Figure 4Stability analysis of indicated proteins in cells treated with shRNAs against various BC - box proteins . Since GFP was tagged at the protein N - terminus and signal peptide and MTSs need to be exposed to be functional , all tested proteins were mislocalized . Protein stability analyses of wild - type ( WT ) and mutant proteins lacking their extreme terminal Gly ( ∆ G ) or having it masked by adding Leu ( + L ) in cells with or without BC - box protein knockdown ( C ) or overexpression ( D ) . Fractionation and Western blot analysis of wild - type and Flag - tagged PDP2 ( top ) , and wild - type and MTS - deleted ( ∆ MTS ) MRPL28 ( bottom ) . Flag - PDP2 and ∆ MTS MRPL28 were mislocalized . W , C , and M denote whole cell lysates , cytosol fraction and mitochondrial fraction , respectively . Tubulin and VDAC serve as fractionation quality controls . Protein abundance analysis of HA - tagged wild - type or mutant secretory proteins with their signal peptide deleted ( ∆ SP ) in cells with or without KLHDC3 or APPBP2 expression . The HA epitope was inserted immediately after the signal peptide ( SP ) , which had no impact on protein secretion .

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Figure 4Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.


Figure 5Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2 , DNCul2 or IMP - 1088 treatments . IMP - 1088 inhibits NMTs . C and M denote cytosol and mitochondrial fractions , respectively . Tubulin and VDAC serve as fractionation quality controls . The relative abundance of cytosolic MIC19 / tubulin was normalized to that of lane 1 and is indicated under each lane in red . Another replicate of this experiment is shown in Fig . EV3B . Abundance comparison of GFP N - or C - terminal tagged with the wild - type or mutant N - terminal ( aa . 1 - 24 ) or C - terminal ( a . a . 218 - 227 ) motif of MIC19 in cells with indicated treatments . Data from sh # 1 is shown here .

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Figure 5Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.


Figure 6Fractionation analysis of wild - type HA - MIC19 in U2OS cells treated with IMP - 1088 and with or without inhibition of ZYG11B or KLHDC2 . The normalized relative abundance of cytosolic HA - MIC19 / tubulin is indicated under each lane in red .

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Figure 6Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.


Figure 7GST pull - down assay using cells expressing GST or GST - tagged wild - type or mutant KLHDC2 and HA - tagged SDE2 , FAU or TUG .

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Figure 7GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.


Figure 8Relative stability analysis of GAPDH C - terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays . The - 3 , - 4 or - 5 residue of SARS - CoV NSP proteins was mutated to Ala ( A ) as indicated . The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2 . Data are presented as the mean value of GFP / RFP + / - standard deviation from triplicate experiments .

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Figure 8Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.


Figure 1D , E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 5 mice / group ) . Areas of MCT2 ( upper panel ) and SMCT1 ( lower panel ) stainings ( D ) are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 . Representative images of MCT2 and SMCT1 stainings ( E ) in tumors from KIC mice treated with βOHB or NaCl . Scale bar : 100 µm .

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Figure 1D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.


Figure 2B - D [ U - 13C ] Glucose ( n = 3 mice ) ( B ) , [ U - 13C ] Glutamine ( n = 5 mice ) ( C ) and [ U - 13C ] Acetate ( n = 6 mice ) ( D ) tracing into βOHB in poorly differentiated PDA explants from KIC mice . Data are expressed as mean ± SEM .

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Figure 2B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6 mice) (D) tracing into βOHB in poorly differentiated PDA explants from KIC mice. Data are expressed as mean ± SEM.


Figure 3G Volcano plot illustrating proteins significantly under - or over - represented in sg - CTRL and sg - HMGCL PANC - 1 spheroids ( n = 3 independent experiments ) . Significance was defined by one - tailed Student ' s t - test . Protein levels with a q - value < 0 . 05 ( horizontal axis ) and a fold change < - 1 . 5 or > + 1 . 5 ( vertical axis ) are considered as significantly down or up - regulated in sg - HMGCL PANC - 1 spheroids .

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Figure 3G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.


Figure 4A , B Quantification of volume with representative images ( A ) and weight ( B ) of sg - CTRL ( n = 15 for A and n = 9 for B ) , sg HMGCL # 2 ( n = 19 for A and n = 8 for B ) , # 3 ( n = 7 for A and B ) pancreatic tumors . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Extracellular matrix quantification following trichrome staining in sg - CTRL and sg - HMGCL # 2 pancreatic tumors sections ( n = 8 and 9 mice / group respectively , left panel ) . Data are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * * : p < 0 . 01 . Representative images of trichrome staining in sg - CTRL and sg - HMGCL pancreatic tumors . Scale bar : 100 µm ( right panel ) . D , E Quantification of volume ( D ) and weight ( E ) of sg - CTRL pancreatic tumors treated with 0 . 9 % NaCl ( i . p . ) ( n = 4 ) , and pancreatic tumors from two different clones of sg - HMGCL treated with 0 . 9 % NaCl ( i . p . ) or βOHB ( 100mg / kg / bi - weekly , i . p . ) ( n = 10 / group ) . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . ns : not significant , * * p < 0 . 01 . H [ U - 13C ] βOHB tracing into TCA intermediate : citrate in sg - CTRL and sg - HMGCL # 2 and # 3 PANC - 1 cells cultured in indicated glucose concentrations . Data are expressed as mean ± SEM . ( n = 2 independent experiments ) . Significance was defined by one - way ANOVA followed by a Bonferroni ' s multiple comparisons test , only significances between sg - HMGCL # 2 , # 3 PANC - 1 cells and sg - CTRL PANC - 1 cells under the same culture condition are mentioned . * * * p < 0 . 001 .

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Figure 4A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.


Figure 5 ( B ) Box - and - whisker plots of ketone bodies ( KB ) metagene score defined as first component of PCA of genes in primary tumors ( n = 728 ) , in all metastases ( n = 76 ) , and in liver metastases specifically ( n = 35 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined at 1 . 5x IQR . Significance was defined by Student ' s t - test . D - F Effect of βOHB treatment on liver metastatic incidence , size and status . Representative HPS staining of liver lobes from metastatic mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 8 mice / group ) . Metastatic areas are separated from liver by yellow dotted lines . Scale bar : 1mm and 500 µm for insets ( D ) . Number and size of metastasis per lobe and classified in small , medium and large size ( E ) . Pathological status of metastasis from mice treated with βOHB or NaCl ( n = 13 or 5 lobes / group , respectively ) . Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis ( F ) .

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Figure 5(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).


Figure 1 ( a , b ) Isolated acini from wild - type ( WT ) and Trpml1 - / - mice ( ML1 - / - ) were used to determine the frequency of Ca2 + oscillations in response to stimulation with 3 and 10 pM CCK ( a ) and the response to high concentrations of CCK ( b ) . The average frequency from multiple experiments is given in the columns as mean ± s . e . m in ( a ) . The traces and columns in ( b ) show the mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 cells . ( c - e ) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations . ( c ) shows exocytosis during the first 5 min of stimulation , ( d ) shows the two phases , first rapid and second sustained phase , of exocytosis and ( e ) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation . The results are averages of 3 - 4 experiments with each mouse line . denotes p < 0 . 01 or better and # denotes p < 0 . 05 or better .

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Figure 1(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.


Figure 2Panel ( a ) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution . Granules size was analyzed in at least 10 images from 3 wild - type and 5 Trpml1 - / - mice . ( b ) Fixed acini were co - stained with Amylase ( granules ) and LAMP1 ( lysosomes ) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) Live wild - type and Trpml1 - / - acini were labeled with mitotracker ( green ) and lysotracker ( red ) . The panels show the merged green , red and DIC images . The large vesicles are marked by white arrows and shown at higher magnification in the inserts . The overlap between lysosomes and granules was determined by ImageJ ( see methods ) . The results are from 14 wild - type and 30 Trpml1 - / - acini each comprising 3 - 7 cells and obtained from 3 wild - type and 4 Trpml1 - / - mice . ( d ) Co - IP of lysosomal LAMP1 and granules VAMP8 and Rab27b . Note the increased lysosomal / granules interaction . ( e ) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . The results are the mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type and 3 Trpml1 - / - mice .

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Figure 2Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice.(b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively.(c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice.(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.


Figure 3 ( a , b ) Plasmaamylase ( a ) and pancreaticMPO ( b ) in 3 mice of each line . ( c ) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H & amp ; amp ; E staining . Shown are examples of images and the average damage in the columns , which show the mean ± s . e . m of 4 experiments with each mouse line . ( d ) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min . Shown are example images and the averages . ( e ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide . The figure shows example images before stimulation and the averaged fluorescence intensity . ( f ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3 . The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as the mean ± s . e . m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment .

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Figure 3(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line.(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.


Figure 4 ( a ) Isolated acini from wild - type ( black ) and Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . The traces are the mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments . ( b , c ) Saliva was collected from wild - type ( black ) and Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like and the results are given as the mean ± s . e . m . ( d ) the saliva collected in ( c ) was used to determine the amount of secreted amylase . ( e , f ) Isolated acini from the parotid glands of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times ( e ) and the indicated isoproterenol concentrations during 30 min stimulation ( f ) . The results are the mean ± s . e . m obtained from two mice of each line .

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

Figure 4(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.(d) the saliva collected in (c) was used to determine the amount of secreted amylase.(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


Figure 5 ( a ) shows example TEM images and ( b ) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild - type mice and 14 images obtained from 4 Trpml1 - / - mice . ( c ) Co - IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b . Input control is actin . ( d ) Acini isolated from the parotid glands of wild - type and Trpml1 - / - mice were incubated with mitotracker ( green ) and lysotracker . The large vesicles in the secretory granules / lysosomal area are marked by white arrows . The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line . ( e ) Shown is the time course ( left panel ) and averaged 10 min secretion ( columns ) of the lysosomal marker acid phosphatase in the saliva collected from wild - type ( black ) and Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol . The mean ± s . e . m is from 4 mice of each line .

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

Figure 5(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


Figure 6 ( a , b ) example traces of mEPSCs recorded from wild - type ( a ) and Trpml1 - / - ( b ) micebrain slices as detailed in Methods . The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of the events at each mEPSC . The lower columns show the distribution of the higher amplitude mEPSCs . ( d ) the frequency of mEPSCs in each line . The results in ( c , b ) are the mean ± s . e . m of 8 experiments each obtained from 4 mice of each line . ( e ) the mean ± s . e . m of the Ca2 + signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild - type and Trpml1 - / - slices , respectively .

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

Figure 6(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


Figure 7 ( a ) example TEM images of synapses in wild - type and Trpml1 - / - micebraincortex . ( b ) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) example TEM images at low magnification of wild - type and Trpml1 - / - micebraincortical slices with the dark PSD profiles marked by white arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 images obtained from 3 mice of each line . ( e ) Glutamate levels and secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) and Trpml1 - / - ( green ) neurons and neurons stimulated by depolarization with 30 mM KCl ( blue and red ) . ( f ) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) and Trpml1 - / - neurons transfected with TRPML1 ( black ) and untransfected ( red ) and TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl . ( g ) averaged Co - IP of SNAP25 and VAMP2 in three brain regions of wild - type and Trpml1 - / - mice . The images of all blots are given in Extended view Figure E4 .

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

Figure 7(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


Figure 1293T cells were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or the indicated constructs . At 24 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , and analyzed by IB using antibodies against PGAM5 and FLAG . Five percent of lysates was analyzed as input . HeLa cells stably expressing FLAG - Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - test ) . MBP or the MBP - Stx17 constructs attached to amylose resin were mixed with GST - PGAM5 , and the proteins bound to the resin were separated by SDS - PAGE and blotted onto PVDF membranes . The blots were detected by an anti - GST antibody ( upper panels ) or stained with Coomassie Brilliant Blue R - 250 ( lower panels ) . Ten percent of the proteins used for each experiment was analyzed as input . Asterisks and double asterisk may represent MBP dimers and degradation products . 293T cells were cotransfected with plasmids encoding FLAG - Stx17 WT and the indicated PGAM5 - GFP constructs , and analyzed293T cells were cotransfected with plasmids encoding FLAG - Stx17 or Stx18 and the C - terminally GFP - tagged transmembrane domain of PGAM5 （ amino acids 1 - 35 ） constructs , and

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

Figure 1293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed


Figure 2HeLa cells were treated with DMSO ( Vehicle ) or 0 . 03 mg / ml digitonin ( + Digitonin ) , fixed and then double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PGAM5 and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' sHeLa cells stably expressing FLAG - Stx17 wild type ( WT ) were transfected with a plasmid encoding Su9 - GFP ( mitochondria ) or Sec61β - GFP ( ER ) . At 24 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PLA dots and Su9 - GFP or Sec61β - GFP . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 ( paired Student ' s t - test ) . HeLa cells were treated with DMSO ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and subjected to Percoll - based fractionation . Equal amounts of proteins were analyzed by IB using the indicated antibodies . PNS , postnuclear supernatant ; MS , microsomes ; Mt , mitochondria . The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment , respectively : PNS ( 6 . 6 mg and 5 . 5 mg ) , cytosol ( 4 . 8 mg and 4 . 9 mg ) , MS ( 2 . 0 mg and 2 . 2 mg ) , MAM ( 0 . 55 mg and 0 . 46 mg ) and Mt ( 0 . 30 mg and 0 . 28 mg ) . Electron microscopic analysis of HeLa cells expressing PGAM5 - GFP and APEX2 - GFP - binding peptide . Arrows indicate the position of 3 , 3 ' - diaminobenzidine reaction at the ER - mitochondria interface . Scale bar , 500 nm . HeLa cells stably expressing FLAG - Stx17 WT were mock - transfected or transfected with siRNA for Mfn1 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) . HeLa cells were mock - transfected or transfected with siRNA for Mfn1 , Stx17 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against Drp1 and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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

Figure 2HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student'sHeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


Figure 3HeLa cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed and double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . 293T cells with mock treatment or depleted of Stx17 were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and then analyzed by IB using the indicated antibodies . 293T cells transiently expressing FLAG - Stx17 wild type ( WT ) or the K254C mutant were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . HeLa cells stably expressing FLAG - Stx17 WT were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' s t - test ) .

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

Figure 3HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).


Figure 4HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted ( KD ) of Stx17 , PGAM5 or Drp1 were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies . HeLa cells stably expressing GFP - Parkin with mock treatment ( Mock ) or depleted of Stx17 , PGAM5 or Drp1 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by immunofluorescence microscopy . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of GFP - Parkin and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) . PINK1 - FLAG and GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( ethanol ) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies . F , full - length PINK1 ; C , cleaved PINK1 .

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

Figure 4HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.


Figure 5GFP - Parkin stably expressing HeLa cells were mock - transfected ( Mock ) or transfected with siRNA ( KD ) for Stx17 or PGAM5 . At 48 h after siRNA transfection , the cells were transfected with a plasmid encoding FLAG - DFCP1 , incubated for 24 h , treated with 20 μM CCCP for 2 h and then immunostained for FLAG . Scale bars , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and FLAG - DFCP1 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) . GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3 . Scale bar , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and LC3 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) . GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by IB using the indicated antibodies .

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Figure 5GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.


Figure 6HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies . GFP - Parkin stably expressing HeLa cells with mock treatment ( Mock ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20 . Scale bar , 5 μm . 293T cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . Three percent of lysates was analyzed as input . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . GFP - Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5 . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * P < 0 . 05 and * * P < 0 . 01 as compared with Mock ( + CCCP ) by paired Student ' s t - test .

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Figure 6HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.


Figure 7A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss . Transmission electron microscope images of the indirect flight muscle in the indicated genotypes ( a , Control ; b , Stx17 - / - ; c , Stx17 - / - and Stx17 overexpression ; d , Stx17 - / - and PGAM5 overexpression ) of 7 - day - old adult flies are shown . The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages ( mean ± SEM ) using the scoring system : Class 0 , normal ; Class 1 , fuzzy or dilated cristae ; Class 2 , fragmented cristae and loss of electron density . * p < 0 . 05 , # p < 0 . 001 vs . the same class of control ( Dunnett ' s test ) . n = 50 - 119 mitochondria from three or four independent samples . Genotypes used were : + / y ; Act5c - GAL4 / + ( a ) , + / y ; Act5c - GAL4 / + ; Stx17LL06330 / Stx17LL06330 ( b ) , + / y ; Act5c - GAL4 / UAS - FLAG - Stx17 ; Stx17LL06330 / Stx17LL06330 ( c ) , + / y ; Act5c - GAL4 / UAS - PGAM5 ; Stx17LL06330 / Stx17LL06330 ( d ) . Scale bars , 200 nm . B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration . a , Stx17 + / - ; b , PGAM5 - / - ; c , Stx17 + / - , PGAM5 - / - . The bar graph on the right represents the mitochondrial phenotypes classified p < 0 . 05 , * * p < 0 . 01 vs . the same class of Stx17 + / - ( Dunnett ' s test ) . n = 62 - 195 mitochondria from three independent samples . Scale bars = 1 µm . Genotypes used were : + / y ; Act5c - GAL4 / + ; Stx17LL06330 / + ( a ) , PGAM51 / y ; Act5c - GAL4 / + ( b ) , PGAM51 / y ; Act5c - GAL4 / UAS - Stx17 - FLAG ; Stx17LL06330 / + ( c ) .

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Figure 7A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm.B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).


Figure 3 ( D ) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 ( n = 3 ) or WT EAAT2 ( n = 3 ) . Cells were intracellularly dialyzed with a 115mM KGluc - based solution and held at 0 mV .

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Figure 3(D) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 (n=3) or WT EAAT2 (n=3). Cells were intracellularly dialyzed with a 115mM KGluc-based solution and held at 0 mV.


Figure 4 ( B ) Representative WT and mutant whole - cell current responses to voltage jumps before and after removal of external K + . Cells were dialyzed with a KNO3 - based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3 . ( C ) Relative changes in fluorescence for WT , D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [ K + ] . Solid lines represent non - parametric fits , and shaded area show bootstrapped 99 % confidence intervals for those fits . Non - overlapping confidence intervals indicate significant difference between WT and mutant binding curves . Values are given as mean ± SD ( n ≥ 3 ) . ( D ) Representative whole - cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline - NO3 - based external solutions or in NaNO3 - based solutions supplemented with 1 mM L - glutamate . Glutamate - induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 ( GltPh D312 and D405 ) are K + - independent , but are functionally expressed in the plasma membrane . ( E ) Current - voltage relationships of steady - state currents from cells dialyzed with a KNO3 - based pipette solution . Currents were measured at external 140 mM KNO3 and normalized to the current at - 170 mV from a consecutive recording of the same cell in K + - free external solutions . Values are given as mean ± SD ( n ≥ 3 )

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Figure 4(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)


Figure 6 ( E ) Representative transport current recordings from cells expressing L448A or L448T ( GltPh A360 ) or WT EAAT1 upon voltage jumps to ˗ 140 mV before and after superfusion with 1 mM l - glutamate . Cells were intracellularly dialyzed with a K + - free solution without permeating anions to isolate K + - independent transport currents . Transient capacitive currents during the first 5 ms after a voltage jump were blanked . ( F ) Current - voltage relationship for net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for choline - based pipette solutions . Values are given as mean ± SD with indicated numbers of experiments . ( G ) Time - dependent changes in L448A EAAT1 ( GltPh A360 ) uptake currents during two repetitive glutamate applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus the time . Glutamate application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace . ( H ) Net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for different pipette solutions , Values are given as mean ± SD with indicated numbers of experiments ( * * * , p < 0 . 001 Student ' s t - test ) .

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Figure 6(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.(G) Time-dependent changes in L448A EAAT1 (GltPh A360) uptake currents during two repetitive glutamate applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus the time. Glutamate application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.(H) Net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for different pipette solutions, Values are given as mean ± SD with indicated numbers of experiments (***, p<0.001 Student's t-test).


Figure 7 ( E ) Representative transport current recordings from a cell expressing R478A EAAT2 ( GltPh R397 ) in the absence of internal K + . Transport currents were elicited by voltage jumps to - 140mV before and after superfusion with 5 mM l - serine . Transient capacitive currents during the first 5 ms after a voltage jump were blanked . ( F ) Time - dependent changes in steady - state R478A EAAT2 ( GltPh R397 ) uptake currents during two consecutive serine applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus time . Serine application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace . ( G ) Current - voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments . ( H ) Mean net transport currents for WT and R478A EAAT2 ( GltPh R397 ) induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments .

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Figure 7(E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.(F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.(G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.(H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.


Figure 1 ( A - B ) Western - Blotting ( WB ) of p53 - negative lung cancer H1299 cells stably expressing ( A ) or transiently expressing ( B ) the indicated constructs . Post - translationally modified Δ133p53 and Δ160p53 isoforms are indicated with * . ( C ) The same WB membrane containing lysates from R273Hp53 - expressing A431cells was incubated with rabbit polyclonal CM - 1 antibody and mouse monoclonal 1801 antibody against the N - terminus of p53 . Detection using anti - rabbit IRDye 680LT ( red ) and anti - mouse IRDye 800CW ( green ) secondary antibodies confirmed that the Δ160p53 band corresponds to a C - terminal isoform of p53 . ( D ) WB of H1299 cells transiently expressing the indicated constructs . ( E ) WB of cell lines expressing wild - type ( HCT116 , U2OS , A549 ) or mutant ( SW480 [ R273H / P309S ] , MDAMB231 [ R280K ] , A431 [ R273H ] , HT29 [ R273H ] and SKBR3 [ R175H ] ) p53 . ( F ) Cell lines harbouring endogenous wild - type ( A549 ) or R273H mutant ( A431 ) p53 were treated or not with control siRNA ( ctl ) or siRNA targeting both p53 transcripts ( ex6 and ex7 ) , full - length mRNA only ( ex2 / 3 ) or Δ133p53 mRNA only ( in4 ) before lysis and WB . ( G ) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO , proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide ( CHX ) . Shown are representative data of three independent experiments . The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α - tubulin or GAPDH and relative to the value indicated in bold and set to 1 . 0 . In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

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Figure 1(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *.(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.(D) WB of H1299 cells transiently expressing the indicated constructs.(E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53.(F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB.(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


Figure 2 ( A ) Cell lines endogenously expressing mutant R273Hp53 ( A431 and HT29 ) were treated with control siRNA ( ctl ) or siRNA targeting exon 7 of p53 , submitted to endoplasmic reticulum ( ER ) stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . Data was normalized against si ctl condition , which was set to 100 . ( B and C ) A549 ( B ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( over - confluency ( O - C ) for A549 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . Also shown is the WB of A549 cells used in ( B ) and stably expressing the indicated constructs . ( B and C ) H1299 ( C ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( thapsigargin ( Th ) for H1299 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . ( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) or cultured under regular conditions ( D ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) . ( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) , DNA - damage by 36h 3 μM etoposide ( Eto ) treatment ( F ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

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

Figure 2(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.(B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


Figure 3 ( A ) Adhesion assay shows that MCF10A cells stably expressing Δ133p53 , Δ160p53 , R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells . Excluding Δ160p53 expression from the mutant background ( M160A / R273Hp53 ) rescued control - cell phenotype . Scale bar represents 200 μm . ( B ) WB of MCF10A cells used in ( A ) and stably expressing the indicated constructs . Shown are representative data of three independent experiments and quantifications . ( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) . ( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) . ( E ) H1299 cells stably expressing empty vector ( control ) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

Figure 3(A) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm.(B) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications.(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


Figure 4 ( A ) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos ( Ctl - 1 or Ctl - 2 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation ( MO ) ( B ) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos ( Ctl - 2 or Ctl - 1 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation site ( MO ) before lysis and WB . wtp53 - expressing A549 cells were used as reference for endogenous wtp53 expression levels . Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells . ( C ) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino ( Ctl - 1 ) or morpholino targeting 160p53 ' s translation initiation site ( MO ) . MO efficiently targets 160p53 in endogenous R273Hp53 cell lines . See also figure EV3 for the raw data . ( D - E ) A431 ( D ) and HT29 ( E ) cells endogenously expressing mutant R273Hp53 were treated with control siRNA ( si Ctl ) or control MO ( MO Ctl - 2 or MO Ctl - 1 ) or siRNA targeting exon 7 of p53 ( si ) or MO targeting Δ160p53 ' s translation initiation site ( MO ) , as indicated , submitted to endoplasmic reticulum stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . wtp53 - expressing A549 cells were similarly control - treated and used as comparison . Data was normalized against A549 cell condition , which was set to 100 . ( F ) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl - 1 or morpholino targeting Δ160p53 ' s translation initiation site ( MO ) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

Figure 4(A) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53's translation initiation (MO)(B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53's translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.(C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53's translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data.(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.(F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53's translation initiation site (MO) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


Figure 1Immunoblot analysis of LC3B and GABARAP lipidation in wild - type ( WT ) and ATG16L1 - KO HEK293A cells , with or without stimulation for 2h as indicated . BafA1 stands for Bafilomycin A1 . Gamma - Tubulin ( γ - Tubulin ) is loading control . Densitometry analysis of lipidated LC3B ( LC3B - II ) to γ - tubulin ( normalized to untreated WT cells ) from immunoblots in ( A ) . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant . LC3B / GABARAP lipidation in ATG16L1 - and ATG5 - KO HEK293A cells treated for 2h as indicated . Vinculin is loading control . Cell lysates were immunoblotted for the specified proteins . Ratio of LC3B - II to Vinculin quantified from immunoblots in ( C ) and normalized to MC3 treated ATG16L1 KO cells . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant . Immunoblot analysis of LC3B and GABARAP lipidation from WT , ATG16L1 - KO ( L1 - KO ) , ATG16L2 - KO ( L2 - KO ) , TECPR1 - KO ( TEC - KO ) , ATG16L1 / L2 - DKO ( L1 / L2 - DKO ) , ATG16L1 / TECPR1 - DKO ( L1 / TEC DKO ) , and ATG16L1 / L2 / TECPR1 - TKO ( L1 / L2 / TEC - TKO ) PC - 3 cells , treated as indicated for 2h , were immunoblotted for the indicated proteins . Vinculin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant . LC3B / GABARAP lipidation in ATG16L1 - KO and ATG16L1 / TECPR1 - DKO HEK293A cells with or without TECPR1 rescue , treated as indicated for 2h . Cell lysates were immunoblotted against the indicated proteins . Vinculin is loading control . Ratio of LC3B - II to Vinculin quantified from immunoblots in ( G ) and normalized to ATG16L1 - KO cells treated with MC3 . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant .

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

Figure 1Immunoblot analysis of LC3B and GABARAP lipidation in wild-type (WT) and ATG16L1-KO HEK293A cells, with or without stimulation for 2h as indicated. BafA1 stands for Bafilomycin A1. Gamma-Tubulin (γ-Tubulin) is loading control. Densitometry analysis of lipidated LC3B (LC3B-II) to γ-tubulin (normalized to untreated WT cells) from immunoblots in (A). Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.LC3B/GABARAP lipidation in ATG16L1- and ATG5-KO HEK293A cells treated for 2h as indicated. Vinculin is loading control. Cell lysates were immunoblotted for the specified proteins. Ratio of LC3B-II to Vinculin quantified from immunoblots in (C) and normalized to MC3 treated ATG16L1 KO cells. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.Immunoblot analysis of LC3B and GABARAP lipidation from WT, ATG16L1-KO (L1-KO), ATG16L2-KO (L2-KO), TECPR1-KO (TEC-KO), ATG16L1/L2-DKO (L1/L2-DKO), ATG16L1/TECPR1-DKO (L1/TEC DKO), and ATG16L1/L2/TECPR1-TKO (L1/L2/TEC-TKO) PC-3 cells, treated as indicated for 2h, were immunoblotted for the indicated proteins. Vinculin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.LC3B/GABARAP lipidation in ATG16L1-KO and ATG16L1/TECPR1-DKO HEK293A cells with or without TECPR1 rescue, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. Vinculin is loading control. Ratio of LC3B-II to Vinculin quantified from immunoblots in (G) and normalized to ATG16L1-KO cells treated with MC3. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.


Figure 2LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO HeLa cells with and without EGFP - TECPR1 or EGFP - ATG16L1β rescue , treated for 2h as indicated . Cell lysates were immunoblotted for the indicated proteins . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( A ) and normalized to untreated WT cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . Confocal images of ATG16L1 / TECPR1 - DKO HeLa cells expressing mScarlet - LC3B , with or without EGFP - TECPR1 , treated for 2h with Cy5 - labeled MC3 particles ( left ) or LLOMe ( right ) as indicated . Scale bars : 10 µm . Confocal time lapse images of ATG16L1 / TECPR1 - DKO HeLa cells , showing localization of mScarlet - LC3B and EGFP - TECPR1 , after treatment with 400 μM LLOMe . Scale bar : 10 µm . Immunoblot analysis of LC3B / GABARAP lipidation in ATG16L1 - KO HEK293A cells , treated with 400µM LLOMe for indicated periods of time . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( E ) and normalized to 2hr ( 120min ) LLOMe treatment . ( Data presented as mean + SEM , n = 4 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ) .

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

Figure 2LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO HeLa cells with and without EGFP-TECPR1 or EGFP-ATG16L1β rescue, treated for 2h as indicated. Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (A) and normalized to untreated WT cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing mScarlet-LC3B, with or without EGFP-TECPR1, treated for 2h with Cy5-labeled MC3 particles (left) or LLOMe (right) as indicated. Scale bars: 10 µm.Confocal time lapse images of ATG16L1/TECPR1-DKO HeLa cells, showing localization of mScarlet-LC3B and EGFP-TECPR1, after treatment with 400 μM LLOMe. Scale bar: 10 µm.Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO HEK293A cells, treated with 400µM LLOMe for indicated periods of time. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (E) and normalized to 2hr (120min) LLOMe treatment. (Data presented as mean + SEM, n = 4 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test).