( 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 .

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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 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 .

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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 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 .

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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 − / − 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 all cells . Fat body was dissected from well - fed larvae and 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 cells . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and 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 body containing a GFP - marked clone of CiCell - expressing cells .

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


( 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 . The arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP fluorescence before and 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 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 .

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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 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 ,

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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 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 .

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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 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 .

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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 proteins in lactacystin and untreated cells . 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 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 .

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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 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 ) .

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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 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 .

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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 ( ± 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 and 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 concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 assay of HIS - tagged C9ORF72 : SMCR8 : WDR41 complex purified from baculovirus infected insect cells .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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

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


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 ) .

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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 . 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 .

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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.


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

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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 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

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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 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

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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 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

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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 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

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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 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

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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 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

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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 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 )

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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 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 )

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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 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

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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 and eosin sections of BAT from WT and CPEB2 - KO female mice at 4 and 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 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

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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


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

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


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

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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 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

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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 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

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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 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

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 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 .

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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 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 ) .

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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 the distributions of fractions of total RPFs and mRNA reads in each reading frame for all 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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 - light Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . The 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O 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 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 O O O B-MEASURED_VAR 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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.