C . CFA with dasatinib or imatinib for H1915 cells with or without tetracycline - inducible expression of ABL1 - T315I . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( control , DMSO vs dasatinib P = 4 . 3 × 10 - 12 vs imatinib P = 6 × 10 - 6 ; dasatinib , control vs tetracycline , P = 9 × 10 - 7 ; imatinib , control vs tetracycline P = 9 . 7 × 10 - 5 ) .

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

C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5).


D - F . Knockdown of mutated ABL1 reduces viability of lung cancer cells . Top , western blot analysis of ABL1 knockdown in ( D ) Beas - 2B , ( E ) H1915 and ( F ) H2110 cells upon transfection with three ABL1 siRNAs , negative control ( scrambled ) and CyclophilinB positive control . Bottom , MTT assay showing viability of ( D ) Beas - 2B , ( E ) H1915 and ( F ) H2110 cells in response to ABL1 knockdown . Error bars represent mean ± S . E . M , n = 9 , * P < 0 . 005 , * * P < 0 . 001 by two - tailed unpaired Student ' s t - test ( Beas - 2B , scrambled vs siABL1 - 3 P = 0 . 002 ; H1915 , scrambled vs siABL1 - 1 P = 0 . 004 , vs siABL1 - 2 P = 0 . 001 , vs siABL1 - 3 P = 0 . 002 ; H2110 scrambled vs siABL1 - 1 P = 5 × 10 - 6 , vs siABL1 - 2 P = 2 × 10 - 5 , vs siABL1 - 3 P = 4 . 2 × 10 - 6 ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Knockdown of mutated ABL1 reduces viability of lung cancer cells. Top, western blot analysis of ABL1 knockdown in (D) Beas-2B, (E) H1915 and (F) H2110 cells upon transfection with three ABL1 siRNAs, negative control (scrambled) and CyclophilinB positive control. Bottom, MTT assay showing viability of (D) Beas-2B, (E) H1915 and (F) H2110 cells in response to ABL1 knockdown. Error bars represent mean ±S.E.M, n=9, *P<0.005, **P<0.001 by two-tailed unpaired Student's t-test (Beas-2B, scrambled vs siABL1-3 P=0.002; H1915, scrambled vs siABL1-1 P=0.004, vs siABL1-2 P=0.001, vs siABL1-3 P=0.002; H2110 scrambled vs siABL1-1 P=5×10-6, vs siABL1-2 P=2×10-5, vs siABL1-3 P=4.2×10-6).


A - C . Average tumor volumes of xenografts derived from H1915 ( A ) , H650 ( B ) and ( C ) H1915 cell line with doxycycline inducible ABL1 gate - keeper mutation ( H1915 - ABL1T315I ) in mice treated with either vehicle or imatinib ( 100mg / kg ) . Error bars represent ± S . D . , n = 6 - 10 mice per group , * P = 0 . 0016 by Mann - Whitney U test .

O O O O O O 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 B-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 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-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.


A and C . Root mean square deviation ( RMSD ) analysis of the movement of ( A ) αC - helix and ( C ) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation ( orange ) compared to the wild type ( WT ) kinase ( black ) . B and D . Time - lapse images show movement within the αC - helix ( B ) and DFG motif ( D ) of the inactive structure of the WT ( black outline ) and R351W ( orange outline ) constructs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs.


E . left , western blot analysis of transient overexpression of wild type ABL1 ( WT ) and ABL1 - R351W , compared to empty vector control ( EV ) in HEK293T cells . Right , quantification of p - CRKL signal based on densitometry analysis of blots from 6 independent experiments . Error bars represent ± S . D . , n = 6 , * P = 0 . 0004 by two - tailed unpaired Student ' s t - test .

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

E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.


F . Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors ( Ding et al , 2008 ) in H157 cells . Values indicate quantified band intensity for p - CRKL , relative to α - tubulin and HA - tagged ABL1 .

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

F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.


A . Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines ; scale bars = 20 μm . B and C Quantification of nuclear versus cytoplasmic localization of ( B ) endogenous ABL1 in NSCLC cell lines and ( C ) tetracycline - inducible expression of ABL1 - WT , ABL1 - R351W and ABL1 - G340L in Beas - 2B cells . Error bars represent ± S . D . , n = 6 - 10 images , * P < 0 . 005 by two - tailed unpaired Student ' s t - test ( Beas - 2B vs H1915 P = 6 . 6 × 10 - 5 , vs H2110 P = 0 . 0001 ; WT vs R351W P = 0 . 003 , vs G340L P = 0 . 002 ) .

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 B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).


Percentage of the total signal intensity attributed to phosphopeptides ( blue bars ; 11 unique peptide ions ) and non - phosphorylated peptides ( grey bars ; 25 unique peptide ions ) following SAX fractionation at ( A ) pH 6 . 0 , ( B ) pH 6 . 8 and ( C ) pH 8 . 0 , with later fractions consisting of up to 100 % phosphopeptide ion signal . Representative pHis - containing myoglobin peptide ( LFTGHPETLEK ; solid line ) , and its non - phosphorylated counterpart ( LFTGHPETLEK ; dashed line ) , quantified across all 16 SAX fractions ; SAX was performed at pH 6 . 0 ( red ) , pH 6 . 8 ( blue ) or pH 8 . 0 ( green ) . % of the total peak area of each individual peptide across the gradient is plotted for each fraction . In order to assess pHis stability and effects of SAX separation in a complex mixture ( black ) , SAX was also repeated at pH 6 . 8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion . Data for the five pHis - containing myoglobin peptides is shown in Fig . EV3 .

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

Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.


siRNA - mediated knockdown of PHPT1 in HeLa cells ( 24 h ) with reference to GAPDH loading control . NT - non - targeting siRNA control .

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

siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) .

Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) . Base peak chromatograms are shown for select SAX fractions following high - resolution LC - MS / MS using an Orbitrap Fusion mass spectrometer : ( D ) fraction 3 ( green ) ; ( E ) fraction 6 ( red ) ; ( F ) fraction 10 ( grey ) . Peptides were fragmented by HCD , with neutral loss of 98 amu from the precursor ions triggering EThCD ( Ferries et al , 2017 ) . Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot . The ptmRS node was used for phosphosite localisation . Analysis was performed on three independent biological replicates for each condition ( NT or PHPT1 siRNA ) .

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

Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).


Comparison of triplet score ( ≥ 5 % S / N ) following HCD of unique phosphopeptides ( 5 % FDR ) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides . Site of phosphorylation ( ptmRS score ≥ 0 . 90 ) is indicated . % of phosphopeptides exhibiting either no neutral loss ( Triplet = 0 ) , or neutral loss of any 1 ( Triplet = 1 ) , 2 ( Triplet = 2 ) , or 3 ions from the precursor ( ∆ 80 , ∆ 98 and / or ∆ 116 amu ) . Distribution of triplet score following HCD fragmentation as a function of site localisation confidence ( ptmRS score ≥ 0 . 75 , ≥ 0 . 90 , ≥ 0 . 95 , ≥ 0 . 99 ) for unique singly phosphorylated peptide as a function of the residue phosphorylated . Numbers of phosphopeptides ( ptmRS ≥ 0 . 90 ) exhibiting different combinations of the three neutral loss species ( ∆ 80 , ∆ 98 and / or ∆ 116 amu from precursor ) for each of the phosphorylated residues .

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

Comparison of triplet score (≥5% S/N) following HCD of unique phosphopeptides (5% FDR) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides. Site of phosphorylation (ptmRS score ≥ 0.90) is indicated. % of phosphopeptides exhibiting either no neutral loss (Triplet =0), or neutral loss of any 1 (Triplet = 1), 2 (Triplet =2), or 3 ions from the precursor (∆80, ∆98 and/or ∆116 amu). Distribution of triplet score following HCD fragmentation as a function of site localisation confidence (ptmRS score ≥ 0.75, ≥ 0.90, ≥ 0.95, ≥ 0.99) for unique singly phosphorylated peptide as a function of the residue phosphorylated. Numbers of phosphopeptides (ptmRS ≥ 0.90) exhibiting different combinations of the three neutral loss species (∆80, ∆98 and/or ∆116 amu from precursor) for each of the phosphorylated residues.


( B ) RAW 264 . 7 cells transfected for 48 h with control ( sc ) , MTMR6 or Jumpy siRNA , were pretreated for 30 min with 100 nM Bafilomycin A1 ( BafA1 ) , 10 μg / ml E64d and 10 μg / ml pepstatin , then incubated for 30 min in full or starvation media in the presence of BafA1 , E64d and pepstatin . Cells were lysed and analysed by immunoblotting with anti - LC3 or anti ‐ actin . Densitometric LC3 ‐ II / actin ratios are shown underneath the blot . Graph : LC3 ‐ II / actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3 ‐ II / actin ratio for control siRNA ( sc ) in starvation medium .

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 O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR 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 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) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.


( C , D ) RAW 264 . 7 cells were transfected for 36 h with control ( scramble ) or Jumpy siRNA , transfected once more with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct overnight and incubated for 2 h in full or starvation media . Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy . ( C ) Representative confocal images of RAW 264 . 7 cells in full or starvation media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . Red and yellow arrows indicate GFP − RFP + and GFP + RFP + puncta , respectively . ( D ) Quantitation of number of LC3 puncta per cell , total puncta per cell ( GFP + RFP + and GFP − RFP + puncta ) GFP + RFP + puncta per cell and GFP − RFP + puncta per cell . Data , mean ± s . e . m . n = 5 independent experiments , 30 cells per experiments . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( t ‐ test ) . Scale bars , 5 μm .

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 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 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 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

(C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm.


( E , F ) C2C12 myoblasts were transfected for 48 h with control ( scramble ) or Jumpy siRNA , followed by a second transfection overnight with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct . Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy . ( E ) Representative confocal images of C2C12 cells in full media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . ( F ) Quantification of the number of LC3 puncta per cell . Data , mean ± SEM for n = 3 ( independent experiments ) , 30 cells per experiments . * P 0 . 05 ( t - test ) . Scale bars , 10 μm .

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

(E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm.


( G , H ) C2C12 cells were transfected for 48 h with control ( sc ) ( lanes 1 , 2 , 5 , 6 ) or Atg5 siRNA ( lanes 3 , 4 , 6 , 7 ) followed by a second transfection for 48 h with same siRNA and control ( lanes 1 , 3 , 5 , 7 ) or Jumpy siRNA ( lanes 2 , 4 , 6 , 8 ) . Cells were incubated for 1 h with or without 100 nM Baf A1 , 10 μg / ml E64d and 10 μg / ml pepstatin in full media , lysed and analysed by immunoblotting with anti ‐ LC3 or anti ‐ actin ( G ) . ( H ) Densitometric LC3 ‐ II / actin ratios for samples treated with BafA1 and protease inhibitors from G ( lanes 5 - 8 ) . Inset shows Atg5 knock ‐ down by immunoblotting .

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

(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.


( A ) Proteolysis of long ‐ lived proteins in C2C12 myoblasts . C2C12 cells were transfected with control ( scramble ) or Jumpy siRNA ( siJumpy ) , labelled overnight in media containing [ 3H ] leucine , washed , incubated for 2 h in complete media ( containing cold leucine ) and incubated for 4 h in full or starvation media . Leucine release was calculated from radioactivity in the tricarboxylic acid ‐ soluble form relative to total cell radioactivity . Results shown represent mean ± s . e . m . for combined data from three 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 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 O O O O O O O O O O O O

(A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments.


( B ) Quantitation of p62 protein levels . C2C12 cells were transfected for 48 h with control ( sc ) or Jumpy siRNA and p62 levels analysed by immunoblotting , quantitated by densitometry and represented as a percentage of control . Data are mean ± s . e . m . ( n = 4 independent experiments ) .

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(B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments).


( C ) C2C12 cells were transfected for 48 h with control ( sc ) or Jumpy siRNA , incubated for 4 h with or without 100 nM Bafilomycin A1 in full media , lysed , probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated ( mean ± s . e . m . , n = 3 ) .

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

(C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3).


( D ) C2C12 cells were transfected for 48 h with control ( sc ) ( lanes 1 , 2 ) or Beclin siRNA ( lanes 3 , 4 ) followed by a second transfection for 48 h with same siRNA and control ( lanes 1 , 3 ) or Jumpy siRNA ( lanes 2 , 4 ) . Cells were lysed , probed for p62 , Beclin and actin by immunoblotting and percentage of p62 were quantitated ( mean ± s . e . m . , n = 5 ) .

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 O O O B-MEASURED_VAR O B-MEASURED_VAR 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

(D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5).


( E - M ) C2C12 cells were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , fixed and immunostained with anti ‐ p62 antibody ( red ) . p62 puncta were quantitated by confocal fluorescence microscopy . Representative confocal images of GFP ( E ) , GFP ‐ Jumpy ( F ) , GFP ‐ MTM1 ( G ) , GFP ‐ MTMR2 ( H ) transfected cells , immunostained for p62 ( I ) , ( J ) , ( K ) and ( L ) , respectively . Scale bars , 5 μm . White lines represent outline of the cells . Quantitation of p62 puncta per cell ( M ) . Bars , SEM ( n = 3 independent experiments , with an average of 30 cells per experiments ) .

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

(E-M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments).


( N , O ) C2C12 cells were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , lysed , analysed for p62 , GFP and actin by immunoblotting ( N ) and percentage of p62 were quantitated ( mean ± s . e . m . , n = 3 ) ( O ) .

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(N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O).


( P , Q ) C2C12 cells were transfected for 48 h with GFP or GFP ‐ Jumpy ( Jumpy ) , incubated with or without 100 nM Bafilomycin A1 ( BafA1 ) for 4 h , lysed , analysed for p62 , GFP and actin by immunoblotting ( P ) and percentage of p62 were quantitated ( mean ± s . e . m . , n = 4 ) ( Q ) . * P0 . 05 , * * * P0 . 001 , † ns ( t ‐ test ) .

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

(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).


( A ) Transient association of Jumpy WT with LC3 + organelles . Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP ‐ Jumpy WT and Cherry ‐ LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope . EBSS was added to the cells and z ‐ stacks collected at 3 ‐ min intervals for a total of 45 min . The collected images were processed to generate a maximum projection ( collapsing a 3D image into an x - y projection ) for each time point . Arrows indicate colocalization of GFP ‐ Jumpy WT with LC3 . Graph : Plots of relative fluorescence intensity ( GFP fluorescence pixel intensity ) over time of GFP ‐ Jumpy WT colocalizing with Cherry ‐ LC3 + ‐ positive puncta indicated by arrows .

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(A) Transient association of Jumpy WT with LC3+ organelles. Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP‐Jumpy WT and Cherry‐LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope. EBSS was added to the cells and z‐stacks collected at 3‐min intervals for a total of 45 min. The collected images were processed to generate a maximum projection (collapsing a 3D image into an x-y projection) for each time point. Arrows indicate colocalization of GFP‐Jumpy WT with LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP‐Jumpy WT colocalizing with Cherry‐LC3+‐positive puncta indicated by arrows.


( B - I ) Association of Jumpy CS mutant with autophagic organelles . C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ G58K ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) and G58K ( white in D , H or blue in E , I ) . Boxed areas ( B - E ) are shown at higher magnification in the corresponding panel below ( F - I ) . Scale bars , 2 μm . White arrows indicate colocalization between Jumpy CS , LC3 but not G58K , blue arrow indicates colocalization between Jumpy CS and G58K but not LC3 .

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(B-I) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐G58K (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B-E) are shown at higher magnification in the corresponding panel below (F-I). Scale bars, 2 μm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow indicates colocalization between Jumpy CS and G58K but not LC3.


( A - H ) C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ Atg16 ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) and Atg16 ( white in B and F or blue in D and H ) . Boxed areas ( A - D ) are shown at higher magnification in the corresponding panel below ( E - H ) . White arrows indicate colocalization among Jumpy CS , LC3 and Atg16 , yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16 , blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3 .

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

(A-H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3.


( I , J ) C2C12 cells were transfected for 24 h with GFP ( I ) , fixed and immunostained with anti ‐ Atg16 ( J ) .

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(I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J).


( K - R ) C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ Atg12 ( M ) antibody . Jumpy CS ( green ) , LC3 ( red ) and Atg12 ( white in L , P or blue in N , R ) . Boxed areas ( K - N ) are shown at higher magnification in the corresponding panel below ( O - R ) . White arrows indicate colocalization among Jumpy CS , LC3 and Atg12 , yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12 , blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3 .

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(K-R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K-N) are shown at higher magnification in the corresponding panel below (O-R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3.


( S - ZA ) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ lamp1 . Jumpy CS ( green ) , LC3 ( red ) and Lamp1 ( white in T , X or blue in V , ZA ) . Boxed areas ( S - V ) are shown at higher magnification in the corresponding panel below ( W - ZA ) . Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1 . Scale bars , 2 μm .

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(S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.


( A - C ) C2C12 were transfected for 48 h with Jumpy siRNA , followed by a second transfection overnight with Jumpy siRNA and GFP ‐ WIPI ‐ 1 , incubated for 4 h in starvation media , fixed and immunostained with anti ‐ Atg16 antibody ( red ) . GFP ‐ WIPI1 ‐ transfected cell ( A ) , endogenous Atg16 immunostaining ( B ) , colocalization of GFP ‐ WIPI ‐ 1 and Atg16 is indicated in yellow in merge image ( C ) . Boxed areas are shown at higher magnification as inset . Scale bars , 10 and 5 μm in insets .

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(A-C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets.


( D - I ) C2C12 cells were transfected for 48 h with control ( scramble ) ( D , E ) or Jumpy siRNA ( F - H ) , followed by a second transfection overnight with corresponding siRNA and GFP ‐ WIPI ‐ 1 . Cells were incubated for 4 h in full ( D , G ) or starvation media ( E , F , H ) in presence ( F ) or absence ( E - H ) of 100 nM wortmannin , fixed and analysed by confocal fluorescence microscopy . Quantitation of WIPI ‐ 1 puncta per cell ( I ) . Bars , s . e . m . ( n = 3 independent experiments , with an average of 30 cells per experiment ) . * P0 . 05 , * * P0 . 01 ( t ‐ test ) .

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

(D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).


( A - T ) C2C12 cells were transfected for 24 h with GFP , GFP ‐ Jumpy WT or GFP ‐ Jumpy C330S ( Jumpy CS ) and Tomato ‐ LC3 ( LC3 ) and HA ‐ Atg9 , fixed and immunostained with anti ‐ HA . GFP ( I ) , Jumpy WT ( M ) , Jumpy CS ( green ) ( A , E , Q ) , LC3 ( red ) ( C , G , J , N , R ) and Atg9 ( white in B , F , K , O , S or blue in D , H , I , P , T ) . Boxed areas ( A - D ) are shown at higher magnification in the corresponding panel below ( E - H ) . White arrows indicate colocalization between Jumpy CS , LC3 and Atg9 , blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9 . White boxes ( I - T ) show LC3 puncta location . Scale bars , 5 μm .

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(A-T) C2C12 cells were transfected for 24 h with GFP, GFP‐Jumpy WT or GFP‐Jumpy C330S (Jumpy CS) and Tomato‐LC3 (LC3) and HA‐Atg9, fixed and immunostained with anti‐HA. GFP (I), Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization between Jumpy CS, LC3 and Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9. White boxes (I-T) show LC3 puncta location. Scale bars, 5 μm.


( U ) Percentage of LC3 puncta colocalizing with Atg9 . n , number of puncta counted .

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(U) Percentage of LC3 puncta colocalizing with Atg9. n, number of puncta counted.


( A - K ) C2C12 cells were transfected for 48 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) , YFP ‐ Jumpy C330S ( Jumpy CS ) , YFP ‐ Jumpy Y462C ( Jumpy YC ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , fixed and immunostained with anti ‐ p62 antibody ( red ) . p62 puncta were quantitated by confocal fluorescence microscopy . Representative confocal images of YFP ( A ) , YFP ‐ Jumpy WT ( B ) , YFP ‐ Jumpy C330S ( C ) , YFP ‐ Jumpy Y462C ( D ) and YFP ‐ Jumpy R336Q ( E ) transfected cells , immunostained for p62 ( F ) , ( G ) , ( H ) , ( I ) and ( J ) , respectively . Scale bars , 5 μm . Quantitation of p62 puncta per cell ( K ) . Bars , s . e . m . ( n = 3 independent experiments , 30 cells per experiments ) . * P0 . 05 ( t ‐ test ) , ns : nonsignificant .

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(A-K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant.


( L ) HeLa cells were transfected for 24 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , incubated with or without 50 ng / μl rapamycin ( BafA1 ( 100 nM ) was present in both control and rapamycin treated cells ) for 2 h , lysed and analysed for LC3 , YFP and actin by immunoblotting . Densitometric LC3 ‐ II / actin ratios are shown underneath the blot .

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(L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.


( C ) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study . The relevant markers are listed to the left of the micrographs . Scale bar : 5 µm . ( D ) Pie charts showing the proportion of specific phenotype mutants ( SPMs ) that have one or more distinct aberrant phenotypes , and non - phenotype - specific mutants for each of the compartments screened .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened.


Representative images of wild - type and mutant cells organized by marker and phenotype ( labels on the left of each panel ) . For each phenotype , three cells labelled for the training set ( labelled single cells ) and cells identified by the 2NN classifier ( identified single cells ) are shown . The table to the right of the images shows ( from left to right ) : [ 1 ] the occurrence of each phenotype in a wild - type population ( % in WT ) ; [ 2 ] the number of specific phenotype mutants ( all ) and stringent specific phenotype mutants ( str ) for each of the 17 mutant morphologies ; [ 3 ] the most significantly enriched GO Slim biological process and ; [ 4 ] the most significantly enriched protein complex . # denotes term below statistical significance .

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Representative images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.


Endocytic patch formation dynamics in the ydl176wΔ ( ipf1Δ ) strain . Patch dynamics were examined using time - lapse fluorescence microscopy of wild - type ( WT ) and ipf1Δ deletion strains carrying reporters for the coat ( Sla1 - GFP ; green ) , and actin ( Sac6 - tdTomato ; red ) modules . Left : Representative kymographs for the WT and ipf1Δ strains . Right : Box plot illustrating the distribution of lifetimes of Sla1 - GFP and Sac6 - tdTomato patches . Significance was determined using unpaired t - tests . Scale bar : 10 seconds .

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Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.


Penetrance as a function of replicative age . Top : Bar graph showing the fraction of outliers in populations of increasing replicative age ( # of divisions ) for wild - type ( WT ) , and 5 mutant strains ( rrd2Δ , rpl20bΔ , cka2Δ , vac8Δ and vac17Δ ) . Data are presented as mean of 3 biological replicates + / - SD . Bottom : Box plot with the distribution of cell sizes for the same populations of cells . Whiskers extend to the 5th and 95th percentile .

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Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.


Micrographs of young ( top row of images ) and older ( bottom row of images ) wild - type ( WT ) cells expressing Vph1 - EGFP ( green vacuole ) and stained with CF640R WGA ( magenta bud scars ) . Scale bar : 5 µm .

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Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.


Combined effect of replicative age and a vacuole inheritance defect on penetrance . Micrographs of wild - type and vac17Δ cells expressing Vph1 - EGFP ( green vacuole ) and Hta2 - mCherry ( red nucleus ) , stained with CF640R WGA ( magenta bud scars ) . Cells with increasing bud - scar staining ( replicative age ) are shown from left to right . Scale bar : 5 µm .

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Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.


Relationship between stress response and penetrance . Single - cell UPRE - RFP levels were measured in ~ 60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology . Cells were binned into equal - sized bins , from low to high stress response , assessed as outlier or inlier , and clustered based on their penetrance profile ( composed from the fraction of outliers in each stress - response bin ) . Each line plot represents a penetrance profile . * denotes the cluster with a profile most similar to wild - type . Insert pie charts show the proportion of mutant strains in each cluster .

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Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.


( C ) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol / kg ( 15 . 4 mg / kg ) oral dose . Shown are unbound fraction ( main graph ) and total levels ( insert ) of CNP520 in the blood and brain , using values for rat plasma protein binding of 97 . 8 % and unspecific binding to brain homogenate of 99 % . Values are mean ± SD , with n = 5 per time point .

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(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.


( A ) Subjective assessment of hair depigmentation ( score 0 - 5 , with 0 = completely black and 5 = completely white ) at 2 doses of either CNP520 or the BACE - 1 / - 2 inhibitor NB - 360 ( mean ± SEM , n = 8 / group ) , statistical comparisons were made for every scoring day , versus vehicle using Kruskal - Wallis with Dunn ' s post - hoc test .

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(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test.


( B ) Summary of doses , steady - state effects on Aβ40 in brain ( % compared to vehicle - treated mice ) and the ratios between total skin concentrations of CNP520 and NB - 360 and their respective IC50 values for inhibition of BACE - 2 in vitro .

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(B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.


( A ) Doses from 1 . 5 mg / kg ( 3 µmol / kg ) to 51 . 3 mg / kg ( 100 µmol / kg ) were given by oral gavage to rats ( n = 5 per dose ) ; animals were killed 4 hours later for determination of Aβ40 in brain tissue . Values are mean ± SEM as determined by ANOVA ( GraphPad Prism ) .

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(A) Doses from 1.5 mg/kg (3 µmol/kg) to 51.3 mg/kg (100 µmol/kg) were given by oral gavage to rats (n = 5 per dose); animals were killed 4 hours later for determination of Aβ40 in brain tissue. Values are mean ± SEM as determined by ANOVA (GraphPad Prism).


( B ) Time - dependent reduction of Aβ40 in rat brain and CSF after a single 15 . 4 mg / kg ( 30 µmol / kg ) oral dose of CNP520 .

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(B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520.


( C ) CNP520 levels in dog blood and CSF , following a 3 . 1 mg / kg ( 6 µmol / kg ) oral dose ( mean ± SD = 4 ) .

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(C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4).


( D ) Time - dependent reduction of Aβ40 and 42 in dog CSF , after a 3 . 1 mg / kg oral CNP520 dose ( mean ± s . d . ) .

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(D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( A ) CNP520 levels in blood and cerebellum .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum.


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( B ) Total cortical plaque area ( normalized to total sample area ( * 10 − 2 ) , stained with antibody NT - 12 .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (B) Total cortical plaque area (normalized to total sample area (*10−2), stained with antibody NT-12.


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( C ) Formic acid - soluble Aβ40 in forebrain . ( D ) Formic acid - soluble Aβ42 in forebrain .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (C) Formic acid-soluble Aβ40 in forebrain. (D) Formic acid-soluble Aβ42 in forebrain.


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( E ) Double - immunofluorescence stained brain sections with NT12 ( against amyloid - β , green ) and Iba1 ( against microglia , red ) or GFAP ( astrocytes , red ) for the different treatment groups as indicated . Scale bar : 500 µm .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (E) Double-immunofluorescence stained brain sections with NT12 (against amyloid-β, green) and Iba1 (against microglia, red) or GFAP (astrocytes, red) for the different treatment groups as indicated. Scale bar: 500 µm.


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( F ) Quantification of total Iba1 positive microglia ( normalized to total area ( * 10 − 2 ) , only a subset of samples analyzed .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (F) Quantification of total Iba1 positive microglia (normalized to total area (*10−2), only a subset of samples analyzed.


Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( G ) Quantification of total GFAP positive astrocyte area ( normalized to total area ( * 10 − 2 ) . Data were analyzed with Dunnett ' s multiple comparison test .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (G) Quantification of total GFAP positive astrocyte area (normalized to total area (*10−2). Data were analyzed with Dunnett's multiple comparison test.


( A ) Mean plasma concentration vs time profiles after single dose .

(A) Mean plasma concentration vs time profiles after single dose.


( B ) Mean plasma concentration profiles for 91 days of dosing and post - dose concentrations up to day 121 .

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(B) Mean plasma concentration profiles for 91 days of dosing and post-dose concentrations up to day 121.


( C ) Time vs CSF concentration profiles after single dose of CNP520 ( Mean ± SEM , n = 6 - 8 ) .

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(C) Time vs CSF concentration profiles after single dose of CNP520 (Mean ± SEM, n = 6-8).


( D ) Dose - CSF concentration profiles after 3 months of dosing ( Mean ± SEM , n = 22 - 24 ) .

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(D) Dose-CSF concentration profiles after 3 months of dosing (Mean ± SEM, n = 22-24).


( A ) Changes in Aβ40 relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± SEM , n = 6 - 8 ) .

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(A) Changes in Aβ40 relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( B ) Changes in sAPPβ relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± SEM , n = 6 - 8 ) .

(B) Changes in sAPPβ relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( C ) Changes in sAPPα relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± SEM , n = 6 - 8 ) .

(C) Changes in sAPPα relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( D ) Dose response of Aβ40 ( mean ± SD ) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing . ( D ) CNP520 was administered daily for 14 days ; CSF was sampled via a lumbar catheter 2 hours before dosing , and 24 hours after the last dose ( mean ± SD , n = 8 in treatment groups , n = 16 in placebo group ) .

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(D) Dose response of Aβ40 (mean ± SD) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing. (D) CNP520 was administered daily for 14 days; CSF was sampled via a lumbar catheter 2 hours before dosing, and 24 hours after the last dose (mean ± SD, n = 8 in treatment groups, n = 16 in placebo group).


( A ) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing . Shown are means ± SD , n = 21 - 25 for pre - dose and day 91 , and n = 2 - 7 for the intermediate time points .

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(A) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing. Shown are means ± SD, n = 21-25 for pre-dose and day 91, and n = 2 - 7 for the intermediate time points.


( B ) Dose - response for CSF Aβ40 at day 91 ( 24 hours after last dose ) and individual percent change from baseline , shown as mean and 95 % confidence interval , n = 21 - 25 per treatment group .

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(B) Dose-response for CSF Aβ40 at day 91 (24 hours after last dose) and individual percent change from baseline, shown as mean and 95% confidence interval, n = 21-25 per treatment group.


( A ) Mouse forebrain Aβ40 levels in homozygous male and female APOE4 - TR mice with and without CNP520 treatment , 4 hours after dosing , shown are mean ± SEM , n = 6 / group .

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(A) Mouse forebrain Aβ40 levels in homozygous male and female APOE4-TR mice with and without CNP520 treatment, 4 hours after dosing, shown are mean ± SEM, n = 6/group.


( B ) Distribution of APOE4 genotypes in the Phase IIa study , vs their baseline Aβ42 / 40 ratios . The dashed line at Aβ42 / 40 ratio of 0 . 09 represents a cutoff , which separates a normal ratio from an abnormally low ratio .

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(B) Distribution of APOE4 genotypes in the Phase IIa study, vs their baseline Aβ42/40 ratios. The dashed line at Aβ42/40 ratio of 0.09 represents a cutoff, which separates a normal ratio from an abnormally low ratio.


( C ) Comparison of CSF Aβ40 reduction in APOE4 non - carriers and APOE4 carriers .

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(C) Comparison of CSF Aβ40 reduction in APOE4 non-carriers and APOE4 carriers.


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(D) Comparison of CSFAβ40 reduction in participants with Aβ42/40 ≥ 0.09 and Aβ42/40 < 0.09.


( E ) Comparison of Aβ42 / 40 ratio at baseline and at day 91 for all participants in the placebo , 35 mg , and 85 mg dose group .

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(E) Comparison of Aβ42/40 ratio at baseline and at day 91 for all participants in the placebo, 35 mg, and 85 mg dose group.


( F ) Change of Aβ42 / 40 ratio for all participants with Aβ42 / 40 ≤ 0 . 09 at baseline ; bars represent mean ± SD . Full circles : APOE4 carrier , open circles : APOE4 non - carrier .

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(F) Change of Aβ42/40 ratio for all participants with Aβ42/40 ≤ 0.09 at baseline; bars represent mean ± SD. Full circles: APOE4 carrier, open circles: APOE4 non-carrier.


293T cells were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or the indicated constructs . At 24 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , and analyzed by IB using antibodies against PGAM5 and FLAG . Five percent of lysates was analyzed as input .

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293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.


HeLa cells stably expressing FLAG - Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).


MBP or the MBP - Stx17 constructs attached to amylose resin were mixed with GST - PGAM5 , and the proteins bound to the resin were separated by SDS - PAGE and blotted onto PVDF membranes . The blots were detected by an anti - GST antibody ( upper panels ) or stained with Coomassie Brilliant Blue R - 250 ( lower panels ) . Ten percent of the proteins used for each experiment was analyzed as input . Asterisks and double asterisk may represent MBP dimers and degradation products .

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MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.


293T cells were cotransfected with plasmids encoding FLAG - Stx17 WT and the indicated PGAM5 - GFP constructs , and

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed


293T cells were cotransfected with plasmids encoding FLAG - Stx17 or Stx18 and the C - terminally GFP - tagged transmembrane domain of PGAM5 （ amino acids 1 - 35 ） constructs , and

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed


HeLa cells were treated with DMSO ( Vehicle ) or 0 . 03 mg / ml digitonin ( + Digitonin ) , fixed and then double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PGAM5 and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student '

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HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's


HeLa cells stably expressing FLAG - Stx17 wild type ( WT ) were transfected with a plasmid encoding Su9 - GFP ( mitochondria ) or Sec61β - GFP ( ER ) . At 24 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PLA dots and Su9 - GFP or Sec61β - GFP . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).


HeLa cells were treated with DMSO ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and subjected to Percoll - based fractionation . Equal amounts of proteins were analyzed by IB using the indicated antibodies . PNS , postnuclear supernatant ; MS , microsomes ; Mt , mitochondria . The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment , respectively : PNS ( 6 . 6 mg and 5 . 5 mg ) , cytosol ( 4 . 8 mg and 4 . 9 mg ) , MS ( 2 . 0 mg and 2 . 2 mg ) , MAM ( 0 . 55 mg and 0 . 46 mg ) and Mt ( 0 . 30 mg and 0 . 28 mg ) .

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HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).


Electron microscopic analysis of HeLa cells expressing PGAM5 - GFP and APEX2 - GFP - binding peptide . Arrows indicate the position of 3 , 3 ' - diaminobenzidine reaction at the ER - mitochondria interface . Scale bar , 500 nm .

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Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.


HeLa cells stably expressing FLAG - Stx17 WT were mock - transfected or transfected with siRNA for Mfn1 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa cells were mock - transfected or transfected with siRNA for Mfn1 , Stx17 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against Drp1 and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed and double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm .

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HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.


293T cells with mock treatment or depleted of Stx17 were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and then analyzed by IB using the indicated antibodies .

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293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.


293T cells transiently expressing FLAG - Stx17 wild type ( WT ) or the K254C mutant were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies .

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293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.


HeLa cells stably expressing FLAG - Stx17 WT were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).


HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted ( KD ) of Stx17 , PGAM5 or Drp1 were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


HeLa cells stably expressing GFP - Parkin with mock treatment ( Mock ) or depleted of Stx17 , PGAM5 or Drp1 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by immunofluorescence microscopy . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of GFP - Parkin and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


PINK1 - FLAG and GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( ethanol ) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies . F , full - length PINK1 ; C , cleaved PINK1 .

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PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.


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

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


GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3 . Scale bar , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and LC3 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) .

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GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).


GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by IB using the indicated antibodies .

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GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.


HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


GFP - Parkin stably expressing HeLa cells with mock treatment ( Mock ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20 . Scale bar , 5 μm .

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GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.


293T cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . Three percent of lysates was analyzed as input . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 .

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293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.


GFP - Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5 . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * P < 0 . 05 and * * P < 0 . 01 as compared with Mock ( + CCCP ) by paired Student ' s t - test .

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GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.


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

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


B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration . a , Stx17 + / - ; b , PGAM5 - / - ; c , Stx17 + / - , PGAM5 - / - . The bar graph on the right represents the mitochondrial phenotypes classified p < 0 . 05 , * * p < 0 . 01 vs . the same class of Stx17 + / - ( Dunnett ' s test ) . n = 62 - 195 mitochondria from three independent samples . Scale bars = 1 µm . Genotypes used were : + / y ; Act5c - GAL4 / + ; Stx17LL06330 / + ( a ) , PGAM51 / y ; Act5c - GAL4 / + ( b ) , PGAM51 / y ; Act5c - GAL4 / UAS - Stx17 - FLAG ; Stx17LL06330 / + ( c ) .

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B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).


A ) Single dose AR - 42 plasma and tissue pharmacokinetic study . Tumor - free CD2F1 mice were administered a single dose of 10 , 20 or 50 mg / kg AR - 42 ( n = 3 ) and plasma ( dashed ) and gastrocnemius ( solid ) tissue were analyzed for AR - 42 content at different times using LC - MS / MS analyses according to the Materials and Methods ( Mean ± SD ) .

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A) Single dose AR-42 plasma and tissue pharmacokinetic study. Tumor-free CD2F1 mice were administered a single dose of 10, 20 or 50 mg/kg AR-42 (n=3) and plasma (dashed) and gastrocnemius (solid) tissue were analyzed for AR-42 content at different times using LC-MS/MS analyses according to the Materials and Methods (Mean±SD).


B ) AR - 42 dose - response . Starting six days after C - 26 cell injection , animals received vehicle or AR - 42 orally at 1 , 3 , 10 or 20 mg / kg daily or 50 mg / kg every other day for 13 days . Individual animal terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor - free controls . Groups : 1mg / kg ( n = 8 ) , 3mg / kg ( n = 8 ) , 10mg / kg ( n = 13 ) , 20mg / kg ( n = 6 ) , 50mg / kg ( n = 5 ) . Dashed reference line ( 100 % ) represents the mean tumor - free control value , solid line represents non - linear fit of dose - response data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) AR-42 dose-response. Starting six days after C-26 cell injection, animals received vehicle or AR-42 orally at 1, 3, 10 or 20 mg/kg daily or 50 mg/kg every other day for 13 days. Individual animal terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor-free controls. Groups: 1mg/kg (n=8), 3mg/kg (n=8), 10mg/kg (n=13), 20mg/kg (n=6), 50mg/kg (n=5). Dashed reference line (100%) represents the mean tumor-free control value, solid line represents non-linear fit of dose-response data.


Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) and tumor - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . C ) : Terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) .

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

Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. C): Terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).


Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . D ) Terminal tumor volumes ( mean ± SD ) .

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

Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) Terminal tumor volumes (mean±SD).