assays with MR and various NBS1 fragments ( 50 nM ) on oligonucleotide - based 5 ' - end labeled dsDNA . The reaction buffer contained 60 mM NaCl and reactions were incubated for 2 h . Quantitation of experiments such as shown in ( A ) . Averages shown ; n ≥ 3 , error bars , SEM .

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Nuclease assays with MR and various NBS1 fragments (50 nM) on oligonucleotide-based 5'-end labeled dsDNA. The reaction buffer contained 60 mM NaCl and reactions were incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n ≥ 3, error bars, SEM.


In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs . Shown are representative images .

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In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs. Shown are representative images.


In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs . Shown are representative images .

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In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs. Shown are representative images.


Quantitation of the experiment shown in ( B ) . At least 100 cells were assessed per condition . The horizontal line represents a median , the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile .

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Quantitation of the experiment shown in (B). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile.


Quantitation of the experiment shown in ( C ) . At least 100 cells were assessed per condition . The horizontal line represents a median , the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile .

Quantitation of the experiment shown in (C). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile.


Quantitation of the single strand annealing ( SSA ) reporter assay in U2OS cells . All cells were treated with DMSO ; where indicated , both PFM03 ( 50 μM ) and PFM39 ( 50 μM ) MRE11 inhibitors were added . Averages shown ; n = 4 , error bars , SEM . Insert , western blot analysis of NBS1 and CtIP protein levels upon depletion with siRNA .

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Quantitation of the single strand annealing (SSA) reporter assay in U2OS cells. All cells were treated with DMSO; where indicated, both PFM03 (50 μM) and PFM39 (50 μM) MRE11 inhibitors were added. Averages shown; n = 4, error bars, SEM. Insert, western blot analysis of NBS1 and CtIP protein levels upon depletion with siRNA.


Quantitation of resection activity in NBS1 ∆ N cells complemented with wild type ( WT ) or NBS1 R28A K160M mutant and either mock depleted ( siCtrl ) or depleted of CtIP ( siCtIP ) . At least 300 cells were assessed per condition . The horizontal line represents a median , the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile . Statistical significance was determined using ordinary one - way ANOVA with Tukey ' s multiple comparison test , * * * * ( p < 0 . 0001 ) .

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Quantitation of resection activity in NBS1∆N cells complemented with wild type (WT) or NBS1 R28A K160M mutant and either mock depleted (siCtrl) or depleted of CtIP (siCtIP). At least 300 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile. Statistical significance was determined using ordinary one-way ANOVA with Tukey's multiple comparison test, **** (p<0.0001).


Nuclease assays with MR and pCtIP on 3 ' - end labeled dsDNA , incubated for 2 h . Quantitation of experiments such as shown in ( A ) . Averages shown ; n = 2 ; error bars , range .

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Nuclease assays with MR and pCtIP on 3'-end labeled dsDNA, incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n = 2; error bars, range.


Nuclease assays with MRN , MR and various concentrations of pCtIP ( mock - treated ) or λCtIP on 5 ' - end labeled dsDNA , incubated for 2 h .

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Nuclease assays with MRN, MR and various concentrations of pCtIP (mock-treated) or λCtIP on 5'-end labeled dsDNA, incubated for 2 h.


Quantitation of experiments such as shown in ( C ) . Averages shown ; n = 3 ; error bars , SEM .

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Quantitation of experiments such as shown in (C). Averages shown; n = 3; error bars, SEM.


Phosphorylated CtIP was mock - incubated ( pCtIP , lane 2 ) or treated with λ phosphatase ( λCtIP , lane 3 ) . The proteins were separated on a 10 % polyacrylamide gel and stained with Coomassie brilliant blue .

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Phosphorylated CtIP was mock-incubated (pCtIP, lane 2) or treated with λ phosphatase (λCtIP, lane 3). The proteins were separated on a 10% polyacrylamide gel and stained with Coomassie brilliant blue.


Quantitation of kinetic nuclease assays with MRN and pCtIP ( mock - treated ) or λCtIP . Averages shown ; n = 3 ; error bars , SEM . Statistical denotations , * * ( p < 0 . 01 ) , two - tailed t - test .

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Quantitation of kinetic nuclease assays with MRN and pCtIP (mock-treated) or λCtIP. Averages shown; n = 3; error bars, SEM. Statistical denotations, ** (p < 0.01), two-tailed t-test.


Quantitation of nuclease assays as in ( F ) , but with MR complex instead of MRN . Averages shown ; n = 3 ; error bars , SEM . Statistical denotations , ns ( p > 0 . 05 ) , two - tailed t - test .

Quantitation of nuclease assays as in (F), but with MR complex instead of MRN. Averages shown; n = 3; error bars, SEM. Statistical denotations, ns (p > 0.05), two-tailed t-test.


Nuclease assays with MR , MRN , pCtIP or pCtIP T847A , as indicated , on 5 ' - end labeled dsDNA . Reactions were incubated for 2 h . Quantitation of experiments such as shown in ( H ) . Averages shown ; n = 4 ; error bars , SEM .

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Nuclease assays with MR, MRN, pCtIP or pCtIP T847A, as indicated, on 5'-end labeled dsDNA. Reactions were incubated for 2 h.   Quantitation of experiments such as shown in (H). Averages shown; n = 4; error bars, SEM.


Interaction between MRE11 and CtIP . MRE11 was immobilized on antibody - coupled Protein G agarose , and incubated with pCtIP or λCtIP ( see cartoon ) . Western blot analysis of bound proteins was performed with anti MRE11 and anti CtIP antibodies .

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Interaction between MRE11 and CtIP. MRE11 was immobilized on antibody-coupled Protein G agarose, and incubated with pCtIP or λCtIP (see cartoon). Western blot analysis of bound proteins was performed with anti MRE11 and anti CtIP antibodies.


Interaction between RAD50 and CtIP . RAD50 - FLAG was immobilized on anti - FLAG affinity resin and incubated with pCtIP and λCtIP ( see cartoon ) . Western blot analysis of bound proteins was performed with anti FLAG and anti CtIP antibodies .

Interaction between RAD50 and CtIP. RAD50-FLAG was immobilized on anti-FLAG affinity resin and incubated with pCtIP and λCtIP (see cartoon). Western blot analysis of bound proteins was performed with anti FLAG and anti CtIP antibodies.


Interaction between MRE11 , MR or MRN and pCtIP or λCtIP , as indicated . Phosphorylated ( pCtIP ) or λ phosphatase - treated CtIP ( λCtIP ) were immobilized on antibody - coupled Protein G agarose , and incubated with MRE11 , MR or MRN complexes ( see cartoon ) . Bound proteins were analyzed by western blot using anti CtIP and anti MRE11 antibodies .

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Interaction between MRE11, MR or MRN and pCtIP or λCtIP, as indicated. Phosphorylated (pCtIP) or λ phosphatase-treated CtIP (λCtIP) were immobilized on antibody-coupled Protein G agarose, and incubated with MRE11, MR or MRN complexes (see cartoon). Bound proteins were analyzed by western blot using anti CtIP and anti MRE11 antibodies.


Representative nuclease assays with MRE11 , MR , MRN and pCtIP , as indicated , on 5 ' - end labeled dsDNA with a single streptavidin - blocked end ( S ) . The substrate contained 5 phosphorothioate bonds ( PTO ) at both 3 ' - ends to prevent exonucleolytic DNA degradation . Reactions were incubated for 2 h .

Representative nuclease assays with MRE11, MR, MRN and pCtIP, as indicated, on 5'-end labeled dsDNA with a single streptavidin-blocked end (S). The substrate contained 5 phosphorothioate bonds (PTO) at both 3'-ends to prevent exonucleolytic DNA degradation. Reactions were incubated for 2 h.


Representative nuclease assays as in ( A ) , but with a substrate containing a nick in the top oligonucleotide ( 20 nt away from the 3 ' - end , see cartoon ) .

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Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).


Quantitation of experiments such as shown in ( A ) , with pCtIP . Averages shown ; n ≥ 4 ; error bars , SEM .

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Quantitation of experiments such as shown in (A), with pCtIP. Averages shown; n ≥ 4; error bars, SEM.


Quantitation of experiments such as shown in ( B ) , with and without pCtIP . Averages shown ; n = 3 ; error bars , SEM .

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Quantitation of experiments such as shown in (B), with and without pCtIP. Averages shown; n = 3; error bars, SEM.


A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup / Cdt1 RNAi , cyclin E RNAi , Cdk2 RNAi , Dacapo overexpression ( + Dacapo ) , and 1 mM HU .

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A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.


B Analysis of Mcm2 ‐ 7 nuclear chromatin association at different points in the cell cycle . Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2 ‐ 7 by Western blot . A non ‐ specific band is indicated by an asterisk ( * ) . C Quantification of the ratio of chromatin ‐ bound Mcm2 ‐ 7 relative to Orc2 ( log10 scale ) for a minimum of 11 replicates ( mean ± SD ) .

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B Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).C Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).


Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h ( top ) . Western blot analysis of Mcm2 ‐ 7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2 ‐ 7 , Orc2 , and Dacapo .

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Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.


B FACS profiles of DNA content for each cell population assayed .

B FACS profiles of DNA content for each cell population assayed.


C Western blot analysis for Orc2 and Mcm2 ‐ 7 in the nuclear chromatin fraction and Orc2 , Mcm2 ‐ 7 , Dup / Cdt1 , and cyclin E in the cytoplasmic fraction for each condition assayed .

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C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.


A Genome ‐ wide analysis of ORC localization by ChIP ‐ chip . ORC enrichment from asynchronous cells is depicted for a 5 ‐ Mb section of chromosome 2L . B Genome ‐ wide analysis of Mcm2 ‐ 7 localization in early G1 by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from cyclin E RNAi ‐ depleted cells is depicted for a 5 ‐ Mb section of chromosome 2L . C Venn diagram depicting the overlap between ORC and Mcm2 ‐ 7 peaks .

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A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.


A Genome ‐ wide analysis of Mcm2 ‐ 7 localization at the G1 / S transition by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from HU ‐ arrested cells is depicted for a 5 ‐ Mb section of chromosome 2L . Inset : transcribed ( green ) and non ‐ transcribed ( red ) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom . B Bimodal distribution of Mcm2 ‐ 7 enrichment over transcribed and non ‐ transcribed genes . Histogram showing the distribution of probe scores found within transcribed ( green ) and non ‐ transcribed ( red ) genes . C " Meta " ‐ gene analysis of Mcm2 ‐ 7 enrichment for different deciles of gene expression and their aggregated probe intensities .

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A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C "Meta"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.


D Mcm2 ‐ 7 is displaced from chromatin by DNA replication . Genome ‐ wide analysis of Mcm2 ‐ 7 localization in late S ‐ phase ( 6 h post HU release ) by ChIP ‐ chip . Mcm2 ‐ 7 enrichment is depicted for a 10 ‐ Mb section of chromosome 2L ( filled gray ) , replication timing profile ( black line ) and early ( yellow ) and late ( purple ) replication timing domains . E Box ‐ plots representing late S ‐ phase Mcm2 ‐ 7 ChIP signal found within early ( 246 ) or late ( 167 ) replication domains .

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D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) and cell lysates ( Lysate ) were collected and immunoblotted with the indicated antibodies （ A ） .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected and ( B - E ) IL - 1β secretion ( B ) , IL - 18 secretion ( C ) and TNF - ɑ secretion ( D ) and LDH release ( E ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . ( F ) HBVLV - Vdr expression in Vdr - / - BMDMs by lentivirus - mediated transduction primed with LPS and infected with 50 MOI S . Typhimurium for 2 h , cell lysates , and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies.


( G ) LPS - primed WT and Vdr - / - BMDMs were treated with 1 µg / ml FLIC ( LFn - Flagellin and PA ) or PrgJ （ 1 µg / ml LFn - PrgJ and PA ） for 1 h , total mixtures ( culture supernatants and cell lysates ) were collected and immunoblotted with the indicated antibodies .

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(G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies.


( H ) LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) and cell lysates were collected and immunoblotted with the indicated antibodies .

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(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies.


LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) were collected and ( I - L ) IL - 1β secretion ( I ) , IL - 18 secretion ( J ) and TNF - ɑ secretion ( K ) and LDH release ( L ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) and Cell lysates were collected and immunoblotted with the indicated antibodies ( A ) .

LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISA


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected antibodies ( A ) . ELISA detected TNF - ɑ ( B ) andIL - 1β ( C ) in supernatants . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) were collected antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium Bacterial burden was detected in spleens ( D ) , PCF ( E ) , and blood ( F ) of WT , Vdr - / - , NLRP3 - / - , and NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.