F ) Dephosphorylation by the PP2A - B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted . Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . Mean and standard deviation from 3 experiments is shown .

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F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.


B ) KD values obtained by ITC measurements with full - length recombinant B56α and indicated ADAM17 variant peptides .

B) KD values obtained by ITC measurements with full-length recombinant B56α and indicated ADAM17 variant peptides.


C ) The indicated full - length murine Myc - ADAM17 derivatives were transfected into HeLa cells stably expressing YFP - B56α . YFP - B56α was purified ( IP ) and ADAM17 binding determined by Western blotting . PP2A - C ; catalytic subunit , PP2A - A ; scaffold subunit .

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C) The indicated full-length murine Myc-ADAM17 derivatives were transfected into HeLa cells stably expressing YFP-B56α. YFP-B56α was purified (IP) and ADAM17 binding determined by Western blotting. PP2A-C; catalytic subunit, PP2A-A; scaffold subunit.


D ) Dephosphorylation by the PP2A - B56α holoenzyme complex of the indicated phosphorylated GST - ADAM17 ( V724 - C827 ) fragments . The GST - ADAM17 ( V724 - C827 ) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . The mean and Standard deviation of 4 independent experiments are shown .

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D) Dephosphorylation by the PP2A-B56α holoenzyme complex of the indicated phosphorylated GST-ADAM17 (V724-C827) fragments. The GST-ADAM17 (V724-C827) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and Standard deviation of 4 independent experiments are shown.


E ) Protein expression of ADAM17 variants in the DLD - 1 Adam17 - / - cell line , determined by Western blot . GAPDH was used as an internal loading control .

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E) Protein expression of ADAM17 variants in the DLD-1 Adam17-/- cell line, determined by Western blot. GAPDH was used as an internal loading control.


F ) Amphiregulin ( AREG ) shedding measured by ELISA of conditioned media from untreated , H2O2 treated or irradiated with x - ray DLD - 1 Adam17 - / - cells ( clone # 1 ) expressing full - length ADAM17 variants ( wt , I762A or LEE ) . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Mean and standard deviation indicated from at least three independent experiments .

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F) Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H2O2 treated or irradiated with x-ray DLD-1 Adam17-/- cells (clone #1) expressing full-length ADAM17 variants (wt, I762A or LEE). Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01, ***p<0.001. Mean and standard deviation indicated from at least three independent experiments.


G ) Exogenous wt and I762A full length ADAM17 was immuno - purified from Adam17 - / - cells treated with H2O2 and subjected to label - free LC - MS / MS to determine differential phosphorylation status of T735 and S808 ( S811 in murine ADAM17 ) .

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G) Exogenous wt and I762A full length ADAM17 was immuno-purified from Adam17-/- cells treated with H2O2 and subjected to label-free LC-MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).


A ) EGFR activation in the ADAM17 variant expressing DLD - 1 Adam17 - / - cells upon H2O2 treatment , determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments .

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A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.


( B , C ) B ) Cell proliferation and C ) Matrigel invasion of the ADAM17 variant expressing DLD - 1 Adam17 - / - cells . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments .

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(B, C) B) Cell proliferation and C) Matrigel invasion of the ADAM17 variant expressing DLD-1 Adam17-/- cells. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.


D ) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17 - / - , determined by Western blot . GAPDH was used as an internal loading control .

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D) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17-/-, determined by Western blot. GAPDH was used as an internal loading control.


E ) Average tumor volume ± SEM of 4T1 Adam17 - / - cells expressing the ADAM17 variants injected into the mammary fat pad of Balb / c mice ( n = 10 for each group ) . Indicated significances are between the I762A and LEE tumors . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 . Mean and standard error of the mean indicated .

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E) Average tumor volume ± SEM of 4T1 Adam17-/- cells expressing the ADAM17 variants injected into the mammary fat pad of Balb/c mice (n=10 for each group). Indicated significances are between the I762A and LEE tumors. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05. Mean and standard error of the mean indicated.


F ) Survival curve of the tumor bearing mice injected with 4T1 Adam17 - / - cells expressing the ADAM17 variants ( n = 10 for each group ) . The indicated significances are between the LEE and wt tumor bearing mice , and the LEE and I762A tumor bearing mice . Log - rank test was applied to test for significant differences . * p < 0 . 05 .

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F) Survival curve of the tumor bearing mice injected with 4T1 Adam17-/- cells expressing the ADAM17 variants (n=10 for each group). The indicated significances are between the LEE and wt tumor bearing mice, and the LEE and I762A tumor bearing mice. Log-rank test was applied to test for significant differences. *p<0.05.


A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells . MIC10 KO cells show a drastic reduction of MIC13 , MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged . MIC60 KO cells show a drastic reduction of protein levels of all MICOS components .

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A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components.


B Representative electron micrographs of WT , MIC10 KO and MIC60 KO HAP1 cells . Scale bar 500 nm .

B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm.


C Quantification of CJs per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) .

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C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).


D Quantification of cristae per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) .

D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).


E Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 034 for basal ( using one sample t - test ) and * P = 0 . 018 for maximum respiration ( using unpaired Student ' s t - test ) .

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E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).


F Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 01 for basal ( using one sample t - test ) , * * * P = 0 . 0004 for maximum respiration and * P = 0 . 012 for non - mitochondrial respiration ( using unpaired Student ' s t - test ) .

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F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).


A Representative STED super - resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies ( top panel ) and TOMM70 ( middle panel ) . Bottom panel shows merged images . Arrowheads show colocalization of MIC60 and TOMM70 punctae . Scale bar 500 nm .

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A Representative STED super-resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies (top panel) and TOMM70 (middle panel). Bottom panel shows merged images. Arrowheads show colocalization of MIC60 and TOMM70 punctae. Scale bar 500 nm.


B Quantification of median interpunctae distance ( IPD ) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Median IPD from n = 10 - 16 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . ns , not significant , unpaired Student ' s t - test .

B Quantification of median interpunctae distance (IPD) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Median IPD from n = 10-16 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ns, not significant, unpaired Student's t-test.


FRAP curves ( curve fitted ) of WT Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown .

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FRAP curves (curve fitted) of WT Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.


C FRAP curves ( curve fitted ) of MIC13 KO Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown .

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C FRAP curves (curve fitted) of MIC13 KO Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.


D Histogram showing average percentage of mobile fractions , obtained from curve fits of all mitochondria expressing GFP - tagged versions of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) in WT and MIC13 KO Hela cells ( data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments , 6 - 10 mitochondria from each experiment ) .

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D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment).


A - D Representative single particle tracks of cells expressing MIC60 - ( A ) and MIC10 - SNAP ( B ) in WT Hela cells and MIC60 - ( C ) and MIC10 - SNAP ( D ) in MIC13 KO Hela cells , stained with silicone - rhodamine dye , and imaged at a rate of 33 ms / frame . Single tracks were colour - coded according to temporal appearance . Scale Bar 5 µm . Insets in each image show zoomed - in images of few representative tracks .

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A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.


E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients ( insD ) in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP stained with silicone - rhodamine . Number of tracks analysed ( obtained from 2 independent experiments ) : n = 2541 , 2540 , 3560 and 1441 in WT Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , MIC13 KO Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , respectively . ( * * * * P ≤ 0 . 0001 for all possible comparisons , unpaired Student ' s t - test ) .

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E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).


F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10 - SNAP . Number of corresponding subtracks analysed ( obtained from 2 independent experiments ) : n = 2561 , 2443 , 3402 , 1360 , 561 and 1110 . Data are mean ± SEM . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired Student ' s t - test .

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F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.


A , C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT ( A ) and MIC13 KO ( C ) Hela cells expressing MIC10 - SNAP stained with silicone - rhodamine . Box in ( A and C ) mark selection shown as a zoom in panel ( B and D ) respectively . Scale bar 500 nm .

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A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.


E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10 - SNAP ( 3 independent experiments , 5 - 8 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * P = 0 . 026 ( merging and splitting events in WT cells ) and * * * * P < 0 . 0001 ( merging and splitting events of WT vs . MIC13 KO ) , unpaired Student ' s t - test .

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E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test.


A Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 2 . 5 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 1 . 3 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing MIC13 - SNAP imaged at a time interval of 1 . 3 s / frame . Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13 - SNAP . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E First image from ( D ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM . Magenta boxes depict intensity maxima at IBM .

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E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.


A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm . B Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in WT Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Green ` Y ´ represent Y - type imminent mergence event . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in WT Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green `Y´ represent Y- type imminent mergence event . Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) showing MIC13 KO Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in MIC13 KO Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s) showing MIC13 KO Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in MIC13 KO Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E First image from ( B ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM . The represented IBM area is superimposed from intensities of MIC13 - SNAP from Figure 6F . Magenta box depicts intensity maxima at cristae membrane apart from IBM .

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E First image from (B) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM. The represented IBM area is superimposed from intensities of MIC13-SNAP from Figure 6F. Magenta box depicts intensity maxima at cristae membrane apart from IBM.


G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I - SNAP ( 3 independent experiments , 3 - 7 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * * * * P < 0 . 0001 , unpaired Student ' s t - test .

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G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I-SNAP (3 independent experiments, 3-7 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. ****P < 0.0001, unpaired Student's t-test.


A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( B ) ( red hot LUT ) . Scale bar 500 nm . B Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green asterisks show merging events and redistribution of TMRM intensity from a high - intensity cristae to a low - intensity cristae ( two such instances are shown at 3 and 7 . 4 s ) . Green arrows pointing inward connected by solid line at 1 . 5 and 5 . 9 s indicate imminent merging events . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (B) (red hot LUT). Scale bar 500 nm. B Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green asterisks show merging events and redistribution of TMRM intensity from a high-intensity cristae to a low-intensity cristae (two such instances are shown at 3 and 7.4 s). Green arrows pointing inward connected by solid line at 1.5 and 5.9 s indicate imminent merging events. Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( D ) ( red hot LUT ) . Scale bar 500 nm . D Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green and magenta asterisks show cristae merging and splitting cycles ( shown at 5 . 9 s and 7 . 4 s ) . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (D) (red hot LUT). Scale bar 500 nm. D Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green and magenta asterisks show cristae merging and splitting cycles (shown at 5.9 s and 7.4 s). Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E Representative time - lapse images of WT Hela cells stained with NAO , and imaged using Airyscan microscope , indicate cristae merging and splitting dynamics . Yellow and magenta asterisks show merging and splitting events of cristae respectively . Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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E Representative time-lapse images of WT Hela cells stained with NAO, and imaged using Airyscan microscope, indicate cristae merging and splitting dynamics. Yellow and magenta asterisks show merging and splitting events of cristae respectively. Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


A Mitochondrion in WT Hela cells expressing ATP5I - PAGFP was photoactivated , in the area shown by a blue box , at 0 s . Inset of white box at 0 . 98 s clearly shows fluorescent crista ( red arrow ) , absent at 0 . 38 s , 0 . 59 s & 0 . 78 s ( red arrowhead showing the area enclosed by white dotted lines ) , confirming merging of non - fluorescent cristae vesicle with the IBM . Threshold images of insets are shown above the whole images . Scale bar 500 nm .

A Mitochondrion in WT Hela cells expressing ATP5I-PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38 s, 0.59 s & 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non-fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm.


B Dual - colour STED super - resolution images of fused mitochondrion in WT Hela cells ( Left Panel ) showing colocalization of cristae marked with ATP5I - GFP ( using anti - GFP antibody ) and ATP5I - SNAP ( stained with silicone - rhodamine ) . ( Right Panel ) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I - GFP ( using anti - GFP antibody ) or ATP5I - SNAP ( stained with silicone - rhodamine ) . Arrowheads show merged cristae while arrows show cristae that maintain their individual identity . Scale bar 500 nm .

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B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm.


( a ) Western blot analysis of HeLa cells treated with siRNA for 84 h .

(a) Western blot analysis of HeLa cells treated with siRNA for 84 h.


( b ) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5 , ATG7 or NDP52 . GAPDH and total protein stained with Coomassie blue serve as loading controls .

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(b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls.


( c ) Western blot analysis of HeLa cells cultured in Hank ' s buffered salt solution ( Starved ) or DMEM + 10 % FBS ( Serum ) .

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(c) Western blot analysis of HeLa cells cultured in Hank's buffered salt solution (Starved) or DMEM+ 10% FBS (Serum).


( d ) Western blot analysis of HeLa cells treated ( 48 h ) with an inhibitor of mTORC1 ( RAP , 20 nM ) or control ( DMSO ) .

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(d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO).


( e ) Western blot analysis comparing the levels of DICER in MDA - 231 , T47D and MDA - 435 cells .

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(e) Western blot analysis comparing the levels of DICER in MDA-231, T47D and MDA-435 cells.


( f ) Western blot analysis of MDA - 231 , T47D and MDA - 435 cells treated ( 48 h ) with inhibitors of mTORC1 ( RAP , 20 nM ) , mTORC1 and 2 ( PP242 , 100 nM ) , or DMSO ( control ) .

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(f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control).


( g ) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h .

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(g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h.


( h ) Western blot analysis of HeLa cells treated with BAF ( 200 nM , 12 h ) , RAP ( 20 nM , 24 h ) , or RAP and BAF . The results in a - h are representative of three experiments and use α - tubulin ( TUBA ) as a loading control .

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(h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a-h are representative of three experiments and use α-tubulin (TUBA) as a loading control.


( i ) Western blot analysis of DICER ( monoclonal antibody 13D6 ) or control ( EEF1A1 ) in HeLa cells treated with control siRNA or siRNA targeting DICER .

(i) Western blot analysis of DICER (monoclonal antibody 13D6) or control (EEF1A1) in HeLa cells treated with control siRNA or siRNA targeting DICER.


( j ) Confocal analysis of the signal from an anti - DICER monoclonal antibody ( clone 13D6 ) or an isotype control monoclonal antibody in HeLa cells . Scale bar , 10 μm .

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(j) Confocal analysis of the signal from an anti-DICER monoclonal antibody (clone 13D6) or an isotype control monoclonal antibody in HeLa cells. Scale bar, 10 μm.


( k , l ) RT - qPCR analysis of AGO1 , AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO , BAF or CQ ( k ) or siRNA for 84 h ( l ) . Error bars show s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k,l) RT-qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and NDP52 in HeLa cells treated with RAP or control . The arrows highlight some examples of co - localization . The lower left insets show higher magnifications of the areas outlined in the main panel . Scale bars , 5 μm . ( b ) Quantification of DICER co - localization with NDP52 . Total fluorescence intensity of DICER co - localized with punctae ( > 0 . 2 μm3 ) of NDP52 was quantified with Volocity software in RAP - treated cells / control - treated cells over several Z - stacked microscope fields per experiment ( n = 4 , error bars s . e . m . ) .

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(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.).


( c ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and HcRed - LC3 in HeLa cells treated with BAF or control . The arrows highlight some examples of co - localization . Insets as in a . Scale bar , 5 μm . ( d ) Quantification of DICER co - localization with HcRed - LC3 was performed as for NDP52 in b .

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(c) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and HcRed-LC3 in HeLa cells treated with BAF or control. The arrows highlight some examples of co-localization. Insets as in a. Scale bar, 5 μm. (d) Quantification of DICER co-localization with HcRed-LC3 was performed as for NDP52 in b.


( e ) Confocal microscopy analysis of GFP - DCP1A and HcRed - LC3wt in HeLa cells . Scale bar , 10 μm .

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(e) Confocal microscopy analysis of GFP-DCP1A and HcRed-LC3wt in HeLa cells. Scale bar, 10 μm.


( f - i ) Localization of DICER ( monoclonal antibody 13D6 ) detected with anti - mouse IgG ( 10 nm gold beads ) by electron microscopy in CQ - ( 20 μM , 12 h ) treated HeLa cells . Mito , mitochondria ; MVB , multivesicular body . The black arrows highlight all gold beads .

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(f-i) Localization of DICER (monoclonal antibody 13D6) detected with anti-mouse IgG (10 nm gold beads) by electron microscopy in CQ- (20 μM, 12 h) treated HeLa cells. Mito, mitochondria; MVB, multivesicular body. The black arrows highlight all gold beads.


( j ) Western blot analysis of fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ; ref . ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body and lysosomal markers are indicated . Material that was pelleted by centrifugation at 100 , 000g ( 100 K pellet ) or that remained in solution after the 100 , 000g spin ( soluble ) was not added to the gradient .

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(j) Western blot analysis of fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV; ref. ). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body and lysosomal markers are indicated. Material that was pelleted by centrifugation at 100,000g (100 K pellet) or that remained in solution after the 100,000g spin (soluble) was not added to the gradient.


( k ) Western blot analysis of UB and p62 in discontinuous Histodenz gradients ( as in j ) of HEK293T cells treated with control ( DMSO ) or CQ . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k) Western blot analysis of UB and p62 in discontinuous Histodenz gradients (as in j) of HEK293T cells treated with control (DMSO) or CQ. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body ( MVB ) and lysosomal markers are indicated .

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(a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated.


( b ) Western blot of GEMIN4 or loading control ( TUBA ) in HeLa cells treated with control siRNA or siRNA targeting ATG5 , ATG6 or ATG7 .

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(b) Western blot of GEMIN4 or loading control (TUBA) in HeLa cells treated with control siRNA or siRNA targeting ATG5, ATG6 or ATG7.


( c ) Western blot analysis of GEMIN4 or control ( TUBA ) in HeLa cells cultured in DMEM 10 % FBS ( Serum ) , Hank ' s buffered salt solution ( Starve ) for 2 h , or treated for 16 h with control ( DMSO ) , BAF ( 200 nM ) , RAP ( 20 nM ) , RAP + BAF , or PP242 ( 100 nM ) .

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(c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank's buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM).


( d ) Top , western blot of GEMIN4 immunoprecipitates ( IP ) from HeLa cells . Bottom , western blot analysis of GEMIN4 in immunoprecipitates of anti - NDP52 , - p62 or control antibody from HeLa cells .

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(d) Top, western blot of GEMIN4 immunoprecipitates (IP) from HeLa cells. Bottom, western blot analysis of GEMIN4 in immunoprecipitates of anti-NDP52, -p62 or control antibody from HeLa cells.


( e ) Western blot of DICER immunoprecipitates from HeLa cells .

(e) Western blot of DICER immunoprecipitates from HeLa cells.


( f ) Western blot of AGO2 immunoprecipitates from HeLa cells .

(f) Western blot of AGO2 immunoprecipitates from HeLa cells.


( g ) Western blot analysis of GEMIN4 , compared with loading controls ( DCP1A , CLN3 ) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4 .

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(g) Western blot analysis of GEMIN4, compared with loading controls (DCP1A, CLN3) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4.


( h ) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3 , GEMIN4 and ATG5 . Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells .

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(h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells.


( i ) Western blot analysis with anti - UB monoclonal antibody ( mono - and polyubiquitylated proteins , FK2 clone ) in Flag immunoprecipitates performed under stringent denaturing conditions . Flag - AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet - inducible Flag - AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs .

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(i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs.


( j ) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h . TUBA , α - tubulin loading control .

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(j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA, α-tubulin loading control.


( k ) Top , RT - qPCR analysis of miR - 16 or let - 7a levels in AGO2 immunoprecipitates from DMSO - and BAF - treated HeLa cells . Error bars represent s . e . m . Bottom , a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Northern blot of miR - 16 and let - 7a in HeLa cells treated with siRNA for 84 h . Levels of U6 , ethidium - bromide - stained total RNA on northern membrane ( loading ) and 28S and 18S ribosomal RNA on 1 % agarose gel are shown as controls for equal loading .

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(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading.


( b - d ) RT - qPCR analysis of let - 7a and miR - 16 ( b ) , pre - let - 7a and pre - miR - 16 ( c ) and let - 7a * and miR - 16 * ( d ) in HeLa cells depleted of NDP52 or ATG5 with siRNA ( 84 h ) . n = 3 independent experiments ; error bars represent s . e . m .

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(b-d) RT-qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h). n = 3 independent experiments; error bars represent s.e.m.


( e ) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let - 7a - let - 7a * labelled on the 3 ′ end of either let - 7 or let - 7a * with FITC . Cells were treated 16 h before with DMSO ( control ) , BAF ( 200 nm ) or geldanamycin ( 10 μM ) . Representative results of one experiment are shown .

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

(e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown.


( f ) Western blot analysis of AGO2 immunoprecipitates ( IP ) used in e .

(f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e.


( g ) Normalized results of four independent experiments ( n = 4 ) identical to e . Error bars represent s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

(g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Renilla luciferase expression controlled by an exogenous siRNA - siRNA * duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA ( black and grey bars correspond to two independent siRNAs ) . Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA - siRNA * duplex . n = 2 independent experiments .

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(a) Renilla luciferase expression controlled by an exogenous siRNA-siRNA* duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA (black and grey bars correspond to two independent siRNAs). Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA-siRNA* duplex. n = 2 independent experiments.


( b ) Renilla luciferase expression controlled by let - 7 in HeLa cells depleted of NDP52 or ATG5 with siRNA ( black and grey bars are independent siRNAs ) . Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let - 7 binding sites . n = 3 independent experiments . The asterisks denote mutations in the let - 7 target sites of the reporter .

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(b) Renilla luciferase expression controlled by let-7 in HeLa cells depleted of NDP52 or ATG5 with siRNA (black and grey bars are independent siRNAs). Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let-7 binding sites. n = 3 independent experiments. The asterisks denote mutations in the let-7 target sites of the reporter.


( c ) Western blot analysis of endogenous miRNA - repressed proteins ( RAS , HMGA2 and CDK6 are repressed by let - 7 ; CDK6 is also repressed by miR - 16 ( bottom panel ) ) or control ( TUBA , α - tubulin ) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were treated in parallel with nonspecific antagomir or let - 7 targeting antagomir .

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(c) Western blot analysis of endogenous miRNA-repressed proteins (RAS, HMGA2 and CDK6 are repressed by let-7; CDK6 is also repressed by miR-16 (bottom panel)) or control (TUBA, α-tubulin) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were treated in parallel with nonspecific antagomir or let-7 targeting antagomir.


( d ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with control antagomir or anti - let - 7 antagomir .

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(d) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with control antagomir or anti-let-7 antagomir.


( e ) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were also treated with nonspecific antagomir or let - 7 targeting antagomir .

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(e) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were also treated with nonspecific antagomir or let-7 targeting antagomir.


( f ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA . All error bars are s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(f) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA. All error bars are s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


A The first 400 nt of all 3 ' UTRs represented in the UPF1 RIP - Seq dataset were analyzed for the occurrence of motifs corresponding to the indicated RNA - binding proteins . Transcripts were divided into groups based on the number of motifs present ( 0 , 1 , more than 1 ) , indicated by the gradient triangle . The dotted line indicates the mean RIP - Seq efficiency for all transcripts used in the analysis . Mean and SD are plotted for all groups .

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A The first 400 nt of all 3'UTRs represented in the UPF1 RIP-Seq dataset were analyzed for the occurrence of motifs corresponding to the indicated RNA-binding proteins. Transcripts were divided into groups based on the number of motifs present (0, 1, more than 1), indicated by the gradient triangle. The dotted line indicates the mean RIP-Seq efficiency for all transcripts used in the analysis. Mean and SD are plotted for all groups.


B CDF plot of relative abundance of transcripts with 3 ' UTRs > 1000 nt in K562 cells under conditions of UPF1 or non - targeting shRNA , for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations . For mRNAs with peaks > 400 nt from the TC or with peaks in the 5 ' UTR or CDS , transcripts with peaks at 3 ' UTR positions 1 - 400 were excluded . Statistical significance was determined by Kruskal - Wallis test with Dunn ' s correction for multiple comparisons and was determined based on comparison to the set with no 3 ' UTR CLIP peaks .

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B CDF plot of relative abundance of transcripts with 3'UTRs > 1000 nt in K562 cells under conditions of UPF1 or non-targeting shRNA, for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations. For mRNAs with peaks > 400 nt from the TC or with peaks in the 5'UTR or CDS, transcripts with peaks at 3'UTR positions 1-400 were excluded. Statistical significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons and was determined based on comparison to the set with no 3'UTR CLIP peaks.


C Representative northern blots assaying the stability of β - globin reporter transcripts containing the SMG5 3 ' UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet - off cells . Half - lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint . Significance was measured compared to the SMG5 3 ' UTR reporter using the sum - of - squares F test .

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C Representative northern blots assaying the stability of β-globin reporter transcripts containing the SMG5 3'UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet-off cells. Half-lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint. Significance was measured compared to the SMG5 3'UTR reporter using the sum-of-squares F test.


D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from ( C ) . " AS " indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3 ' UTR in the same position as the RSE . The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation . * indicates P = 0 . 0146 in two - tailed Student ' s t - test . The reporter containing the GAP 3 ' UTR used here as a transfection control contains a predicted hnRNP L binding site , allowing its consistent recovery in the IPs .

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D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from (C). "AS" indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3'UTR in the same position as the RSE. The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation. * indicates P = 0.0146 in two-tailed Student's t-test. The reporter containing the GAP 3'UTR used here as a transfection control contains a predicted hnRNP L binding site, allowing its consistent recovery in the IPs.


A RNA - seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1 - dependent manner . B CDF plot of transcript 3 ' UTR lengths for subsets identified in ( A ) . Statistical significance was determined by two - sided K - S test . P value for decreased set is from comparison to the set of total transcripts . P value for rescued set is from comparison to decreased set .

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A RNA-seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1-dependent manner. B CDF plot of transcript 3'UTR lengths for subsets identified in (A). Statistical significance was determined by two-sided K-S test. P value for decreased set is from comparison to the set of total transcripts. P value for rescued set is from comparison to decreased set.


A qPCR of selected transcripts found by RNA - Seq to decrease in abundance in the absence of hnRNP L . Values represent the mean and standard deviation of 3 independent biological replicates . Significance of hnRNP L KD and UPF1 KD were compared to NT , and the double knockdown was compared to hnRNP L KD . For clarity , only a single star is used to indicate significance ; in all cases P ≤ 0 . 025 in two - tailed Student ' s t - tests .

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A qPCR of selected transcripts found by RNA-Seq to decrease in abundance in the absence of hnRNP L. Values represent the mean and standard deviation of 3 independent biological replicates. Significance of hnRNP L KD and UPF1 KD were compared to NT, and the double knockdown was compared to hnRNP L KD. For clarity, only a single star is used to indicate significance; in all cases P ≤ 0.025 in two-tailed Student's t-tests.


B Half - lives of selected transcripts under conditions of non - targeting or hnRNP L knockdown . Measurements were calculated based on proportional recovery of transcript labeled with 5 - EU in 1 hour versus total transcript . Significance was determined using two - tailed Student ' s t - test : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 . Values represent mean and standard deviation of 3 independent replicates .

B Half-lives of selected transcripts under conditions of non-targeting or hnRNP L knockdown. Measurements were calculated based on proportional recovery of transcript labeled with 5-EU in 1 hour versus total transcript. Significance was determined using two-tailed Student's t-test: *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Values represent mean and standard deviation of 3 independent replicates.


C The stability of β globin reporters with the CSRP1 3 ' UTR , along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated , were evaluated in HeLa Tet - off cells . The schematic indicates areas of 3 ' UTR CA - enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3 ' UTR , where putative hnRNP L binding elements are in red typeface . The northern blot is a representative example from 3 independent replicates . Mean values plotted with SD at each timepoint were used to quantify decay ( right panel ) . Significance is measured compared to the WT reporter using the sum - of - squares F test .

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C The stability of β globin reporters with the CSRP1 3'UTR, along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated, were evaluated in HeLa Tet-off cells. The schematic indicates areas of 3' UTR CA-enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3'UTR, where putative hnRNP L binding elements are in red typeface. The northern blot is a representative example from 3 independent replicates. Mean values plotted with SD at each timepoint were used to quantify decay (right panel). Significance is measured compared to the WT reporter using the sum-of-squares F test.


D The stability of the WT and L mut 1 - 200 CSRP1 reporters in HeLa Tet - off cells treated with non - targeting RNAi or siRNA against UPF1 was evaluated . The northern blot is a representative example from at least 3 biological replicates . Decay quantification and statistics were performed as in ( C ) , with significance measured compared to the non - targeting condition .

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D The stability of the WT and L mut 1-200 CSRP1 reporters in HeLa Tet-off cells treated with non-targeting RNAi or siRNA against UPF1 was evaluated. The northern blot is a representative example from at least 3 biological replicates. Decay quantification and statistics were performed as in (C), with significance measured compared to the non-targeting condition.


A Motif analysis of recovered UPF1 - associated transcripts analyzed by RIP - Seq Transcripts were binned into three 3 ' UTR length classes , which were then further subdivided based on hnRNP L motif density . Each box plot represents the median and interquartile range with extensions from 10 % to 90 % of the indicated subset . Dotted line represents the median RIP - Seq efficiency of the class of transcripts with the shortest 3 ' UTR and no hnRNP L binding motifs . Significance was computed using Kruskal - Wallis tests with Dunn ' s correction for multiple comparisons . B CDF plots of selected transcript classes from ( A ) . Log2 ( FC ) of UPF1 knockdown as compared to the non - targeting control indicates NMD sensitivity of each class . Significance was determined by Kruskal - Wallis test with Dunn ' s correction for multiple comparisons Data information : * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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A Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq Transcripts were binned into three 3'UTR length classes, which were then further subdivided based on hnRNP L motif density. Each box plot represents the median and interquartile range with extensions from 10% to 90% of the indicated subset. Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and no hnRNP L binding motifs. Significance was computed using Kruskal-Wallis tests with Dunn's correction for multiple comparisons. B CDF plots of selected transcript classes from (A). Log2(FC) of UPF1 knockdown as compared to the non-targeting control indicates NMD sensitivity of each class. Significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons Data information: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C Motif analysis of recovered UPF1 - associated transcripts analyzed by RIP - Seq Transcripts are divided into tertiles by hnRNP L and PTBP1 binding motif density and 3 ' UTR length . Data are represented and analyzed as in ( A ) . Dotted line represents the median RIP - Seq efficiency of the class of transcripts with the shortest 3 ' UTR and low PTBP1 / hnRNP L motif density . D CDF plots of selected transcript classes from ( C ) analyzed as in ( B ) . Data information : * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

C Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq Transcripts are divided into tertiles by hnRNP L and PTBP1 binding motif density and 3'UTR length. Data are represented and analyzed as in (A). Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and low PTBP1/hnRNP L motif density. D CDF plots of selected transcript classes from (C) analyzed as in (B). Data information: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C qPCR of selected transcripts in SU - DHL - 4 cells under conditions of non - targeting RNAi or hnRNP L knockdown with or without 50 μg / mL cycloheximide treatment ( for 4 hours ; n ≥ 3 ) . Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO , and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO . Bars represent the mean ± SD . For clarity , only a single star was used to indicate significance ; in all cases P ≤ 0 . 05 in two - tailed Student ' s t - tests .

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C qPCR of selected transcripts in SU-DHL-4 cells under conditions of non-targeting RNAi or hnRNP L knockdown with or without 50 μg/mL cycloheximide treatment (for 4 hours; n ≥ 3). Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO, and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO. Bars represent the mean ± SD. For clarity, only a single star was used to indicate significance; in all cases P ≤ 0.05 in two-tailed Student's t-tests.


E Half - life determination from RT - qPCR detection of reporter constructs in HeLa Tet - off cells transfected with non - targeting or UPF1 siRNAs . For plots comparing non - targeting with UPF1 knockdown , significance is compared to the non - targeting condition using the the sum - of - squares F test . The fourth plot compares the non - targeting decay curves of the three reporters , and significance is compared to the decay of the fusion transcript . Points represent the mean ± SD from qRT - PCR at each time point of at least 3 independent experiments .

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E Half-life determination from RT-qPCR detection of reporter constructs in HeLa Tet-off cells transfected with non-targeting or UPF1 siRNAs. For plots comparing non-targeting with UPF1 knockdown, significance is compared to the non-targeting condition using the the sum-of-squares F test. The fourth plot compares the non-targeting decay curves of the three reporters, and significance is compared to the decay of the fusion transcript. Points represent the mean ± SD from qRT-PCR at each time point of at least 3 independent experiments.


A qPCR of selected transcripts in SU - DHL - 4 cells under conditions of BCL2 knockdown , hnRNP L knockdown , or non - targeting control siRNA Nucleofection ( n ≥ 4 independent experiments ) . Bars indicate mean ± SD . Significance was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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A qPCR of selected transcripts in SU-DHL-4 cells under conditions of BCL2 knockdown, hnRNP L knockdown, or non-targeting control siRNA Nucleofection (n ≥ 4 independent experiments). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


Quantification of protein reduction in knockdown experiments and a representative western blot . Each point represents a trial ( n ≥ 5 for a given condition ) . Mean with standard deviation are indicated on the plot . Target protein levels from all biological replicates were normalized to MATRIN3 , with statistical analysis as in panel ( A ) . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

Quantification of protein reduction in knockdown experiments and a representative western blot. Each point represents a trial (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot. Target protein levels from all biological replicates were normalized to MATRIN3, with statistical analysis as in panel (A). Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C FACS - based apoptosis analysis of SU - DHL - 4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours . Quantification of relative change in each quadrant is provided where points represent individual experiments ( n ≥ 5 for a given condition ) . Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel ( A ) . A representative FACS experiment is shown with the percentage of cells in each gate indicated . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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C FACS-based apoptosis analysis of SU-DHL-4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours. Quantification of relative change in each quadrant is provided where points represent individual experiments (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel (A). A representative FACS experiment is shown with the percentage of cells in each gate indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


B SU - DHL - 4 and SU - DHL - 6 cells were treated with varying doses of BCL2 inhibitor . Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells . Traces were generated from at least 5 independent biological replicates . Points represent mean ± SD at each dose . P value from comparison of dose response between SU - DHL - 4 and SU - DHL - 6 cells calculated using sum - of - squares F test is given in the graph key . Significance of individual points calculated using two - tailed Student ' s t - tests is marked using asterisks on the graph . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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B SU-DHL-4 and SU-DHL-6 cells were treated with varying doses of BCL2 inhibitor. Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells. Traces were generated from at least 5 independent biological replicates. Points represent mean ± SD at each dose. P value from comparison of dose response between SU-DHL-4 and SU-DHL-6 cells calculated using sum-of-squares F test is given in the graph key. Significance of individual points calculated using two-tailed Student's t-tests is marked using asterisks on the graph. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


C qPCR of selected transcripts in SU - DHL - 6 cells under conditions of hnRNP L knockdown or non - targeting control ( n = 5 independent experiments ) . Bars indicate mean ± SD . Significance of hnRNP L KD was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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C qPCR of selected transcripts in SU-DHL-6 cells under conditions of hnRNP L knockdown or non-targeting control (n = 5 independent experiments). Bars indicate mean ± SD. Significance of hnRNP L KD was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


D Representative amplification of the region of chromosomal deletion in edited cells . PCR products were run on a 1 % agarose gel to resolve the amplicons from edited and unedited chromosomes . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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D Representative amplification of the region of chromosomal deletion in edited cells. PCR products were run on a 1% agarose gel to resolve the amplicons from edited and unedited chromosomes. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


E qPCR of selected transcripts in edited SU - DHL - 6 cells ( n = 4 independent experiments ) . Significance was determined as compared to control Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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E qPCR of selected transcripts in edited SU-DHL-6 cells (n = 4 independent experiments). Significance was determined as compared to control Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells . Target protein levels from all biological replicates were normalized to MATRIN3 . Each point represents a trial ( n = 4 ) . Bars indicate mean ± SD . Significance was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells. Target protein levels from all biological replicates were normalized to MATRIN3. Each point represents a trial (n = 4). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


G FACS - based apoptosis analysis of edited SU - DHL - 6 . Quantification of relative change in each quadrant is provided , where points represent individual experiments ( n = 6 for a given condition ) . Mean with standard deviation are indicated on the plot , analyzed as in ( F ) . Shown is a representative FACS experiment . Percentage of cells in each gate is indicated . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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G FACS-based apoptosis analysis of edited SU-DHL-6. Quantification of relative change in each quadrant is provided, where points represent individual experiments (n = 6 for a given condition). Mean with standard deviation are indicated on the plot, analyzed as in (F). Shown is a representative FACS experiment. Percentage of cells in each gate is indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8 were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) ,

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A),


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) , or were visualized by fluorescence microscopy ( B )

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)


( C ) Cells ( TOS038 ) expressing Atg1 - GFP were grown to mid - log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min . Cells were washed , shifted to SD - N for 4 h , and then visualized by fluorescence microscopy . Scale bar , 5 μm .

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(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( BY4741 ) and pep4 ∆ ( TOS015 ) strains were grown to mid - log phase and preincubated in YPD with cerulenin or DMSO for 30 min . Cells were pulse - labeled for 10 min with [ 35S ] methionine and cysteine and chased for the indicated time periods . Cell lysates were subjected to immunoprecipitation with anti - Ape1 antibodies followed by SDS ‒ PAGE and X - ray film to detect radioactive signals . prApe1 , premature Ape1 ; mApe1 , mature Ape1 . (

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

(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (


( E ) Cells of the fas1 ∆ ( TOS029 ) strain expressing GFP ‒ Atg8 were grown to mid - log phase in YPD + 0 . 1 mM palmitic / stearic / myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min . Cells were then shifted to SD - N for the indicated times . Cell lysates were subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies .

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

(E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.


( F ) WT ( BY4741 ) cells were grown to mid - log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin ( 25 , 50 ) or with DMSO ( - ) in rich medium for 30 min . Cells were washed and shifted to SD - N in the presence of cerulenin ( 25 , 50 ) or DMSO ( - ) . Cells were lysed and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type YPD , complete medium .

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

(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.


( A ) Cells ( TN124 strain ) were grown to mid - log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods ( Cer preincubation ) or incubated with DMSO without preincubation ] ( - ) . Cells were then shifted to SD - N for 3 h with the addition of DMSO or 50 µM cerulenin ( cer ) . Autophagic activity was measured by alkaline phosphatase assay . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) Cells were grown as in ( A ) , stained with BODIPY , and visualized by fluorescence microscopy . Scale bar , 5 μm .

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(B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm.


( C ) WT ( SCY62 ) and tagΔsteΔ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 3 h . Cells were stained with Nile red and visualized by fluorescence microscopy . Scale bar , 5 μm .

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(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cell lysates were subjected to SDS − PAGE , followed by western blot analysis using anti - GFP , anti - Ape1 ( prApe1 , premature Ape1 ; mApe1 , mature Ape1 ) , and anti - Fas1 and anti - Pgk1 antibodies .

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

(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.


( E ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type , YPD , complete medium .

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

(E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.


( A ) GAL - DGA1GAL - ARE2lro1Δ are1Δ ( FYS118 strain ) was grown on SC ( synthetic minimal medium without dextrose ) + raffinoseovernight , diluted to OD 0 . 4 , and grown to mid - log phase in either SC + glucose or SC + galactose medium . Cells were shifted to SD - N for 2 h , stained with BODIPY , and visualized by fluorescencemicroscopy . Scale bar , 5 μm .

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

(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.


( B ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 were grown as in A . Cells were lysed at the indicated times and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies . (

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(B) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (


( C , D ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 ( C ) or GFP − Atg1 ( D ) were grown as in A and visualized by fluorescence microscopy after 2 h in SD - N . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild - type .

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(C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 6 h . Cells were stained with BODIPY and visualized by fluorescence microscopy . Scale bar , 5 μm .

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(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.