Figure 2 ( 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 ) . ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).(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).(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).(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).


Figure 3 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4 ( 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 ) . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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).


Figure 6 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


f1 ( a ) MEFs stably expressing EGFP - Parkin were exposed to CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis ( IB ) with antibodies to the indicated proteins . The asterisk indicates a nonspecific band . ( b ) SH - SY5Y cells were exposed to CCCP ( 10 μM ) for the indicated times and then subjected to immunoblot analysis . ( c ) MEFs stably expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope . The fluorescence of EGFP - Parkin was monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band.(b) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis.(c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


f2 ( a , c ) MEFs ( a ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively . ( a , c ) HeLa cells ( c ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively . ( e ) NIH 3T3 cells stably expressing EGFP - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , after which whole - cell homogenates as well as cytosolic , ER and mitochondrial fractions were prepared and subjected to immunoblot analysis . Scale bar ( a - d ) , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.(a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.(e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.


f3 ( a ) MEFs stably expressing EGFP - tagged wild - type ( WT ) or T240R mutant forms of Parkin as well as FLAG - tagged VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP was also monitored directly . ( b ) MEFs as in a were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . ( c ) MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) MEFs stably expressing EGFP - Parkin were incubated for 12 h in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) as indicated and then subjected to immunoblot analysis . Scale bar ( a , c ) , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) MEFs stably expressing EGFP-tagged wild-type (WT) or T240R mutant forms of Parkin as well as FLAG-tagged VAP-A were incubated in the absence or presence of CCCP (30 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP was also monitored directly.(b) MEFs as in a were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis.(c) MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP-Parkin was also monitored directly.(d) MEFs stably expressing EGFP-Parkin were incubated for 12 h in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) as indicated and then subjected to immunoblot analysis. Scale bar (a,c), 10 μm.


f4 ( b ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were treated with CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . Scale bar , 10 μm . ( c ) MEFs stably expressing mCherry - tagged Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

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f4(b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm.(c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


f5 ( a ) MEFs stably expressing FLAG - Parkin and either EGFP - Bcl - 2 or EGFP - Bcl - xL were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( c ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of Bcl - 2 , Bcl - xL , Bcl - 2 / xL or Bcl - xL / 2 were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a) MEFs stably expressing FLAG-Parkin and either EGFP-Bcl-2 or EGFP-Bcl-xL were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.(c) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of Bcl-2, Bcl-xL, Bcl-2/xL or Bcl-xL/2 were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


f7 ( a ) MEFs stably expressing EGFP - FKBP38 ( N402K ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - FKBP38 ( N402K ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set . ( b ) MEFs stably expressing EGFP - FKBP38 ( N402K ) , mCherry - FKBP38 and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG ( M2 ) . The fluorescence of EGFP - FKBP38 ( N402K ) and mCherry - FKBP38 was also monitored directly . ( c ) MEFs stably expressing FLAG - Parkin and either EGFP - FKBP38 or EGFP - FKBP38 ( N402K ) were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( d ) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in ( c ) . Data are expressed relative to the corresponding control value ( CCCP 0 h ) and are means ± s . d . , from three independent experiments . * * P0 . 01 ( Student ' s t test ) . ( e ) MEFs stably expressing EGFP - Bcl - 2 ( H235R ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - Bcl - 2 ( H235R ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set . ( g ) HeLa cells stably expressing FLAG - Parkin were transiently transfected with vectors for KikGR - FKBP38ΔN47 or KikGR - FKBP38ΔN47 ( N402K ) , exposed to UV , incubated in the absence or presence of CCCP ( 10 μM ) for 4 h , and then monitored for red and green KikGR fluorescence . Scale bar ( a , b , e , g ) , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.(b) MEFs stably expressing EGFP-FKBP38(N402K), mCherry-FKBP38 and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG (M2). The fluorescence of EGFP-FKBP38(N402K) and mCherry-FKBP38 was also monitored directly.(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).(e) MEFs stably expressing EGFP-Bcl-2(H235R) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-Bcl-2(H235R) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.(g) HeLa cells stably expressing FLAG-Parkin were transiently transfected with vectors for KikGR-FKBP38ΔN47 or KikGR-FKBP38ΔN47(N402K), exposed to UV, incubated in the absence or presence of CCCP (10 μM) for 4 h, and then monitored for red and green KikGR fluorescence. Scale bar (a,b,e,g), 10 μm.


f8 ( a ) Wild - type ( Mfn2 + / + ) and Mfn2 knockout ( Mfn2 - / - ) MEFs stably expressing mCherry - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c . The fluorescence of mCherry - Parkin was also monitored directly . ( b ) MEFs as in a but expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . The asterisk indicates a nonspecific band . ( c ) Wild - type MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) or nocodazole ( 10 μM ) for 6 h as indicated , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c , to FLAG and to FKBP38 . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) Higher - magnification views of the boxed areas in ( c , e ) . Wild - type MEFs stably expressing FLAG - Parkin and EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 10 h and then subjected to immuno - electron microscopic analysis with antibodies to GFP . Black arrowheads indicate the mitochondria . White arrowheads indicate small vesicle . ( f ) Wild - type ( Fkbp38 + / + ) and Fkbp38 knockout ( Fkbp38 − / − ) MEFs stably expressing FLAG - Parkin and either EGFP or EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then stained with allophycocyanin ( APC ) - labelled annexin V and propidium iodide ( PI ) for flow cytometric analysis of the percentage of apoptotic cells ( annexin V + and PI + cells ) . Data are means ± s . d . , from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f8(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly.(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.(c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle.(f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments.


Figure 1A Location of missense variants of PDIA1 and ERp57 identified in ALS cases . PDI primary structure : catalytic a and a ' domains containing the active site motif CXXC sequence ( dark purple and dark green ) , non - catalytic domains b and b ' containing ligand binding sites ( light purple and light green ) , and x - linker region ( light gray ) . The constructs used in this study contained a V5 - tag ( orange ) at the C - terminus that was inserted previous to the ER - retention signal ( dark gray ) . B Expression of PDI variants in zebrafish . Zebrafish embryos at 1 - 2 cell stage were injected with sense mRNA coding for the indicated PDIs ( PDIA1WT , PDIA1D292N and PDIA1R300H : 200 pg / embryo ; ERp57WT , ERp57D217N and ERp57Q481K : 80 pg / embryo ) . Protein expression was confirmed by Western blot analysis using anti - V5 antibody in total embryo extracts at 24 h post - fertilization ( hpf ) . Actin was used as a loading control . In the left panel 80 µg protein and in the right panel 120 µg protein extract were used . C and D , Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively . C and D , Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively . E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg ( Huc : Kaede ) embryos injected with the indicated PDIA1 ( D ) and ERp57 ( E ) mutants . E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg ( Huc : Kaede ) embryos injected with the indicated PDIA1 ( D ) and ERp57 ( E ) mutants . G Touch - evoked escape responses of 48 hpf zebrafish embryos injected with ALS - linked PDI mutants ( Kabashi et al , 2011 ; Kabashi et al , 2009 ) . The number of touches necessary to evoke an escape response ( top ) and speed ( bottom , in mm / s ) of the escape response was determined for each condition . The total number of analyzed embryos is indicated in parenthesis . Experiments of D - F were performed in selected animals sharing normal overall embryo morphology and viability , to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and / or animal behavior . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . Abbreviations : A ( anterior ) , D ( dorsal ) , P ( posterior ) , V ( ventral ) . Scale bars represent 500 m ( C and D : a , d . g , j ) , 200 m ( C and D : b , e , h , k ) , 100 m ( C and D : c , f , i , l ) , and 25 µm ( G - H ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Location of missense variants of PDIA1 and ERp57 identified in ALS cases. PDI primary structure: catalytic a and a' domains containing the active site motif CXXC sequence (dark purple and dark green), non-catalytic domains b and b' containing ligand binding sites (light purple and light green), and x-linker region (light gray). The constructs used in this study contained a V5-tag (orange) at the C-terminus that was inserted previous to the ER-retention signal (dark gray).B Expression of PDI variants in zebrafish. Zebrafish embryos at 1-2 cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1WT, PDIA1D292N and PDIA1R300H: 200 pg/embryo; ERp57WT, ERp57D217N and ERp57Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti-V5 antibody in total embryo extracts at 24 h post-fertilization (hpf). Actin was used as a loading control. In the left panel 80 µg protein and in the right panel 120 µg protein extract were used.C and D, Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.C and D, Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (D) and ERp57 (E) mutants.E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (D) and ERp57 (E) mutants.G Touch-evoked escape responses of 48 hpf zebrafish embryos injected with ALS-linked PDI mutants (Kabashi et al, 2011; Kabashi et al, 2009). The number of touches necessary to evoke an escape response (top) and speed (bottom, in mm/s) of the escape response was determined for each condition. The total number of analyzed embryos is indicated in parenthesis. Experiments of D-F were performed in selected animals sharing normal overall embryo morphology and viability, to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and/or animal behavior. Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Abbreviations: A (anterior), D (dorsal), P (posterior), V (ventral). Scale bars represent 500 m (C and D: a,d.g,j), 200 m (C and D: b,e,h,k), 100 m (C and D: c,f,i,l), and 25 µm (G-H).


Figure 2A NSC34cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIs . After 48 h , overexpressed PDI variants was assessed under reducing conditions in an 8 % SDS - PAGE . An antibody detecting total PDIA1 ( endogenous mousePDIA1 and exogenous humanV5 - tagged PDIA1 ) ( first panel ) . A second antibody detects only humanPDIA1 , therefore only V5 - tagged PDIA1 appears ( second panel ) . A mouse and human specific antibody was used to detect total ERp57 ( third panel ) . Anti - V5 was used to detect the exogenous PDI variants . Anti - β - actin was used as a loading control . B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid . Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . Increased neurite outgrowth is indicated with white arrow heads . B Quantification of the average primary neurite lengths was performed , all cells from three independent experiments were compiled . A minimum of 100 cells per experiment was analyzed . In addition , C the percentage of cells with neurites was quantified in the three independent experiments ( right panel ) . B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid . Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . Increased neurite outgrowth is indicated with white arrow heads . B Quantification of the average primary neurite lengths was performed , all cells from three independent experiments were compiled . A minimum of 100 cells per experiment was analyzed . In addition , C the percentage of cells with neurites was quantified in the three independent experiments ( right panel ) . D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro ( DIV ) transfected with GFP alone or together with the indicated PDIs constructs . Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons . Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified . Results are compiled from 3 independent experiments . E Human motoneurons were differentiated from the human embryonic stem cell ( ESC ) HuESC 3 Hb9 : : GFP line . Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs - expressing plasmids . Transduced cells were cultured for another 10 days . To identify motoneurons after lentiviral transduction , immunocytochemistry analyses were performed . Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained . Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons ( see methods ) . Four independent experiments were performed . F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . The percentage of cells with neurites was quantified in the three independent experiments . A minimum of 100 cells per experiment was analyzed . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A NSC34cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mousePDIA1 and exogenous humanV5-tagged PDIA1) (first panel). A second antibody detects only humanPDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro (DIV) transfected with GFP alone or together with the indicated PDIs constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified. Results are compiled from 3 independent experiments.E Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs-expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained. Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons (see methods). Four independent experiments were performed.F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment was analyzed.Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


Figure 3A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot . B Analysis of the PDIA1 structure to model the effects of the R300H mutation . The close association between Arg300 located to the b ' domain of PDIA1 with Trp396 located to the a ' domain adjacent to the active site motif CGHC ( designated as AS in yellow ) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues . A potential stabilization of the interaction between the b ' and a ' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396 . C ALS - linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of PDIA1WT and mutants are shown . D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants . E Representative electrophoresis analysis of Proteinase K - treated recombinant PDIA1 variants . Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples ( arrows indicate band 1 and band 2 ) indicated differences in the removal of the x - linker region . Bottom panel : Ratios of band 1 to band 2 were quantified in 4 independent experiments . F Representative fluorescence spectra of recombinant PDIA1b ' xWT fragments and the equivalent ALS - linked mutants ( n = 6 ) . Ratios from the two peak areas and change compared to PDIA1 b ' xWT . Both mutants analyzed show a significant shift in peak position compared to wild - type , but in opposite directions , suggesting that PDIA1D292N shifts equilibrium towards the capped version ( x region over the binding pocket ) and the PDIA1R300H mutant towards the uncapped version ( Nguyen et al , 2008 ) . G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis . The percentages of different BPTI species was calculated ( 6H , fully reduced ; 1S , one disulfide bond ; 2S , two disulfide bonds ; 3S , three disulfide bonds ) at time point 2 . 5 min in four independent experiments . H Measurement of H2O2 levels at the ER lumen of living NSC34 . Left panel : Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen ( O2 ) , generating hydrogen peroxide ( H2O2 ) as a product of PDIs activity . Right panel : NSC34 cells were transiently co - transfected with ER luminal HyPer sensor and indicated PDIs . After 48 h the 490 / 420 nm fluorescence ratio was recorded for 2 min under basal conditions . Means and SEM derived from all cells per condition ( n = 55 - 74 ) monitored in four independent experiments are shown . I HEK293T cells were transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous ERO1Lα was analyzed by western blot . The inputs and elutions are shown as control . Right panel : Quantification of the degree of interaction is presented .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIA1. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.B Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg300 located to the b' domain of PDIA1 with Trp396 located to the a' domain adjacent to the active site motif CGHC (designated as AS in yellow) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues. A potential stabilization of the interaction between the b' and a' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396.C ALS-linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of PDIA1WT and mutants are shown.D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants.E Representative electrophoresis analysis of Proteinase K-treated recombinant PDIA1 variants. Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x-linker region. Bottom panel: Ratios of band 1 to band 2 were quantified in 4 independent experiments.F Representative fluorescence spectra of recombinant PDIA1b'xWT fragments and the equivalent ALS-linked mutants (n=6). Ratios from the two peak areas and change compared to PDIA1 b'xWT. Both mutants analyzed show a significant shift in peak position compared to wild-type, but in opposite directions, suggesting that PDIA1D292N shifts equilibrium towards the capped version (x region over the binding pocket) and the PDIA1R300H mutant towards the uncapped version (Nguyen et al, 2008).G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.I HEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant PDIA1, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by western blot. The inputs and elutions are shown as control. Right panel: Quantification of the degree of interaction is presented.


Figure 4A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot . B ALS - linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown . C A thermal denaturation curve of ERp57WT and mutants was performed . D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins . E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P - domain . KD values are expressed as percentage of ERp57WT . Values are derived from four independent experiments . For absolute KD values please see Appendix Table S2 . FHEK293T NSC34 were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous calnexin ( CNX ) and calreticulin ( CRT ) was analyzed by Western blot . The inputs and elutions are shown . Right panel : Quantification of the degree of interaction is presented . G The gain of an N - glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT / S consensus sequence . Neuro2a cells were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector and treated with 1 µg / ml tunicamycin ( Tm ) for 20 h to inhibit N - glycosylation . Alternatively , protein extracts were digested with PNGase F and the possible removal of the N - glycosylation was analyzed by Western blot using anti - V5 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.C A thermal denaturation curve of ERp57WT and mutants was performed.D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.FHEK293T NSC34 were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.


Figure 5A ERp57flox / flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals . The levels of ERp57 protein in the spinal cord were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 levels were determined as a loading control . Right panel : Quantification of ERp57 levels was performed relative to Hsp90 levels . B Body weight measurements were performed for indicated time points in ERp57WT ( n = 50 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 19 ) mice . C Rotarod performance was performed ERp57WT ( n = 20 ) , ERp57Nes + / - ( n = 15 ) and ERp57Nes - / - ( n = 8 ) mice . D Hanging test performance was assessed in ERp57WT ( n = 41 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 12 ) mice . E Kaplan - Meier survival curve for ERp57Nes - / - mice ( N = 19 ) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days . Mean survival of this subgroup of animals was 57 days . ERp57WT ( n = 50 ) and ERp57Nes + / - ( n = 32 ) mice are shown as a reference . F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes - / - mice in three animals per group using indirect immunofluorescence . The nucleus was stained with Hoechst . Representative images from one mouse per group are shown . Scale bar : 50 μm . G Stereological analysis of the spinal cord from ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 4 ) and ERp57Nes - / - ( n = 4 ) mice . Alternate series of sections from the spinal cord of the mice were either stained for Nissl ( top row images ) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase ( ChAT , bottom row images ) . The nucleoli of the motoneurons , as stained in the Nissl series , were counted inside the motoneurons pools previously defined using the adjacent ChAT series ( contours not shown ) . Cell densities of the three genotypes are shown on the right plots . No significant differences were found between the genotypes . Scale bar represents 200 μm and 50 μm on large and inset images , respectively . H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes - / - mice .

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Figure 5A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.


Figure 6A Representative EMG recordings of ERp57WT ( n = 16 ) , ERp57Nes - / + ( n = 12 ) and ERp57Nes - / - ( n = 5 ) mice is presented . The presence of positive sharp waves ( PSW ) in ERp57Nes + / - and ERp57Nes - / - mice indicated muscle denervation . B Analysis of NMJ and muscle morphologies were performed from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) . Quantifications of endplate width , measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile . Twenty to forty measurements per diaphragm were obtained per animal . C Whole - mounted diaphragms from ERp57WT and ERp57Nes - / - mice were co - stained with anti - neurofilament ( red ) and α - BTX to reveal the postsynaptic densities ( green ) . 3D reconstructions ( lower panel ) of higher magnification . ERp57WT NMJs are fully innervated pretzel - like , whereas ERp57Nes - / - NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern ( Movies EV4 - 7 ) . D Representative 3D reconstructions of z - stacks showing the five categories in which NMJs morphologies were grouped : Pretzel , fragmented , O shaped , C shaped , and dismantled NMJs . Histogram of the distribution frequency of > 200 different NMJ per animal is shown . E Quantitative morphometry of > 60 NMJs in the different genotypes analyzed .

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Figure 6A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.


Figure 7A The levels of ERp57 protein in the muscle were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 was determined as loading control . B Tibialis anterior muscles from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) , and ERp57Nes - / - ( n = 4 ) mice were dissected and cryosectioned ( 20 µm ) for the analysis of several parameters of muscle physiology . Upper panel : hyaluronic acid ( HA ) staining was performed to assess the gross morphology of the muscle . Middle panel : Bismarck brown staining to monitor skeletal muscles fibrosis . Lower panel : Cryosections were then double stained with wheat germ agglutinin ( WGA ) to visualize plasmamembrane and nuclei . The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration / regeneration cycles . Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles . C Tibialis anterior cryosections ( 20 µm ) from ERp57WT , ERp57Nes + / - , and ERp57Nes - / - mice were stained for NADH - TR activity to identify oxidative myofibers ( upper panel ) . Representative micrographs of transversal cryosections show variable proportions of light , intermediate and dark positive staining in the different genotypes : ( middle panel ) NADH - TR positive ( slow - twitch ) and negative ( fast - twitch ) myofibers were quantified as percentage of total for every genotype ( lower panel ) The mean diameter of slow - and fast - twitch muscle fibers was evaluated at the light microscope using the ImageJ software . Data represent the average ± SEM of > 300 fibers per animal . Scale bars represent 50 m in B and C .

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Figure 7A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasmamembrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.Scale bars represent 50 m in B and C.


Figure 8A The levels of synaptic proteins SV2 , NMDAR2A , PSD95 and Synaptophysin in the cortex were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 and β - Actin were determined as loading controls . B Quantification of SV2 monomer ( left panel ) and SV2 high molecular weight species ( right panel ) . β - Actin was used as the reference .

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Figure 8A The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the cortex were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 and β-Actin were determined as loading controls.B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.


Figure 1Western blot detection of PAX6 isoforms in human fetal forebrain and retina . The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform . # 1 , # 2 are # 3 are from ~ 8 - week - old human embryos . # 4 is from ~ 12 - week - old human fetus . qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of PAX6A & B isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR of PAX6D isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs . D10 indicates day 10 of differentiation . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform .

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Figure 1Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus.qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.


Figure 2qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation . Immunostaining of PAX6 in Wildtype and PAX6 KO cells . C ' - PAX6 antibody detects PAX6A , B and D . N ' - PAX6 antibody detects PAX6 A and B but not D . Scale bars : 50 µm . qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells . Samples are collected at day 6 , 10 , 14 and 19 after the start of differentiation .

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Figure 2qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation.Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm.qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.


Figure 3qPCR verification of PAX6D KO efficiency . PAX6A & B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells . qPCR quantification of retinal gene VSX2 in PAX6D KO cells ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene SIX6 in PAX6D KO cells ( n = 3 biological replicates for each group ) . Immunostaining of VSX2 in wildtype and PAX6D KO cells . Scale bars : 100 µm . Samples were from day 30 differentiating retinal cells .

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Figure 3qPCR verification of PAX6D KO efficiency. PAX6A&B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells.qPCR quantification of retinal gene VSX2 in PAX6D KO cells (n=3 biological replicates for each group).   qPCR quantification of retinal gene SIX6 in PAX6D KO cells (n=3 biological replicates for each group).   Immunostaining of VSX2 in wildtype and PAX6D KO cells. Scale bars:100 µm. Samples were from day 30 differentiating retinal cells.


Figure 4Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues . Scale bars : 50 µm . Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines . Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines . Quantification of SOX1 - / SOX2 + ratio in panel C ( n = 3 biological replicates for each sample ) . qPCR quantification of SOX1 in wildtype , PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation ( n = 3 biological replicates for each sample ) .

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Figure 4Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues. Scale bars:50 µm.Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines. Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.Quantification of SOX1-/SOX2+ ratio in panel C (n=3 biological replicates for each sample).qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).


Figure 5qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX ( n = 3 biological replicates for each sample ) . Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX . qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells ( n = 3 biological replicates for each group ) . qPCR quantification of retinal marker expression in PAX6D TetOn cells w / o DOX treatment ( n = 3 biological replicates for each group ) . Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry . SOX1 / SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate .

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Figure 5qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.


Figure 6Verification of PAX6D target genes using PAX6D TetOn cells . Two sets of genes ( retinal and non - retinal neural genes ) are directly regulated by PAX6D in opposite directions ( n = 3 biological replicates for each group ) .

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Figure 6Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).


Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation ( n = 3 biological replicates for each group ) . Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat , control shRNA lentivirus , WNT8B shRNA lentivirus . qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 ( n = 3 biological replicates for each group ) .

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Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus.qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).


Figure 1B , C . Distribution of the m6A peaks across host mRNAs ( B ) and EBV mRNAs ( C ) was analyzed based on the MeRIP - seq data .

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Figure 1B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.


Figure 2B . The BZLF1 m6A levels were measured using MeRIP - qPCR in CNE2EBV , Raji and B95 - 8 cells with latent EBV infection or lytic reactivation . TPA ( 30 ng / ml ) and NaB ( 2 mM ) were used to treat the cells for 24 h to reactivate EBV . The cellular RNA was harvested for MeRIP - qPCR assays . The fold enrichment was determined by calculating the 2 - ΔCt of the MeRIP sample relative to the input sample . The mean value of the results in mock - treated cells was normalized to 1 .

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Figure 2B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.