A ) TDP - 43 ALS - linked acid substitutions K181E , A315T / E , A321G , Q331K , M337V , A382T . Boxed in C - terminal domain is α - helical structure within conserved region ( a . a . 320 - 343 ) . Droplets formed by TDP - 43 wild - type ( WT ) different mutants ( 4 μM ) observed by brightfield microscopy in presence of no control or A ( GU ) 18 ( 3 . 9 μM ) at 250 mM NaCl . Mutations A321G M337V , showing decreased liquidity of condensates in presence of A ( GU ) 18 , are boxed in red . Representative for 3 biological replicates using 3 different preparations of WT M337V , 2 preparations of A321G , Q331K , K181E one preparation of A315T / E A382T . Scale bars , 10 μm .

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A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.


B ) Phase separation of WT ( 4 μM , 10 % Oregon green - labeled ) M337V ( 4 μM , 10 % Cy - 3 - labeled ) observed by brightfield microscopy in presence of A ( GU ) 18 ( 3 . 9 μM ) at 250 mM NaCl . middle panel shows mixing of these WT ( 2 μM ) M337V ( 2 μM ) samples . Representative for 3 biological replicates using 2 preparations . Scale bars , 10 μm .

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B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.


A ) Binding of TDP - 43 to GU - rich promotes liquid properties of TDP - 43 condensates , as shown by fusion of small condensates into larger droplets . This fluidity is impaired by loss of binding affinity or disease - linked mutations that alter phase separation , such as M337V A321G , which could explain increased TDP - 43 aggregation .

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A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.


stoichiometry ' s for B56α measured by ITC . Global direct fitting shown for one experiment ( reverse ) . Each dot is integrated heat per injection error bars represent uncertainty with this integrated value . experiment was done in both direct ( B56 in ) reverse ( B56 in syringe ) with similar results .

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stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results.


C ) Time from envelope breakdown ( NEBD ) to mitotic exit of expressing indicated B56 inhibitors with each circle representing a single . Only with similar expression of various B56 inhibitor constructs were analysed . Median time is indicated by red . A representative result from at least three independent experiments is shown . At least 25 were counted per condition in experiment shown . A Mann - Whitney U ‐ was used for statistical ( ns : non ‐ significant , * * * P ≤ 0 . 001 ) .

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C) Time from Nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analysed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann-Whitney U‐test was used for statistical analysis (ns: non‐significant, ***P ≤ 0.001).


D ) Volcano plot representing mass spectrometry identified co - purifying with B56 inhibitor versus control inhibitor from HeLa . PP2A - B56 subunits co - purifying with B56 inhibitor are indicated .

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D) Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated.


E ) F ) Competition in HeLa stably expressing RFP - tagged B56 inhibitor ( LxxIxE ) or control inhibitor ( AxxAxA ) . YFP - B56α was transfected into subsequently purified from these lines . Loss of binding of indicated determined by either mass spectrometry ( pink - B56 SLIM containing known B56 interactor ; blue - known B56 interactor , green - B56 SLIM containing ) ( E ) or Western blotting ( F ) .

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E) and F) Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink - B56 SLIM containing protein and known B56 interactor; blue - known B56 interactor, green - B56 SLIM containing protein) (E) or Western blotting (F).


A - B ) Schematic of synchronisation protocol for G1 / S ( A ) or mitotic ( B ) arrested accompanying volcano plot of phosphorylation sites quantified . phosphorylation sites showing an increase in presence of B56 inhibitor are shown in grey . Phosphorylation sites in a containing an LxxIxE motif are coloured pink .

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A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink.


A ) Michaelis - Menten kinetic parameters of purified PP2A - B56α PP2A - B55α holoenzymes were determined against indicated phospho - peptides . Mean standard deviation shown in plots as black bars ( n = 3 independent experiments )

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A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)


B - D ) In vitro dephosphorylation by PP2A - B55α PP2A - B56α holoenzymes of panels of phosphorylated peptides as indicated . Mean standard deviation shown in plots as black bars ( n = 3 independent experiments )

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B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)


B ) indicated YFP - FoxO3 constructs were transfected into HeLa , purified using YFP resin PP2A - B56 binding determined by Western blotting . PP2A - A ; scaffold subunit .

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B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit.


C ) - D ) YFP - FoxO3 constructs were transfected into HeLa lysates ( pS413 ) or YFP purifications ( pS253 ) were subjected to Western Blotting probed with indicated phosphoantibodies . Quantifications arise from five ( pS413 ) or three ( pS253 ) independent experiments . Mean standard deviation shown in plots as black bars .

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C) - D) YFP-FoxO3 constructs were transfected into HeLa cells and lysates (pS413) or YFP purifications (pS253) were subjected to Western Blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars.


E ) Steady - state localization of indicated YFP - FOXO3 variants in HeLa . Each data point represents from a single . A representative result from at least three independent experiments is shown . At least 25 were counted per condition in experiment shown . Scale bar is 10 μm . Mann - Whitney significance values ; ∗ p < 0 . 02 , ∗ ∗ ∗ p < 0 . 0002 .

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E) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann-Whitney test significance values; ∗p < 0.02, ∗∗∗p < 0.0002.


F ) Dephosphorylation by PP2A - B56α holoenzyme of substrates with increasing between phosphorylation sites binding motifs as depicted . Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 incubated with PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . Mean 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 - recombinant B56α indicated ADAM17 variant peptides .

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


C ) indicated full - murine Myc - ADAM17 derivatives were transfected into HeLa stably expressing YFP - B56α . YFP - B56α was purified ( IP ) 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 PP2A - B56α holoenzyme of indicated phosphorylated GST - ADAM17 ( V724 - C827 ) fragments . GST - ADAM17 ( V724 - C827 ) substrates was phosphorylated with radioactive ATP using Kinase A incubated with PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . mean 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 ) expression of ADAM17 variants in DLD - 1 Adam17 - / - , 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 - / - ( clone # 1 ) expressing full - ADAM17 variants ( wt , I762A or LEE ) . Two sided , unpaired Students t - was applied to for significant differences * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Mean 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 I762A full ADAM17 was immuno - purified from Adam17 - / - treated with H2O2 subjected to label - free LC - MS / MS to determine differential phosphorylation status of T735 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 ADAM17 variant expressing DLD - 1 Adam17 - / - upon H2O2 treatment , determined by Western blot quantified below as of EGFR autophosphorylated at Tyr1068 to total EGFR . Two sided , unpaired Students t - was applied to for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean 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 ) proliferation C ) Matrigel invasion of ADAM17 variant expressing DLD - 1 Adam17 - / - . Two sided , unpaired Students t - was applied to for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean 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 ) expression of ADAM17 variants in mouse breast cancer 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 volume ± of 4T1 Adam17 - / - expressing ADAM17 variants injected into mammary fat pad of Balb / c mice ( n = 10 for each group ) . Indicated significances are between I762A LEE tumors . Two sided , unpaired Students t - was applied to for significant differences * p < 0 . 05 . Mean standard error of 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 bearing mice injected with 4T1 Adam17 - / - expressing ADAM17 variants ( n = 10 for each group ) . indicated significances are between LEE wt bearing mice , LEE I762A bearing mice . Log - rank was applied to 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 of lysates from WT MIC10 KO or MIC60 KO HAP1 . MIC10 KO show a drastic reduction of MIC13 , MIC26 MIC27 while of other MICOS components remain unchanged . MIC60 KO show a drastic reduction of of 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 of WT , MIC10 KO MIC60 KO HAP1 . Scale bar 500 nm .

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


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

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

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 numbers by Hoechst ) of basal , proton leak , maximum non - mitochondrial respiration in WT MIC10 KO obtained from 3 independent experiments . data is normalized to basal respiration from HAP1 WT . Bar error bar represent mean ± from 3 independent experiments . * P = 0 . 034 for basal ( using one sample t - ) * P = 0 . 018 for maximum respiration ( using unpaired Student ' s t - ) .

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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 numbers by Hoechst ) of basal , proton leak , maximum non - mitochondrial respiration in WT MIC60 KO obtained from 3 independent experiments . data is normalized to basal respiration from HAP1 WT . Bar error bar represent mean ± from 3 independent experiments . * P = 0 . 01 for basal ( using one sample t - ) , * * * P = 0 . 0004 for maximum respiration * P = 0 . 012 for non - mitochondrial respiration ( using unpaired Student ' s t - ) .

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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 of WT MIC10 KO HAP1 immunostained with MIC60 or MIC10 antibodies ( top panel ) TOMM70 ( middle panel ) . Bottom panel shows merged . Arrowheads show colocalization of MIC60 TOMM70 . 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 of median interpunctae ( IPD ) per mitochondrion between MIC60 or MIC10 in indicated lines represented by boxplots . Boxplots show median interquartile range from 25 to 75 percentile whiskers represent minimum maximum value . Median IPD from n = 10 - 16 mitochondria ( from 2 independent experiments ) are shown as data points in boxplots . ns , not significant , unpaired Student ' s t - .

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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 expressing GFP - tagged fusion of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) 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 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 expressing GFP - tagged fusion of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) 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 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 of mobile fractions , obtained from curve fits of mitochondria expressing GFP - tagged versions of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) ATP5I ( CM ) in WT MIC13 KO Hela ( data calculated from FRAP curves shown in Figure 3B 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 expressing MIC60 - ( A ) MIC10 - SNAP ( B ) in WT Hela MIC60 - ( C ) MIC10 - SNAP ( D ) in MIC13 KO Hela , stained with silicone - rhodamine dye , 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 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 MIC13 KO Hela expressing MIC60 - MIC10 - SNAP stained with silicone - rhodamine . Number of tracks analysed ( obtained from 2 independent experiments ) : n = 2541 , 2540 , 3560 1441 in WT Hela expressing MIC60 - SNAP MIC10 - SNAP , MIC13 KO Hela expressing MIC60 - SNAP MIC10 - SNAP , respectively . ( * * * * P ≤ 0 . 0001 for possible comparisons , unpaired Student ' s t - ) .

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

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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 - STED super - resolution ( t = 0 s ) showing WT ( A ) MIC13 KO ( C ) Hela expressing MIC10 - SNAP stained with silicone - rhodamine . Box in ( A C ) mark selection shown as a zoom in panel ( B 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 of merging splitting events of CJs in WT MIC13 KO Hela expressing MIC10 - SNAP ( 3 independent experiments , 5 - 8 mitochondria for each experiment ) represented as boxplots . Boxplots show median interquartile range from 25 to 75 percentile whiskers represent minimum maximum value . * P = 0 . 026 ( merging splitting events in WT ) * * * * P < 0 . 0001 ( merging splitting events of WT vs . MIC13 KO ) , unpaired Student ' s t - .

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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 - STED super - resolution ( t = 0 s ) , showing WT Hela expressing MIC13 - SNAP , from a time - series of 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 - STED super - resolution ( t = 0 s ) , showing WT Hela expressing MIC13 - SNAP , from a time - series of 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 magenta asterisks show cycles of cristae merging splitting marked by MIC13 - SNAP . Green arrows pointing inward connected by solid magenta arrows pointing outward connected by dotted show sites of imminent merging 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 profiles along of three cristae ( numbered 1 - 3 marked with arrows ) in ( F ) . F profiles of lines drawn across of cristae show increased intensities at edges of scans representing IBM . Magenta boxes depict 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 - STED super - resolution ( t = 0 s ) showing WT Hela 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 ( 2 . 5 s / frame ) . Green magenta asterisks show merging splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid magenta arrows pointing outward connected by dotted show sites of imminent merging 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 - STED super - resolution ( t = 0 s ) showing MIC13 KO Hela 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 ( 2 . 5 s / frame ) . Green magenta asterisks show merging splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid magenta arrows pointing outward connected by dotted show sites of imminent merging 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 profiles along of three cristae ( numbered 1 - 3 marked with arrows ) in ( F ) . F profiles of lines drawn across of cristae show increased intensities in middle of scans representing CM . represented IBM is superimposed from intensities of MIC13 - SNAP from Figure 6F . Magenta box depicts maxima at cristae 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 of cristae merging splitting events in WT MIC13 KO Hela expressing ATP5I - SNAP ( 3 independent experiments , 3 - 7 mitochondria for each experiment ) represented as boxplots . Boxplots show median interquartile range from 25 to 75 percentile whiskers represent minimum maximum value . * * * * P < 0 . 0001 , unpaired Student ' s t - .

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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 - STED super - resolution ( t = 0 s ) showing WT Hela 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 redistribution of TMRM from a high - cristae to a low - cristae ( two such instances are shown at 3 7 . 4 s ) . Green arrows pointing inward connected by solid at 1 . 5 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 - STED super - resolution ( t = 0 s ) showing WT Hela 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 magenta asterisks show cristae merging splitting cycles ( shown at 5 . 9 s 7 . 4 s ) . Green arrows pointing inward connected by solid magenta arrows pointing outward connected by dotted show sites of imminent merging 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 of WT Hela stained with NAO , imaged using Airyscan microscope , indicate cristae merging splitting dynamics . Yellow magenta asterisks show merging splitting events of cristae respectively . Yellow arrows pointing inward connected by solid magenta arrows pointing outward connected by dotted show sites of imminent merging 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 expressing ATP5I - PAGFP was photoactivated , in shown by a blue box , at 0 s . Inset of box at 0 . 98 s clearly shows crista ( red arrow ) , absent at 0 . 38 s , 0 . 59 s & 0 . 78 s ( red arrowhead showing enclosed by dotted lines ) , confirming merging of non - cristae vesicle with IBM . Threshold of insets are shown above whole . Scale bar 500 nm .

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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 of fused mitochondrion in WT Hela ( Left Panel ) showing colocalization of cristae marked with ATP5I - GFP ( using anti - GFP antibody ) ATP5I - SNAP ( stained with silicone - rhodamine ) . ( Right Panel ) Fused mitochondrion in MIC13 KO Hela 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 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 of HeLa treated with siRNA for 84 h .

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


( b ) Western blot of HeLa treated with a second independent siRNA targeting ATG5 , ATG7 or NDP52 . GAPDH total 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 of HeLa 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 of HeLa 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 comparing of DICER in MDA - 231 , T47D MDA - 435 .

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


( f ) Western blot of MDA - 231 , T47D MDA - 435 treated ( 48 h ) with inhibitors of mTORC1 ( RAP , 20 nM ) , mTORC1 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 of HeLa 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 of HeLa treated with BAF ( 200 nM , 12 h ) , RAP ( 20 nM , 24 h ) , or RAP BAF . results in a - h are representative of three experiments 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 of DICER ( monoclonal antibody 13D6 ) or control ( EEF1A1 ) in HeLa treated with control siRNA or siRNA targeting DICER .

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(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 of signal from an anti - DICER monoclonal antibody ( clone 13D6 ) or an isotype control monoclonal antibody in HeLa . 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 of AGO1 , AGO2 DICER mRNA in HeLa treated for 12 h with DMSO , BAF or CQ ( k ) or siRNA for 84 h ( l ) . Error bars show s . e . m . Uncropped 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 ) NDP52 in HeLa treated with RAP or control . arrows highlight some examples of co - localization . lower left insets show higher magnifications of areas outlined in main panel . Scale bars , 5 μm . ( b ) of DICER co - localization with NDP52 . Total of DICER co - localized with ( > 0 . 2 μm3 ) of NDP52 was quantified with Volocity software in RAP - treated / control - treated 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 ) HcRed - LC3 in HeLa treated with BAF or control . arrows highlight some examples of co - localization . Insets as in a . Scale bar , 5 μm . ( d ) 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 of GFP - DCP1A HcRed - LC3wt in HeLa . 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 . Mito , mitochondria ; MVB , multivesicular . black arrows highlight 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 of fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( AV ) autophagolysosomes ( heavy AV ; ref . ) . HEK293T were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular lysosomal markers are indicated . Material that was pelleted by centrifugation at 100 , 000g ( 100 K pellet ) or that remained in solution after 100 , 000g spin ( soluble ) was not added to 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 of UB p62 in discontinuous Histodenz gradients ( as in j ) of HEK293T treated with control ( DMSO ) or CQ . Uncropped 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 of GEMIN4 in fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( AV ) autophagolysosomes ( heavy AV ) . HEK293T were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular ( MVB ) 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 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 of GEMIN4 or control ( TUBA ) in HeLa 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 . Bottom , western blot of GEMIN4 in immunoprecipitates of anti - NDP52 , - p62 or control antibody from HeLa .

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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 .

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


( f ) Western blot of AGO2 immunoprecipitates from HeLa .

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


( g ) Western blot of GEMIN4 , compared with loading controls ( DCP1A , CLN3 ) in HeLa treated with control siRNA or siRNA targeting GEMIN3 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 of HeLa treated with indicated combinations of control siRNA or siRNA targeting GEMIN3 , GEMIN4 ATG5 . Western blot of CLN3 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 with anti - UB monoclonal antibody ( mono - polyubiquitylated , FK2 clone ) in Flag immunoprecipitates performed under stringent denaturing conditions . Flag - AGO2 was induced for 24 h with tetracycline in HEK293T with Tet - inducible Flag - AGO2 stably integrated that had been treated 24 h before with 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 of HeLa 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 of miR - 16 or let - 7a in AGO2 immunoprecipitates from DMSO - BAF - treated HeLa . Error bars represent s . e . m . Bottom , a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments . Uncropped 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 let - 7a in HeLa treated with siRNA for 84 h . of U6 , ethidium - bromide - stained total on northern ( loading ) 28S 18S ribosomal 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 of let - 7a miR - 16 ( b ) , pre - let - 7a pre - miR - 16 ( c ) let - 7a * miR - 16 * ( d ) in HeLa 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 ) of signal in AGO2 immunoprecipitates from HeLa extracts incubated with duplexes of let - 7a - let - 7a * labelled on 3 ′ end of either let - 7 or let - 7a * with FITC . were treated 16 h before with DMSO ( control ) , BAF ( 200 nm ) or geldanamycin ( 10 μM ) . Representative results of one experiment are shown .

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(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 of AGO2 immunoprecipitates ( IP ) used in e .

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(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 of blots are shown in Supplementary Fig . S3 .

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(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 depleted of ATG5 or ATG7 with siRNA ( black grey bars correspond to two independent siRNAs ) . Results are normalized to firefly luciferase Renilla luciferase expression in 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 depleted of NDP52 or ATG5 with siRNA ( black grey bars are independent siRNAs ) . Results are normalized to firefly luciferase a version of Renilla luciferase construct with mutated let - 7 binding sites . n = 3 independent experiments . asterisks denote mutations in let - 7 target sites of 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 of endogenous miRNA - repressed ( RAS , HMGA2 CDK6 are repressed by let - 7 ; CDK6 is also repressed by miR - 16 ( bottom panel ) ) or control ( TUBA , α - tubulin ) in HeLa treated with control siRNA or either of two independent siRNAs targeting ATG7 . 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 DICER 3 ′ UTR in HeLa 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 of DICER in HeLa treated with control siRNA or either of two independent siRNAs targeting ATG7 . 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 DICER 3 ′ UTR in HeLa treated with either of two independent siRNAs targeting ATG5 or a control siRNA . error bars are s . e . m . Uncropped 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 first 400 nt of 3 ' UTRs represented in UPF1 RIP - Seq dataset were analyzed for occurrence of motifs corresponding to indicated - binding . Transcripts were divided into groups on number of motifs present ( 0 , 1 , more than 1 ) , indicated by gradient triangle . dotted indicates mean RIP - Seq efficiency for transcripts used in . Mean SD are plotted for 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 under conditions of UPF1 or non - targeting shRNA , for transcripts with hnRNP L eCLIP peaks in indicated intervals Each trace represents a category of CLIP peak locations . For mRNAs with peaks > 400 nt from TC or with peaks in 5 ' UTR or CDS , transcripts with peaks at 3 ' UTR positions 1 - 400 were excluded . Statistical significance was determined by Kruskal - Wallis with Dunn ' s correction for multiple comparisons was determined on comparison to 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 stability of β - globin reporter transcripts containing SMG5 3 ' UTR alone or supplemented with cassettes containing indicated RSE variants in HeLa Tet - off . Half - lives were determined from at least 3 independent experiments decay curves are shown with points representing mean standard deviation at each timepoint . Significance was measured compared to SMG5 3 ' UTR reporter using sum - of - squares F .

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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 immunoprecipitation experiments evaluating hnRNP L association with deletion replacement reporter transcripts from ( C ) . " AS " indicates a reporter in which antisense of RSE was inserted in SMG5 3 ' UTR in same position as RSE . northern blot is representative of 3 independent experiments is presented in accompanying bar graph with mean standard deviation . * indicates P = 0 . 0146 in two - Student ' s t - . reporter containing GAP 3 ' UTR used here as a transfection control contains a predicted hnRNP L binding site , allowing its consistent recovery in 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 - seq of treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L 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 . P value for decreased set is from comparison to 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 - Seq to decrease in abundance in absence of hnRNP L . Values represent mean standard deviation of 3 independent biological replicates . Significance of hnRNP L KD UPF1 KD were compared to NT , double knockdown was compared to hnRNP L KD . For clarity , only a single star is used to indicate significance ; in cases P ≤ 0 . 025 in two - Student ' s t - .

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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 on proportional recovery of transcript labeled with 5 - EU in 1 hour versus total transcript . Significance was determined using two - Student ' s t - : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 . Values represent mean standard deviation of 3 independent replicates .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 stability of β globin reporters with CSRP1 3 ' UTR , along with several variants in which putative hnRNP L motifs in indicated intervals were mutated , were evaluated in HeLa Tet - off . schematic indicates areas of 3 ' UTR CA - enrichment in blue includes of first 200 nucleotides of CSRP1 3 ' UTR , where putative hnRNP L binding elements are in red typeface . 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 WT reporter using sum - of - squares F .

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

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

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-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

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

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

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 of recovered UPF1 - associated transcripts analyzed by RIP - Seq Transcripts are divided into tertiles by hnRNP L PTBP1 binding motif density 3 ' UTR . Data are represented analyzed as in ( A ) . Dotted represents median RIP - Seq efficiency of class of transcripts with shortest 3 ' UTR 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 .

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

C 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 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 NT + CHX were compared to NT + DMSO , hnRNP L KD + CHX was compared to hnRNP L KD + DMSO . Bars represent mean ± SD . For clarity , only a single star was used to indicate significance ; in cases P ≤ 0 . 05 in two - Student ' s t - .

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

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 transfected with non - targeting or UPF1 siRNAs . For plots comparing non - targeting with UPF1 knockdown , significance is compared to non - targeting condition using sum - of - squares F . fourth plot compares non - targeting decay curves of three reporters , significance is compared to decay of fusion transcript . Points represent mean ± SD from qRT - PCR at each time point of at least 3 independent experiments .

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 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

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 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 - Student ' s t - . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

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.


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

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

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

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

C 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 SU - DHL - 6 were treated with varying doses of BCL2 inhibitor . viability was measured after 24 hours of drug treatment using an for ATP as a proxy for live . 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 SU - DHL - 6 calculated using sum - of - squares F is given in graph key . Significance of points calculated using two - Student ' s t - is marked using asterisks on graph . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 - Student ' s t - . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

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

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

D Representative 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 ( n = 4 independent experiments ) . Significance was determined as compared to control Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of reduction western blot of lysates from a representative experiment of edited . Target from 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 - Student ' s t - . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

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

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

G 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 ) expressing GFP − Atg8 were grown to mid - log - phase in YPD were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic or with DMSO ( - ) in rich medium for 30 min . were washed shifted to nitrogen - starvation medium ( SD - N ) for 4 h in presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic or DMSO . were then lysed subjected to SDS − PAGE , followed by western blot using anti - GFP 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 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR 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

(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),