( g ) U20S stably expressing HaloTag - p62 were labelled with HaloTag ligand for 15 min washed out followed by treatment with purmorphamine . After 48 h , were lysed , run on a SDS - polyacrylamide gel electrophoresis HaloTag was visualized using a Typhoon 8600 variable mode imager . was normalized to total , detected on same gels by Kryton stain . A representative gel its quantitated normalized are shown . In panels , graphs show mean values error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - unless indicated : * * * P0 . 001 ; * * P0 . 01 ; * P0 . 05 . SDS - PAGE , SDS - polyacrylamide gel electrophoresis .

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(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.


( a ) HeLa were transiently transfected with pcDNA ( vector ) , Ptch1 or Ptch2 constructs for 48 h . Where indicated , were treated with bafA1 for last 4 h . LC3 - II were detected by western blotting were quantified by densitometric relative to tubulin shown in graph .

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(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.


( b ) HeLa were transfected with Ptch1 or Ptch2 together with HA - HttQ74 of transfected with was assessed by HA immunofluorescence . P - values were calculated by odds .

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(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.


( c ) Atg5 + / + or Atg5 − / − MEFs were transfected with GFP - HttQ74 for 48 h , were fixed of transfected with was scored by direct .

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(c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence.


( d , e ) Endogenous LC3 - II were detected in HeLa transfected with control , Ptch1 or Ptch2 siRNA either left untreated or treated with 100 mM trehalose in last 24 h ( d ) statistical is shown in graphs .

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.


( d , e ) Endogenous LC3 - II were detected in HeLa transfected with control , Ptch1 or Ptch2 siRNA either left untreated or treated with400 nM bafA1 for 4 h ( e ) . statistical is shown in

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs


( f ) siRNA - targeting Ptch2 was transfected into HeLa stably expressing mRFP - GFP - LC3 representative confocal microscopy are shown . Scale bars represent 26 μm . of GFP - positive vesicles ( autophagosomes ) , RFP - positive vesicles ( total number of vesicles ) or of autolysosomes ( calculated by subtracting numbers of GFP from RFP vesicles ) , quantified using a Cellomics array scan , was calculated relative to control siRNA - transfected . Although an increase in autophagosome biogenesis generally increases both number of GFP + vesicles number of GFP - / RFP + vesicles as a result of an enhanced autophagy flux , defects in autophagosome degradation , such as in bafA1 - treated , increase GFP + vesicles but not GFP − / RFP + structures , as GFP remains intact . Graph shows mean value obtained from four independent experiments in triplicate with control conditions set to 100 . In panels , unless indicated , graphs show mean values error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - : * * P0 . 01 ; * P0 . 05 ; NS , not significant .

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(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.


( a ) Smo siRNA - treated were transfected with HA - HttQ74 for last 24 h of transfected with scored in HA - positive by immunofluorescence . Graphs show mean values error bars represent s . d . of a representative experiment performed in triplicate . P - values were calculated by odds .

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(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio.


( b ) siRNA - targeting Smo was transfected into HeLa stably expressing mRFP - GFP - LC3 . Representative confocal microscopy are shown . Bars represent 26 μm . number of vesicles was analyzed in a Cellomics array scan was calculated relative to control siRNA - transfected . Statistical were performed on data from three independent experiments in triplicate . * P0 . 05 ; * * * P0 . 001 .

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(b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.


( a ) Wild - type Gli + / + or Gli1 − / − , Gli2 − / − Gli3 − / − MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total lysates were subjected to immunoblot with anti - LC3 anti - actin antibodies .

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(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( b ) Gli2 − / − MEFs were transfected for 48 h with either empty vector or Gli2 construct treated as in a . with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total lysates were subjected to immunoblot with anti - LC3 anti - actin antibodies .

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(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( c ) LC3 - II were detected by western blotting of total lysates from wild - type Gli2 − / − mice ( E12 . 5 ) . For , LC3 - II from a total of five wild - type five Gli2 − / − were normalized to actin , wild - type was set to 100 . Graphs show mean values error bars represent s . d . Statistical were performed by two - tail Student ' s t - * * P0 . 01 . Pmph , purmorphamine .

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(c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.


( a , b ) Control ( a ) or hh [ ts2 ] ( b ) larvae were incubated at permissive ( 18 ° C ) to third instar stage , then cultured at non - permissive ( 29 ° C ) in well - fed conditions for 24 h . Dissected fat tissue was incubated in Lysotracker Red DAPI imaged unfixed .

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(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed.


( c ) Lysotracker Red - labelled fat containing single - flipout clones ( GFP positive ) overexpressing patched . Fat was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI imaged unfixed .

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(c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( d ) Confocal image of a GFP - positive patched - expressing clone , with mCherry - Atg8a expressed in . Fat was dissected from well - fed larvae imaged after formaldehyde fixation .

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(d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation.


( e ) GFP - marked flipout clones of CiCell - expressing . Fat was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI imaged unfixed .

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(e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( f ) Confocal image of fixed mCherry - Atg8a expressing fat containing a GFP - marked clone of CiCell - expressing .

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(f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells.


( g ) homozygous for null costal2 [ 2 ] allele ( marked by lack of GFP ) were generated by flp / FRT - mediated recombination in mCherry - Atg8a expressing fat . Larvae were starved 4 h before dissection fixation .

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(g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCherry-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation.


( a ) Graph representing fold change in mRNA in treated with Shh against untreated . Each circle represents an autophagy gene ; those with largest fold - changes are indicated in red . central represents no changes in expression ; above central , genes whose expression is increased : below , those with reduced . Grey lines indicate 2 - fold increase or decrease . ( b ) Table showing 10 genes with higher fold changes in expression values obtained .

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(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained.


( c ) of PERK were assessed by western blotting in HeLa treated with 10 μM purmorphamine for 24 h . Actin was used as a loading control .

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(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.


( d ) HeLa were treated with purmorphamine for 24 h either left in rich media or in HBSS for last 4 h . of S51 phoshorylation total of eIF2α were detected by western blotting using LI - COR infra - red imager .

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(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.


( e , f ) Wild - type PERK − / − ( e ) or eIF2α S51A / S51A MEFs ( f ) were treated with DMSO or 10 μM purmorphamine for 24 h , where indicated , were treated with bafA1 for last 4 h . tubulin - II tubulin or tubulin , as a loading control , were detected a representative blot is shown . HBSS , Hank ' s balanced salt solution .

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(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.


A ) Differential interference contrast ( DIC ) image of cortical neurons in culture . arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP before after exposure to lactacystin ( 10µM ) .

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A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).


C ) A neuron coexpressing Ub - R - GFP CFP before ( left ) 10 hours after exposure to lactacystin ( right ) . Top row - GFP ; bottom row - CFP . Bars : 50µm ( A , B ) , 20 µm ( C ) . D ) Changes in GFP CFP following exposure to lactacystin at t = 1 hour ( vertical dashed ) . Changes normalized to initial in same . 9 neurons from 5 experiments ; averages . E ) Same as in D , except that here neurons were exposed only to carrier solution . 11 neurons from 5 experiments ; averages ( barely observable ) . F ) Comparison of GFP accumulation rates . Linear fits shown as black lines ; fit parameters shown next to fits . Same data as in D , E .

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C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.


A ) Hippocampal neurons in culture immunolabeled against three synaptic ( postsynaptic scaffold molecule Dlg4 / PSD - 95 , presynaptic active zone molecule RIM presynaptic vesicle SV2A ) following 10 hour incubations in carrier solution ( control , upper panels ) or Lactacystin ( 10 μM ; bottom panels ) . Scale bar ,

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A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm


B ) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution . Numbers within bars represent number of fields of view analyzed for each condition ( ~ 170 synapses / field of view , on average ) . Error bars , ; P values - two unpaired t - . Note that a statistically significant accumulation was observed only for RIM , whereas immunolabeling of other was either unchanged or reduced .

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B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.


C ) Hippocampal neurons in culture were grown in presence of Lactacystin ( 10 μM ) , fixed at two hour intervals immunolabeled against Shank3 / ProSAP2 . Two representative fields of view at t = 0 t = 24h . Scale bar , 10μm . D ) Average synaptic Shank3 / ProSAP2 immunofluorescence at increasing times in presence of Lactacystin . was normalized to mean at t = 0 . Numbers under data points reflect numbers of fields of view analyzed at each time point . Error bars , .

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C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.


B ) Comparisons of H / L ratios obtained for 1409 in lactacystin untreated . H / L ratios are averages of 2 experiments .

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B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.


A ) Fold changes in H / L ratios ( ) measured for each in experiments described in Fig . 3 ( expressed as log2 ( H / L ) Lactacystin - log2 ( H / L ) control ) . H / L ratios used here are average of ratios obtained in two experiments for each each condition . were sorted according to fold change in H / L ratios - from whose ratios were most strongly reduced ( ~ 8 - fold reduction ) to whose H / L ratios increased to greatest extent ( > 8 - fold increase ) . Red circles indicate location of synaptic along this sorted list . names of 34 synaptic located at left most region of sorted list are provided in order of their appearance . B ) apparent fold - change in half - lives under certain assumptions described in Results . See Materials Methods for further details on calculation of apparent fold change in half - lives .

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A) Fold changes in the H/L ratios ( ) measured for each protein in the experiments described in Fig. 3 (expressed as log2(H/L)Lactacystin - log2(H/L)control). H/L ratios used here are the average of ratios obtained in two experiments for each protein and each condition. Proteins were sorted according to the fold change in H/L ratios - from the proteins whose ratios were most strongly reduced (~8-fold reduction) to proteins whose H/L ratios increased to the greatest extent (>8-fold increase). Red circles indicate the location of synaptic proteins along this sorted list. The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance. B) The apparent fold-change in protein half-lives under certain assumptions described in Results. See Materials and Methods for further details on the calculation of the apparent fold change in half-lives.


A ) Average log2 ( H / M ) ratios for 1 , 750 at four time points in three separate experiments . Each time point shows average standard deviation of three mean log2 ( H / M ) values obtained in three separate experiments . Each mean , in turn , represents mean log2 ( H / M ) value measured in population of 1 , 750 in that experiment time point . B ) Distribution of log2 ( H / M ) values for 1 , 750 at t = 0 . log2 ( H / M ) value of each represents average value for that in three experiments . In principle , log2 ( H / M ) for at t = 0 should be 0 , but in practice they are distributed around 0 as shown here . H / M ratios for 3 standard deviation limits are shown beneath X axis . C , D ) same as in A B but for two experiments in which Epoxomicin was used ( 1 , 823 ) . E , F ) same as in A B but for combined data from experiments ( 3 experiments in which lactacystin was used 2 experiments in which epoxomicin was used ; 1 , 469 ) .

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A) Average log2(H/M) ratios for 1,750 proteins at four time points in three separate experiments. Each time point shows the average and standard deviation of three mean log2(H/M) values obtained in three separate experiments. Each mean, in turn, represents the mean log2(H/M) value measured in the population of 1,750 proteins in that experiment and time point. B) Distribution of log2(H/M) values for 1,750 proteins at t=0. The log2(H/M) value of each protein represents the average value for that protein in the three experiments. In principle, log2(H/M) for all proteins at t=0 should be 0, but in practice they are distributed around 0 as shown here. The H/M ratios for 3 standard deviation limits are shown beneath the X axis. C, D) The same as in A and B but for two experiments in which Epoxomicin was used (1,823 proteins). E, F) The same as in A and B but for the combined data from all experiments (3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used; 1,469 proteins).


A ) log2 ( H / M ) values for 5 synaptic for which degradation rates were significantly slowed down in presence of lactacystin . Each point represents average log2 ( H / M ) from 3 experiments . B ) log2 ( H / M ) values for same 5 synaptic in experiments in which epoxomicin was used . Each point represents average log2 ( H / M ) from 2 experiments . C ) Average ( ± ) log2 ( H / M ) ratios for same 5 synaptic , on five experiments ( 3 lactacystin , 2 epoxomicin ) . P values - two - Welch ' s t - , comparing five log2 ( H / M ) measurements at t = 24 to those measured for same at t = 0 .

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A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.


D , E , F ) Average ( ± ) Log2 ( H / M ) values for three groups of synaptic ( synaptic vesicle , presynaptic , postsynaptic ) , on five experiments . None of changes observed for these were statistically significant . temporal changes common to groups most likely represent slight imperfections in H / M normalization process ( Compare with Fig . 6E ; see Results for further details ) .

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D,E,F) Average (± SEM) Log2(H/M) values for three groups of synaptic proteins (synaptic vesicle, presynaptic, postsynaptic), based on all five experiments. None of the changes observed for these proteins were statistically significant. The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H/M normalization process (Compare with Fig. 6E; see Results for further details).


G ) log2 ( H / M ) values at t = 24 hours for a group of well - studied synaptic , sorted from highest to lowest . P values ( two - Welch ' s t - ) of comparisons with log2 ( H / M ) values for same at t = 0 are shown in parentheses . that exhibited statistically significant changes ( P ≤ 0 . 05 ) at t = 24h are shown in red . for which ( 0 . 05 < P ≤ 0 . 10 ) at t = 24h are shown in blue / red . gray region depicts three standard deviations of log2 ( H / M ) values at t = 0 ( Fig . 6F ) , to provide a measure of method ' s sensitivity .

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G) log2(H/M) values at t=24 hours for a group of well-studied synaptic proteins, sorted from highest to lowest. P values (two-tailed Welch's t-test) of comparisons with log2(H/M) values for the same proteins at t=0 are shown in parentheses. Proteins that exhibited statistically significant changes (P≤0.05) at t=24h are shown in red. Proteins for which (0.05 < P ≤ 0.10) at t=24h are shown in blue/red. The gray region depicts three standard deviations of log2(H/M) values at t=0 (Fig. 6F), to provide a measure of the method's sensitivity.


Measured log2 ( H / M ) values at four time points as a function of their half - lives estimated here elsewhere ( Cohen et al , 2013 ) . log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - Welch ' s t - ) are shown in red . Expected log2 ( H / M ) values on equation 4 are plotted as blue lines . To minimize masking of potential dependencies by slight imperfections in H / M normalization process ( which introduces small offsets along Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from measured Log2 ( H / M ) values . Data for 1 , 416 for which half - life estimates were available .

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Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.


Measured Log2 ( H / M ) values at four time points as a function of their half - lives estimated here elsewhere ( Cohen et al , 2013 ) . log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - Welch ' s t - ) are shown in red . Expected log2 ( H / M ) values on equation 4 are plotted as blue lines . To minimize masking of potential dependencies by slight imperfections in H / M normalization process ( which introduces small offsets along Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from measured Log2 ( H / M ) values . Data for 174 synaptic for which half - life estimates were available .

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Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.


( A ) Silver of extracted from N2A mouse neuronal expressing Flag - HA - tagged C9ORF72 captured through consecutive anti - Flag anti - HA affinity purification steps .

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(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps.


( B ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 / or Flag - tagged SMCR8 / or Flag - tagged WDR41 .

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(B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.


( C ) Coomassie blue of Nickel - NTA affinity purification of HIS - C9ORF72 , SMCR8 WDR41 co - expressed in baculovirus infected insect .

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(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.


( D ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 HA - tagged SMCR8 with various Flag - tagged Rab GTPases .

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(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.


( E ) Immunoblot against endogenous C9orf72 , Wdr41 , Rab8a , Rab39b , Rab5 Rab7 of control ( IgG alone ) or endogenous Smcr8 immunoprecipitated from adult mouse brain .

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(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.


( F ) αP32 - radiolabelled GDP release from GST - tagged purified RAB8a as a function of increased of either recombinant purified C9ORF72 alone or in with SMCR8 WDR41 .

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(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.


( G ) Identical GDP release as in ( F ) but using recombinant purified GST - tagged RAB39b instead of RAB8a . Error bars indicate s . e . m . Experiments were repeated 3 times ( n = 3 ) .

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(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).


( A ) Left panel , representative of organotypic cultures of E18 mouse cortical neurons transfected with GFP - RFP - LC3B transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA treated or not with Torin . Right panel , of LC3B .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.


( B ) Upper panel , immunoblot of endogenous LC3B ( Map1lc3b ) , C9orf72 control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA treated or not with Torin . Lower panel , real time RT - qPCR of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA .

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(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.


( C ) Left panel , representative of immunofluorescence labeling of endogenous P62 ( Sqstm1 ) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , of P62 .

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(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.


( D ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting C9orf72 plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , of P62 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot of HA - immunoprecipitated lysate of HEK293 co - expressing HA - tagged C9ORF72 HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD .

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(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.


( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase in presence of γP32 - radiolabelled ATP . were separated by SDS page migration phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) .

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(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).


( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase of HIS - tagged C9ORF72 : SMCR8 : WDR41 purified from baculovirus infected insect .

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(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.


( D ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting 3 ' UTR of Smcr8 mRNA plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A T796A ; TD , S402D T796D ; UD , S400D , S492D , S562D T666D ) . Right panel , of P62 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.


( E ) Left panel , representative of immunofluorescence labeling of transfected constructs ( Green ) endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal transfected with siRNA targeting Tbk1 plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , of P62 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot of endogenous Tdp - 43 , C9orf72 control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA .

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(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.


( B ) Left panel , representative of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , of Tdp - 43 .

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(B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.


( C ) Left panel , representative of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , of with of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - , * * * indicates p < 0 . 001 . n = 3 .

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(C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( A ) Left panel , representative of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , of ATXN2 . Scale bars , 10 µm . Nuclei were counterstained with DAPI .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.


( B ) viability ( tetrazolium ) of GT1 - 7 neuronal co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - , * * * indicates p < 0 . 001 . n = 3 .

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(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( C ) Tracing of swimming trajectories of 48 hours post - fertilization zebrafish larvae following touch . ( D - F ) of touch - evoked swimming ( D ) , average velocity ( E ) , maximum velocity attained ( F ) shows significant functional impairment of zebrafish injected with HA - tagged ATXN2 with intermediate of polyQ ( Q30x ) antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to sole injection of AMO against C9orf72 .

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(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.


( G ) Representative of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) of motor neuron axonal demonstrates a significant decrease of axonal in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . n = 3 .

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(G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


E F . TLC of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . FL - hcGAS was 0 . 265 mg / mL . tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , 0 . 85 mg / mL . extracted total concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , 0 . 5 mg / mL . NC indicates negative control that has only FL - hcGAS . dsDNA ISD at a of 0 . 05 mg / mL was used as a positive control . samples were prepared in 20 mM HEPES at pH 7 . 5 150 mM NaCl .

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E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


F . Western blot of OptiPrep gradient fractions with or without DNase RNase treatments .

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F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.


G . three on left showing signals of endogenous cGAS in HeLa ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . scale bar represents 10 µm . scatterplot on right plots signal intensities of cGAS versus at each pixel in indicated ( yellow circle ) . H . three on left showing signals of mCherry in HEK293T ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . scale bar represents 10 µm . scatterplot on right plots signal intensities of mCherry versus at each pixel in indicated ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) of signals of cGAS vs . ( n = 13 , biological replicates ) , mCherry vs . ( n = 9 , biological replicates ) . median values were calculated used for evaluation of significance . * * * P < 0 . 001 ( two - Mann - Whitney ) .

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G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


D . microscopy showing replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative that is 1 / 16 of raw image is displayed . scale bars represent 20 µm . E . microscopy showing replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative that is 1 / 16 of raw image is displayed . scale bars represent 20 µm . F . of replacement in " D " . For each square , raw image used was evenly divided into four parts . signal ratios of Cy5 - ISD over FTSC - tRNA in four parts were calculated , averaged plotted . G . of replacement in " E " . For each square , raw image used was evenly divided into four parts . signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in four parts were calculated , averaged plotted .

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D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.


3 ' - UTR of mUcp1 . Canonical CPEs ( UUUUA1 - 2U in bold ) , reported BRF1 - binding site ( UAUUUAU , gray box ) predicted IMP2 - binding motifs ( AYAYA YA2YA , underlined ) are denoted . Ucp1L Ucp1S carrying long short 3 ' - UTRs , respectively , are resulted from alternative use of two polyadenylation signals ( Hex : AAUAAA , open box ) . ORF , open reading frame . arrowhead marks end of Ucp1S . Northern blots of Ucp1 using 5 µg BAT total denoted radiolabeled probes

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The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes


Schematic comparison of mUcp1L hUcp1 . Poly ( A ) signal ( Hex , open box ) CPE ( black box ) are denoted . RT - PCR results from human , WT ( Ucp1 + / + ) UCP1 - KO ( Ucp1 - / - ) BAT samples

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Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples


Dual luciferase reporter RT - qPCR . reporter plasmids , Fluc - mUcp1S , - mUcp1L or - hUcp1L 3 ' - UTR , Rluc were co - transfected into differentiated HIB1B adipocytes . transfected were used for luciferase activity RT - qPCR . Data are mean ± SD from 3 independent experiments . Translation efficiency was defined as normalized activity ( Fluc / Rluc ) divided by normalized level ( Fluc / Rluc ) . * * * p < 0 . 001 compared with Fluc - mUcp1S , one - way ANOVA

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Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA


Northern blotting of Ucp1 18S rRNA RT - PCR of Ucp1L Ucp1S using total RNAs from denoted BAT samples ( 2 mice per genotype , 2 - 3 mo old ) . short PCR fragments were isolated for sequencing

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Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing


Western blot using WT Ucp1 ∆ L BAT lysates . level of UCP1 was normalized to GAPDH ( 4 mo old , n = 4 per group ) . * * p < 0 . 01 , Student ' s t

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Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test


Dual luciferase reporter . reporter plasmids , Fluc - mUcp1L 3 ' - UTR , Rluc were co - transfected with plasmid expressing EGFP , or myc - tagged CPEB ( immunoblots shown at left ) into differentiated HIB1B adipocytes ( Data from 3 independent experiments ) . * p < 0 . 05 compared with EGFP , one - way ANOVA

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Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA


Western blot using BAT lysates prepared from various CPEB - WT KO ( 4 mo old , n = 4 per group ) . * p < 0 . 05 , Student ' s t

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Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test


Northern blotting of Ucp1L Ucp1S in BAT from CPEB2 - WT - KO mice at room or after 5 - h cold exposure ( CE ) at 4oC ( 5 mice per genotype , 4 - 7 mo old )

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Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)


Western blot RT - qPCR of mRNA of UCP1 GAPDH in WT KO BAT ( 13 mo old , n = 3 - 4 per group )

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Western blot analysis and RT-qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT (13 mo old, n = 3-4 per group)


Western blot of UCP1 GAPDH in BAT lysates from WT KO mice at 4 12 mo old ( n = 3 per group ) . were normalized to that of GAPDH . RT - qPCR of Ucp1 mRNA level in BAT collected from ( B ) relative to Gapdh mRNA

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Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old (n = 3 per group). Protein levels were normalized to that of GAPDH. RT-qPCR analysis of Ucp1 mRNA level in BAT collected from (B) relative to Gapdh mRNA level


Hematoxylin eosin sections of BAT from WT CPEB2 - KO mice at 4 12 mo old . Scale bars , 1

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Hematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mm


Lipid droplet number ( i . e . , number of nuclei ) in WT KO BAT ( representative shown in 4D ) were quantified displayed as arbitrary units . Three tissue sections from each BAT 3 mice per group were

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Lipid droplet size and cell number (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzed


representative of WT KO BAT . Three mice per group were

Weight and representative images of WT and KO BAT. Three mice per group were analyzed


CPEB2 - WT - KO mice were transduced with adeno - associated ( AAV ) expressing full - ( AAV _ CP2 ) or - binding domain ( AAV _ RBD ) of CPEB2 . Immunostaining of CPEB2 UCP1 in KO BAT transduced with AAV _ CP2 for 2 , with Hoechst of nuclei . Scale bar , 10 µm . See more in Fig EV4D

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CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D


Western blot of UCP1 in BAT lysates from WT KO mice 2 after AAV transduction ( n = 6 mice per group ) . * p < 0 . 05 , Student ' s t

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Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction (n = 6 mice per group). *p < 0.05, Student's t test


BAT of WT KO mice after 2 of AAV transduction measured during 12 - h time ( n = 7 mice per group ) . * p < 0 . 05 , Student ' s t

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BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12-h light time (n = 7 mice per group). *p < 0.05, Student's t test


BAT in WT KO mice , of AAV _ CP2 - transduced WT KO mice ( n = 7 mice per group ) before after i . p . injection of CL316243 ( CL ) . * p < 0 . 05 , repeated - measures two - way ANOVA

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BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA


Normalized RPF profile averaged for E . coli transcripts . Profiles generated for grown in absence presence of IPTG ( 1 mM ) . Start stop codons are shaded .

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Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.


Bar chart of measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for grown in absence presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for grown in absence presence of IPTG ( 1 mM ) .

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Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).


Comparison of synthesis rate of endogenous E . coli genes measured using Ribo - seq from this study ( in molecules / s units ) from that by Li et al . ( Li et al , 2014 ) ( in molecules / generation units ) . Each point corresponds to a single gene color denotes of transcription initiation rate to translation initiation rate ( giving RNAP / ribosome ) capturing whether transcription ( yellow ) or translation ( blue ) is more dominant .

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Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.


Transcription ( bottom ) translation ( top ) profiles for yggN , rpoH greA , computed from - seq Ribo - seq data without induction . Positions of genetic parts gene are shown below profiles . ( C ) Promoter strengths in RNAP / s units RBS initiation rates in ribosome / s units .

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Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units.


Transcription ( bottom ) translation ( top ) profiles for lacZ . Profiles are shown for in absence presence of IPTG ( 1 mM ) . Position of genetic parts gene is shown below profiles . RBS is omitted from genetic design due to its . ( E ) Measured promoter strength in RNAP / s units , RBS initiation rate in ribosomes / s units , transcriptional terminator translation termination efficiency for lacZ . Data shown for in absence presence of IPTG ( 1 mM ) .

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Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).


Translation profiles for PK - LacZ construct in cultured in absence ( bottom ) presence ( top ) of IPTG ( 1 mM ) . gene10 , middle , lacZ regions are labelled above profiles . Shaded region denotes PK , dashed lines denote start codon stop codons of gene10 LacZ .

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Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.


Fraction of total RPFs mRNA reads in each reading frame for gene10 , PK or middle , lacZ regions schematically shown below are of PK - LacZ construct . Data shown separately for cultured in absence presence of IPTG ( 1 mM ) .

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Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).


Violin plots of distributions of fractions of total RPFs mRNA reads in each reading frame for E . coli transcripts .

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Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E. coli transcripts.


Change in expression of chromosomal genes in E . coli following induction of PK - lacZ expression ( 1 mM IPTG ) . Each point represents a transcript . Differentially expressed genes ( mRNA : P < 0 . 001 absolute log2 fold - change > 1 . 37 ; translation efficiency : P < 0 . 01 ) are highlighted in color by an alternative point shape ( transcriptional regulation : purple cross ; translational regulation : orange open circle ) .

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Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).


Translation initiation rates for E . coli RBSs in harboring LacZ PK - LacZ constructs in absence presence of IPTG ( 1 mM ) . Solid shows same initiation rate for both conditions . Dotted lines denote linear regressions for data with no offset .

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Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.


Fractions of mRNA reads RPFs mapping to each synthetic expression construct ( LacZ PK - LacZ ) E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , other functions . Data shown for cultured in absence presence of IPTG ( 1 mM ) .

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Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


B HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.


C HeLa were infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for p62 . scale bar represents 5 μm . Graph represents mean % ± of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.


D Control ( CTRL ) or isopropanol - treated x - Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . scale bar represents 5 μm .

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D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.


E HeLa were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , then labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


B HeLa stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , then tested with Co - IP . Empty vector / or uninfected HeLa were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . lysates were immunoblotted for GAPDH as control for .

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

B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


A HeLa were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed labelled for endogenous SEPT7 for confocal microscopy . scale bar represents 0 . 5 μm . See also Fig EV3A .

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A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.


B HeLa were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . scale bar represents 1 μm .

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B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.


C HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , processed for CLEM . SEPT6 is shown in green , mitochondria in red , Shigella - mCherry in blue . Septin cages identified by microscopy ( FLM ) were processed for . right - most image is enlarged from boxed region in image , shows bacterium ( Sf ) surrounded by a double of autophagy ( Au ) mitochondrial ( Mt ) . scale bar represents 1 μm . See also Fig EV3B .

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C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.


D HeLa stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around bacterium . Each frame was acquired every 2 min . scale bar represents 1 μm . See also Movie EV1 .

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D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.


E HeLa were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole lysates were immunoblotted for Drp1 or Mfn1 to show efficiency of depletion . GAPDH was used as loading control . siRNA - treated were infected with S . flexneri for 4 h 40 min , then fixed for microscopy stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents mean % ± of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

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

E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


A HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . scale bar represents 5 μm .

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A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.


B HeLa were fixed labelled with antibody against SEPT7 secondary antibody coupled to HRP , processed for . right image is enlarged from boxed region in image , shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with mitochondrial ( Mt ) fission site . scale bar represents 100 nm . See also Fig EV4 .

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 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

B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.


C HeLa expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . scale bar represents 1 µm . See also Movie EV3 .

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C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.


D HeLa expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 SEPT6 association with location of mitochondrial fission . scale bar represents 1 μm .

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D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.


E HeLa were transfected with Mito - BFP ActA - SEPT7 - mCherry , fixed labelled with antibody to Drp1 for widefield immunofluorescence microscopy . scale bar represents 5 μm . Inset highlight Drp1 SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± ) shows that mitochondria in ActA - SEPT7 transfected recruit significantly more Drp1 than control ( Mito - BFP transfected alone ) . Student ' s t - , * = p < 0 . 05 .

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

E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


A HeLa were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole lysate of siRNA - treated were immunoblotted for GAPDH , SEPT7 , or Drp1 to show efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated were labelled with MitoTracker Red CMXRos , fixed for confocal microscopy . scale bar represents 5 μm .

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

A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.


B HeLa were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , fixed for quantitative confocal microscopy . Graph represents distribution in ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated from 3 independent experiments ( of at least 100 measurements per biological replicate ) . Student ' s t - , * * * = p < 0 . 001 .

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B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.


C HeLa stably expressing SEPT6 - GFP were harvested , then tested with Co - IP . HeLa expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from , extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . lysates were immunoblotted for GAPDH as control for .

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

C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


D HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , whole lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents mean % ± of relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 quantified by densitometry from 4 independent experiments . Student ' s t - , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 .

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

D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.


E HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

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 B-MEASURED_VAR O 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

E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


F HeLa were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative shown here . scale bar represents 1 μm . recruitment of Mfn1 to mitochondria was significantly increased as compared to control as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± , of at least 250 per biological replicate ) . Student ' s t - , * = p < 0 . 05 .

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 B-MEASURED_VAR 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

F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


A HeLa stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages fused mitochondria . scale bar represents 1 μm . See also Movie EV3 .

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A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.


B HeLa were treated with Drp1 siRNA for 72 h , then infected with x - Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , then samples were labelled with antibody for SEPT7 . Graph represents mean % ± of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - , * * * = p < 0 . 001 .

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B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.