Figure 4 ( C ) Degree of conformational changes in CM induced by 60b linear ssDNA . Blue bar , Arm / Smc5 - Nse2 ( wild - type ) ; grey bar , Arm / Smc5 - Nse2 ( K333E / K344E ) ; green bar , Arm / Smc5 - Nse2 ( K764E ) ; pink bar , Arm / Smc5 - Nse2 ( K333E / K344E / K764E ) ; red bar , Arm / Smc5 - Nse2 ( K743E / K745E ) . Reactions were performed at least in three different independent experiments . Data values are mean ± s . d . and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * * * * P < 0 . 0001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001


Figure 5 ( B ) Growth test analysis of wild - type , nse2 - CH and the indicated smc5 - KE mutants ; 10 - fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours ( C ) Same as in ( B ) but using the nse2 - CH , smc5 - 3KE , smc5 - K743 , 745E , smc5 - K743 , 745R , smc5 - 7KE and smc5 - 7KR mutants

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours  (C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants


Figure 6 ( A ) Protein extracts from exponentially growing wild - type , smc5 - 3KE , smc5 - K743 , 745E , smc5 - 4KE and smc5 - K743 , 745R cells were prepared under denaturing conditions ; 6xHis - Flag tagged SUMO ( HF - SUMO ) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot . A strain with no HF - tag was used as control ; arrow points to unmodified form of the protein , vertical bar to sumoylated forms ( B ) Left , same as in ( A ) , but using wild - type and two independent clones of smc5 - 7KE ; right , same as in ( A ) , but using wild - type , smc5 - 7KE and smc5 - 7KR mutant cells ( C ) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5 - KE mutants , relative to wild - type controls from at least three independent experiments . Boxes , 25 - 75 % data range ; whiskers , total data range ; black bar , median ; grey cross , mean . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 . 1 - way ANOVA , n = number of samples analyzed ( D ) Co - immunoprecipitation analysis of the Smc5 - Nse2 interactions . NSE2 - 6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles , and protein extracts from exponentially growing cells subjected to anti - HA immunoprecipitation . A strain with no HA tag was used as control ( E ) Same as in ( D ) , but using SMC6 - 6HA cells ( F ) Quantification of Smc5 - 6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes . The mean value on wild - type spreads was arbitrarily set to 1 ; each dot represents one nucleus ; red line , median . 1 - way ANOVA , n = number of nuclei analyzed . * * * * P < 0 . 0001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6 (A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms  (B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells  (C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed  (D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control  (E) Same as in (D), but using SMC6-6HA cells  (F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001


figf1a , Northern blot analysis of lamp - 2 expression . Total RNA was hybridized using a lamp - 2 cDNA2 probe and a murine glyceraldehyde - 3 - phosphate dehydrogenase probe . b , Western blot analysis of LAMP - 2 expression . In control liver and kidney extracts the glycosylated LAMP - 2 molecules were detected . In LAMP - 2 - / - tissues no LAMP - 2 product was found . c , Increased mortality in LAMP - 2 deficient outbred ( C57B6 / Jx129SV ) mice . Forty - two control ( closed circles ) and LAMP - 2 - deficient mice ( open circles ) were monitored over 300 days . d , Loss of weight in early dying ( n = 7 males ) and surviving LAMP - 2 - deficient mice ( n = 11 males ) in comparison with control mice ( n = 12 males ) monitored for 60 days after birth .

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 O O O O O 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 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

figf1a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe.b, Western blot analysis of LAMP-2 expression. In control liver and kidney extracts the glycosylated LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2 product was found.c, Increased mortality in LAMP-2 deficient outbred (C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient mice (open circles) were monitored over 300 days.d, Loss of weight in early dying (n = 7 males) and surviving LAMP-2-deficient mice (n = 11 males) in comparison with control mice ( n = 12 males) monitored for 60 days after birth.


figf2a , Ultrastructure of autophagic vacuoles ( arrowheads ) in pancreatic acinar gland cells from a 6 . 5 - month - old LAMP - 2 - deficient mouse . b , Autophagic vacuoles containing glycogen ( g ) , cytosol and cellular constituents in a hepatocyte of an 8 - day - old LAMP - 2 - / - mouse . m , mitochrondria ; bc , bile canaliculus . c , Accumulation of numerous early autophagic vacuoles in cultured LAMP - 2 - / - hepatocytes . Arrow , large late autophagic vacuole containing partially degraded material . Insert , magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents . Scale bars : a , 3 . 5 µm ; b , 0 . 2 µm ; c , 1 . 3 µm ; insert , 0 . 9 µm .

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 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

figf2a, Ultrastructure of autophagic vacuoles (arrowheads) in pancreatic acinar gland cells from a 6.5-month-old LAMP-2-deficient mouse.b, Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria; bc, bile canaliculus. c, Accumulation of numerous early autophagic vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late autophagic vacuole containing partially degraded material. Insert, magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents. Scale bars: a, 3.5 µm; b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.


figf3a , Vacuole in a fibre of the soleus muscle . b , Ultrastructure of a vacuole in a cardiomyocyte . Scale bars : a , 1 µm ; b , 1 . 5 µm . c , Typical original twitch recordings of developed force of heart muscles from control ( thin line ) and LAMP - 2 - deficient ( thick line ) mice . d , Average values of developed force . Preparations of LAMP - 2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz ; P 0 . 01 .

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 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

figf3a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm; b, 1.5 µm.c, Typical original twitch recordings of developed force of heart muscles from control (thin line) and LAMP-2-deficient (thick line) mice. d, Average values of developed force. Preparations of LAMP-2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.


figf4a , Volume density of early ( Avi ) and late ( Avd ) autophagic vacuoles from four independent cultures . Examples of Avi and Avd are shown in b and c , respectively . Bars represent 0 . 4 µm . d , Labelling density of cathepsin - D as estimated in two independent experiments . e , Labelling density of LAMP - 1 . f , Degradation of long - lived proteins followed over 24 h in amino acid and fetal - calf - serum - containing medium . g , Effect of amino - acid ( AA ) deprivation and 3 - methyladenine ( MA ) on protein degradation during 4 h of chase . The error bars in a , d , e and g represent s . e . m .

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 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 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

figf4a, Volume density of early (Avi) and late (Avd) autophagic vacuoles from four independent cultures. Examples of Avi and Avd are shown in b and c, respectively. Bars represent 0.4 µm.d, Labelling density of cathepsin-D as estimated in two independent experiments. e, Labelling density of LAMP-1.f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.


Figure 1B ) Percentage specific binding of [ 3H ] dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence ( control ) or presence of 10 and 50 µM of LUF compounds . The specific binding of [ 3H ] dofetilide in the absence of test compounds was set as Bcontrol , while the specific binding in their presence was set as B . * = p < 0 . 05 vs control ; N : 3 - 4 . C ) Representative traces of hERG activation ( left ) , inactivation ( middle ) , and deactivation ( right ) measured in HEK293 hERG cells under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . Insets : voltage clamp protocols . D ) Steady - state activation ( empty symbols ) and inactivation ( filled symbols ) curves for IKr under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . The corresponding Boltzmann ' s fittings are superimposed to data points . * = p < 0 . 05 vs baseline . N : 11 - 14 . E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fittings under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 vs respective baseline . N : 8 .

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 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

Figure 1B) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4.C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols.D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann's fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14.E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.


Figure 2B ) Baseline QT interval ( top ) , baseline RR interval ( middle ) , and baseline QT interval corrected with the Bazett ' s formula ( bottom ) . Bar graphs are divided by isogenic pairs ( LQT1R594Q and JLNSR594Q , left ; LQT2corr , LQT2N996I , hESCWT , and hESC - LQT2N996I , middle ; LQT1corr and LQT1R190Q , right ) and in each graph the unrelated WT is shown as a comparison . N : 17 - 53 . * = p < 0 . 05 . The colour of the symbol indicates comparisons and relative statistical significance . C ) Representative traces of IKr steady state activation in WT hiPSC - CMs in Tyrode ( left ) , in the presence of 5 µM LUF7346 ( middle ) and after the application of 5 µM E4031 to selectively block IKr ( right ) . Inset : voltage clamp protocol . D ) Average I / V relationships ( left ) and steady - state activation curves with superimposed Boltzmann ' s fittings ( right ) under baseline conditions ( black ) and in the presence of 5 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 19 . E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fits ( left ) and representative examples ( right ) under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 17 - 21 .

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 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 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

Figure 2B) Baseline QT interval (top), baseline RR interval (middle), and baseline QT interval corrected with the Bazett's formula (bottom). Bar graphs are divided by isogenic pairs (LQT1R594Q and JLNSR594Q, left; LQT2corr, LQT2N996I, hESCWT, and hESC-LQT2N996I, middle; LQT1corr and LQT1R190Q, right) and in each graph the unrelated WT is shown as a comparison. N: 17-53. * = p<0.05. The colour of the symbol indicates comparisons and relative statistical significance.C) Representative traces of IKr steady state activation in WT hiPSC-CMs in Tyrode (left), in the presence of 5 µM LUF7346 (middle) and after the application of 5 µM E4031 to selectively block IKr (right). Inset: voltage clamp protocol.D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19.E) Plot of the time constants (τ) of deactivation derived from biexponential fits (left) and representative examples (right) under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p<0.05. N: 17-21.


Figure 3A ) Representative MEA trace ( left ) showing the effect of increasing concentrations of LUF7346 on FP contour , measured in LQT2N996I - CMs . Average data ( right ) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs baseline . Colour in the heatmap defines the magnitude of QT shortening ( blue ) after treatment with increasing concentrations of LUF7346 . N : 7 - 14 . B ) Representative AP from LQT2corr - , LQT2N996I - , LQT1corr - , and LQT1R190Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of increasing concentrations of LUF7346 ( colour - code is shown ) . C ) Effect of LUF7346 on APD90 , APD70 , and APD50 , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , LQT1R190Q - ( dark blue ) paced at 1 Hz ; * = p < 0 . 05 vs baseline . The colour of the asterisk indicates comparisons and respective statistical significance . ° = p < 0 . 05 vs the genetically - matched corrected control . # = p < 0 . 05 LQT1corr vs LQT2corr . N : 12 - 15 . D ) Effect of LUF7346 on APA and Ediast , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , and LQT1R190Q - CMs ( dark blue ) paced at 1 Hz . N : 12 - 15 .

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

Figure 3A) Representative MEA trace (left) showing the effect of increasing concentrations of LUF7346 on FP contour, measured in LQT2N996I-CMs. Average data (right) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs baseline. Colour in the heatmap defines the magnitude of QT shortening (blue) after treatment with increasing concentrations of LUF7346. N: 7-14.B) Representative AP from LQT2corr-, LQT2N996I-, LQT1corr-, and LQT1R190Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of increasing concentrations of LUF7346 (colour-code is shown).C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.D) Effect of LUF7346 on APA and Ediast, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), and LQT1R190Q-CMs (dark blue) paced at 1 Hz. N: 12-15.


Figure 4A ) Representative MEA trace ( left ) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour , measured in LQT1R594Q - CMs . Average data ( right ) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs respective baseline . ° = p < 0 . 05 vs AST . Colour in the heatmap defines the magnitude of QT prolongation ( red ) and QT shortening ( blue ) , respectively , after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST . N : 5 - 10 . B ) Representative AP from WT - , LQT1R594Q - and JLNSR594Q - CMs , paced at 1 Hz , under baseline conditions ( black ) , after application of 100 nM AST ( red ) , and after addition of increasing concentrations of LUF7346 in the presence of AST ( colour - code is shown ) . C ) Effect of AST and LUF7346 in the presence of AST on APD90 , APD70 , and APD50 in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . * = p < 0 . 05 vs respective baseline ; ° = p < 0 . 05 vs AST . N : 11 - 16 . D ) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . N : 11 - 16 .

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 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 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

Figure 4A) Representative MEA trace (left) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour, measured in LQT1R594Q-CMs. Average data (right) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs respective baseline. ° = p<0.05 vs AST. Colour in the heatmap defines the magnitude of QT prolongation (red) and QT shortening (blue), respectively, after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST. N: 5-10.B) Representative AP from WT-, LQT1R594Q- and JLNSR594Q-CMs, paced at 1 Hz, under baseline conditions (black), after application of 100 nM AST (red), and after addition of increasing concentrations of LUF7346 in the presence of AST (colour-code is shown).C) Effect of AST and LUF7346 in the presence of AST on APD90, APD70, and APD50 in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. * = p<0.05 vs respective baseline; ° = p<0.05 vs AST. N: 11-16.D) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. N: 11-16.


Figure 5A : Representative AP traces measured in JLNSR594Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of either AST alone ( red ) , AST + LUF7346 3 µM ( green ) and AST + LUF7346 5 µM ( blue ) . B : STV values calculated on spontaneously beating hiPSC - CM clusters over 30 consecutive beats as measured with MEA . N = 7 - 24 . * = p < 0 . 05 vs respective isogenic pair . C : STV changes ( left ) and representative examples of QT interval oscillations plotted over 30 consecutive beats ( right ) in the presence of AST and AST + LUF7346 20 µM . * = p < 0 . 05 vs Baseline . ° = p < 0 . 05 vs AST . N = 7 - 24 .

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Figure 5A: Representative AP traces measured in JLNSR594Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of either AST alone (red), AST+LUF7346 3 µM (green) and AST+LUF7346 5 µM (blue).B: STV values calculated on spontaneously beating hiPSC-CM clusters over 30 consecutive beats as measured with MEA. N = 7-24. * = p<0.05 vs respective isogenic pair.C: STV changes (left) and representative examples of QT interval oscillations plotted over 30 consecutive beats (right) in the presence of AST and AST+LUF7346 20 µM. * = p<0.05 vs Baseline. ° = p<0.05 vs AST. N = 7-24.


Figure 1Dose - response curves representing 72 - h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib . Ranges of estimated IC50 and Emax for the selected lines are shown . Data are represented as mean ± SD . Pairwise distribution and correlation of estimated IC50 and Emax values for responses ( 72 - h viability ) of 8 cell lines to four RAF inhibitors and one MEK inhibitor . Each dot represents one drug / cell line combination . Sensitive cell lines are arbitrarily defined based on their IC50s ( the most widely used metric for evaluation of drug response ; log10 [ IC50 ] < − 6 . 5 ) .

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Figure 1Dose-response curves representing 72-h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib. Ranges of estimated IC50 and Emax for the selected lines are shown. Data are represented as mean ± SD.Pairwise distribution and correlation of estimated IC50 and Emax values for responses (72-h viability) of 8 cell lines to four RAF inhibitors and one MEK inhibitor. Each dot represents one drug/cell line combination. Sensitive cell lines are arbitrarily defined based on their IC50s (the most widely used metric for evaluation of drug response; log10[IC50] < −6.5).


Figure 3c - Jun and p - c - Jun ( Ser63 ) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h ( or 5 h in the case of WM115cell line ) and 48 h . B , C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( B ) Dose - response profiles for apoptosis induction with vemurafenib and JNK - IN - 8 combination . C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( C ) Excess over the predicted Bliss independence ( EOBI ) calculated for different combined doses of vemurafenib and JNK - IN - 8 . D c - Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non - targeting controls quantified in triplicate 48 h after transfection . E , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of vemurafenibE , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of selumetinib ( F )

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Figure 3c-Jun and p-c-Jun(Ser63) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h (or 5 h in the case of WM115cell line) and 48 h.B, C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (B) Dose-response profiles for apoptosis induction with vemurafenib and JNK-IN-8 combination.C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (C) Excess over the predicted Bliss independence (EOBI) calculated for different combined doses of vemurafenib and JNK-IN-8.D c-Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non-targeting controls quantified in triplicate 48 h after transfection.E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of vemurafenibE, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of selumetinib (F)


Figure 4Time - dose - response plots indicating changes in RPPA measurements for p27 versus p - histone H3 for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control . B - D Time - dose - response plots indicating changes in RPPA measurements for pERK ( Thr202 / Tyr204 ) versus pAKT ( Ser473 ) for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control . Time - dose - response plots indicating changes in RPPA measurements for pS6 ( Ser235 / 236 ) versus total c - Jun levels for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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Figure 4Time-dose-response plots indicating changes in RPPA measurements for p27 versus p-histone H3 for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.B-D Time-dose-response plots indicating changes in RPPA measurements for pERK(Thr202/Tyr204) versus pAKT(Ser473) for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.Time-dose-response plots indicating changes in RPPA measurements for pS6(Ser235/236) versus total c-Jun levels for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


Figure 5Covariate single - cell analysis of Ki - 67 ( left ) and pRb ( Ser807 / 811 ) ( right ) versus p - cJun ( Ser73 ) in WM1552C cells before and 24 h after exposure to 0 . 8 μM vemurafenib . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The vertical lines were used to gate p - cJunHigh versus p - cJunLow cells . The horizontal lines were used to gate Ki - 67High versus Ki - 67Low cells , and pRbHigh versus pRbLow cells . Analysis of drug dose - dependent changes in proportion of pRbLow / p - cJunHigh and pRbHigh / p - cJunHigh subpopulations in four melanoma cell lines ( WM115 , WM1552C , LOXIMVI , COLO858 ) after exposure to vemurafenib for 24 h . These subpopulations were gated as shown in ( A ) . Data are represented as mean ± SD for two replicatesCovariate single - cell analysis of pRb ( Ser807 / 811 ) versus pS6 ( Ser235 / 236 ) following 24 - h treatment of WM1552C cells with different doses of vemurafenib alone ( top ) and vemurafenib and JNK - IN - 8 together ( bottom ) . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The horizontal and vertical lines were used to gate pS6High versus pS6Low cells , and pRbHigh versus pRbLow cells , respectively . Selected immunofluorescence images of pS6 ( Ser235 / 236 ) and Hoechst staining in WM1552C cells in a DMSO - treated control and 24 h after exposure to 5 μM of vemurafenib , JNK - IN - 8 , and their combination . Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines ( LOXIMVI , WM115 and WM1552C ) as a function of drug concentration for single - drug vemurafenib and JNK - IN - 8 treatments and their combination treatment . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . pS6High population of cells was gated as indicated in ( C ) . Data are represented as mean ± SD for two replicates . Data comparison between vemurafenib treatment and vemurafenib / JNK - IN - 8 combined treatment was made by using two - way analysis of variance ( ANOVA ) . Fraction of c - JunHighcells ( as measured by single - cell ( immunofluorescence microscopy ) after 48 h JUN knockdown followed by 24 - h treatment with vemurafenib . Fold changes are shown relative to control - treated cells . Data are presented as mean ± SD . Single - cell pS6 ( Ser235 / 236 ) levels in the c - JunHigh and c - JunLow fractions of cells ( as measured by single - cell multiplex immunofluorescence microscopy ) after 48 h of JUN knockdown and 24 - h treatment with 0 . 32 μM vemurafenib . Single - cell pS6 data are presented as box - and - whisker plots with median signal intensities and interquartile ranges ; bars extending to 1 . 5 × the interquartile range are shown for each condition as a measure of variance . P - values were calculated using a two - sided nonparametric Mann - Whitney U - test .

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Figure 5Covariate single-cell analysis of Ki-67 (left) and pRb(Ser807/811) (right) versus p-cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p-cJunHigh versus p-cJunLow cells. The horizontal lines were used to gate Ki-67High versus Ki-67Low cells, and pRbHigh versus pRbLow cells.Analysis of drug dose-dependent changes in proportion of pRbLow/p-cJunHigh and pRbHigh/p-cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicatesCovariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively.Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO-treated control and 24 h after exposure to 5 μM of vemurafenib, JNK-IN-8, and their combination.Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single-drug vemurafenib and JNK-IN-8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK-IN-8 combined treatment was made by using two-way analysis of variance (ANOVA).Fraction of c-JunHighcells (as measured by single-cell (immunofluorescence microscopy) after 48 h JUN knockdown followed by 24-h treatment with vemurafenib. Fold changes are shown relative to control-treated cells. Data are presented as mean ± SD.Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.


Figure 6Pairwise Spearman ' s correlation between the 1 - h changes in pERK ( Thr202 / Tyr204 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the seven dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study . Comparison of the level of 1 - h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720 . Comparison of the 72 - h measured relative viability AUC for 8 / 10 cell lines treated with SB590885 versus PLX4720Comparison of the level of 1 - h pERK inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor . Comparison of the 72 - h measured relative viability AUC between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor . Pairwise Spearman ' s correlation between the 24 - h changes in pS6 ( Ser235 / 236 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study . Pairwise Spearman ' s correlation between the 24 - h p - cJun levels as measured by immunofluorescence microscopy and 72 - h relative viability after treatment with PLX4720 for 8 cell lines . p - cJun levels are averaged over five doses ( 0 . 1 - 5 μM ) , and viability is represented by the AUC of the dose - response curves . Comparison of the level of 24 - h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720 . Comparison of the level of 24 - h pS6 inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK

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Figure 6Pairwise Spearman's correlation between the 1-h changes in pERK(Thr202/Tyr204) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the seven dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.Comparison of the level of 1-h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720.Comparison of the 72-h measured relative viability AUC for 8/10 cell lines treated with SB590885 versus PLX4720Comparison of the level of 1-h pERK inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.Comparison of the 72-h measured relative viability AUC between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.Pairwise Spearman's correlation between the 24-h changes in pS6(Ser235/236) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.Pairwise Spearman's correlation between the 24-h p-cJun levels as measured by immunofluorescence microscopy and 72-h relative viability after treatment with PLX4720 for 8 cell lines. p-cJun levels are averaged over five doses (0.1-5 μM), and viability is represented by the AUC of the dose-response curves.Comparison of the level of 24-h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720.Comparison of the level of 24-h pS6 inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor


Figure 1DRiP labelling in GFP - NCL HeLa Kyoto cells that were either left untreated or treated with OP - puro ( 25 µM ) for 2 h . Where indicated cells were allowed to recover in drug - free medium ( control ) or in presence of MG132 ( 10 µM ) for 5 h . Scale bars : 5 µm . GFP - NCL HeLa Kyoto cells were treated as described in A , with the exception that a lower concentration of OP - puro was used ( 5 µM ) . The distribution of DRiPs and GFP - NCL was analyzed by STED super - resolution microscopy . Scale bars : 5 µm . Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin ( 25 µM ) for 2 h . Puromycylated proteins were detected by immunoblotting ( right panel ) . The left panel shows the purity of isolated nucleoli by western blotting , where fibrillarin was used as nucleolar marker , FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker .

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Figure 1DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with OP-puro (25 µM) for 2 h. Where indicated cells were allowed to recover in drug-free medium (control) or in presence of MG132 (10 µM) for 5 h. Scale bars: 5 µm. GFP-NCL HeLa Kyoto cells were treated as described in A, with the exception that a lower concentration of OP-puro was used (5 µM). The distribution of DRiPs and GFP-NCL was analyzed by STED super-resolution microscopy. Scale bars: 5 µm.Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin (25 µM) for 2 h. Puromycylated proteins were detected by immunoblotting (right panel). The left panel shows the purity of isolated nucleoli by western blotting, where fibrillarin was used as nucleolar marker, FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker.


Figure 2HeLa cells were left untreated or treated with heat shock ( HS ) at 42 ˚ C for 3 h , alone or with cycloheximide ( CHX ; 50 μg / ml ) , or Actinomycin D ( Act . D ; 3 µM ) followed by staining with the dye Amylo - glo . DRiP labelling in GFP - NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42 ˚ C and OP - puro ( 25 µM ) for 2 h . Where indicated , cells were cotreated with CHX ( 50 μg / ml ) . HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42 ˚ C for 2 h , alone or combined with CHX , in GFP - NCL HeLa Kyoto cells . DRiP labelling in HeLa cells treated with MG132 ( 10 µM ) alone or with CHX ( 50 μg / ml ) for 8 h ; where indicated , OP - puro ( 25 µM ) was added during the last 45 min , prior to cell fixation . Amylo - glo staining of HeLa cells left untreated or exposed to MG132 ( 10 µM ) alone or combined with CHX ( 50 μg / ml ) for 8 or 13 h . Amylo - glo staining of HeLa cells left untreated or subjected to transcriptional stress ( MG132 10 µM and Act . D 4 µM ) for 6 hrs . Where indicated , translation was concomitantly inhibited with CHX ( 50 μg / ml ) . Amylo - Glo staining is also shown in HeLa cells treated with Act . D alone or with CHX for 6 hrs , as control . Amylo - glo staining of HeLa cells exposed to HS at 42 ˚ C for 3 h and let to recover for 3 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated cells were treated with VER alone ( VER 3 h ) . Amylo - glo staining of HeLa cells treated with MG132 ( 10 µM ) for 13 h and let to recover for 5 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated cells were treated with VER alone ( VER 5 h ) . HeLa cells were transfected with vectors encoding for GFP , GFP - GR50 or RPL23a - GFP . 24 hrs post - transfection cells were fixed and stained with Amylo - glo . The percentage of transfected cells with Amylo - Glo positive nucleoli is shown . Number of cells counted / condition : GFP , 412 ; RPL23a - GFP , 482 ; GFP - GR50 , 359 , in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 7 , ± s . e . m .

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

Figure 2HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo.DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml).HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells.DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation.Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h.Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control.Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h).Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h).HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.


Figure 3DRiPs , polyUb proteins ( FK1 ) and PML labelling in HeLa cells treated with OP - puro ( 25 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h ; where indicated cycloheximide was also added ( CHX ; 50 μg / ml ) . Scale bars : 5 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa cells treated with a low concentration of OP - puro ( 5 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h . PML - NBs were visualized using a PML specific antibody , followed by incubation with alexa fluor 647 . For convenience the alexa fluor 647 signal is shown in green . Scale bars : 5 µm . PolyUb proteins ( FK1 ) labelling in HeLa cells left untreated or treated with HS at 42 ˚ C for 2 h , alone or combined with CHX ( 50 μg / ml ) . Scale bars : 5 µm . STED super - resolution microscopy showing colocalization of endogenous polyUb proteins ( FK1 ) and PML - NBs in PML - GFP HeLa cells treated with HS at 42 ˚ C for 2 h . Scale bars : 5 µm . Quantitation of cells shown in A - C . Number of cells counted / condition : 712 - 1343 in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 5 , ± s . e . m . DRiPs , polyUb proteins ( FK1 ) and PML labelling in HeLa cells treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h ; where indicated , cycloheximide was also added ( CHX ; 50 μg / ml ) . Asterisks ( * ) indicate nucleoli . Scale bars : 10 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa cells treated with a low concentration of OP - puro ( 5 µM ) and MG132 ( 10 µM ) for 4 hrs . Scale bars : 5 µm . Quantitation of cells shown in E . Number of cells counted / condition : 298 - 1713 in three independent experiments ; statistical significance via One - way ANOVA ; p ≤ 0 . 001 , ± s . e . m . Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Asterisks ( * ) indicate nucleoli . Scale bars : 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 O O O O O O O O O O O O O O O O O O O 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 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 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 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 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

Figure 3DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm.PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm.Quantitation of cells shown in A-C. Number of cells counted/condition: 712 - 1343 in three independent experiments; statistical significance via One-way ANOVA; p < 10-5, ± s.e.m.DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm. Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m.Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.


Figure 4HeLa cells were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , total proteins were extracted and expression levels of PML were analyzed by immunoblotting . TUBA4A was used as loading control . HeLa cells were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , cells were treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Left panel : automated quantitation of the number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 334 - 364 ; p < 10 - 10 . Right panel : automated quantitation of the number of DRiP foci / nucleus . Cell number analyzed : 334 - 364 ; p < 10 - 10 . Automated DRiP segmentation is based on azide signal . DRiPs and polyUb ( FK1 ) protein distribution in cells treated as described in ( A ) . Automated DRiP segmentation is based on azide signal . Quantitation of the number of DRiP foci enriched for polyUb ( > 2 ) is shown ; n = 590 - 2114 ; statistical significance via 2 ‐ tailed Student ' s t ‐ test ; p < 10 - 10 . Scale bars : 10 µm . Immunoblotting on protein extracts from HeLa cells lipofected with non - targeting siRNA control or a specific siRNA against Ubc9 . A dilution curve of the control sample is shown . TUBA4A was used as loading control . Quantitation of the number of PML - NBs / nucleus from cells treated as described in ( D ) . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal . Cells lipofected for 72 h with non - targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Automated quantitation of the number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 151 - 225 ; p < 10 - 10 ( see panel G ) . Quantitation of the number of PML - NBs / nucleus from cells treated as described in ( D ) . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal . Representative pictures showing that Ubc9 - depleted cells cannot efficiently induce the self - assembly of PML - NBs upon treatment with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Scale bars : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O 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 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

Figure 4HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control.HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal.DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm.Immunoblotting on protein extracts from HeLa cells lipofected with non-targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control. Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal.Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G).Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.


Figure 5PolyUb protein ( FK1 ) labelling in PML - GFP HeLa Kyoto cells that were either left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Cells were either immediately fixed or let to recover for 5 h in drug - free medium ( Control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Scale bars : 10 µm . Quantitation of the % of cells with polyUb ( FK1 ) nuclear foci shown in ( A ) . Number of cells counted / condition : 310 - 1258 in three independent experiments ; statistical significance via One - way ANOVA ; p = 0 . 0003 or 10 - 6 , ± s . e . m . Quantitation of the number of PML - NBs enriched for polyUb ( > 2 ) ; n = 611 - 2612 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal . Quantitation of the % of cells with polyUb proteins ( FK1 ) sequestered in nuclear foci . HeLa cells were treated with OP - puro ( 25 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h . Then , cells were either immediately fixed or let to recover for 3 h in drug - free medium ( control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Number of cells counted / condition : 593 - 1016 in three independent experiments ; statistical significance via One - way ANOVA ; p = 10 - 3 , ± s . e . m . Quantitation of the number of PML - NBs enriched for polyUb ( > 2 ) in HeLa cells treated as described in ( D ) but with a recovery time of 2 h ; n = 1155 - 3283 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal . Amylo - Glo staining of PML - GFP HeLa Kyoto cells that were subjected to the following treatments : untreated ( Control ) ; MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h , alone or combined with CHX ( 50 μg / ml ) . Where indicated cells were allowed to recover for 5 hrs in drug - free medium ( Control ) or with VER ( 40 µM ) . Confocal images showing Amylo - Glo and PML - GFP . Scale bars : 5 µm . Amylo - Glo staining of PML - GFP HeLa Kyoto cells that were subjected to heat shock ( HS ) at 42 ˚ C for 2 hrs . Confocal images showing Amylo - Glo and PML - GFP . Scale bars : 5 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 O O O O O O O O O O O O O O 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 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 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 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

Figure 5PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm. Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m.Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m.Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.


Figure 6PML - GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry - VHL . 24 h post - transfection , cells were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Where indicated ( OP - puro + MG132 + CHX ) , translation was inhibited during stress with cycloheximide ( CHX ; 50 μg / ml ) . In the lower panel , cells were allowed to recover for 5 h in drug - free medium ( Control ) , or with VER ( 40 µM ) . The subcellular distribution of PML - GFP and mCherry - VHL was studied by live - cell confocal imaging . Representative pictures are shown . Scale bars : 5 µm . Quantitation of the fluorescence intensity recovery after bleach of PML - GFP . PML - GFP HeLa Kyoto overexpressing mCherry - VHL for 24 h and treated as described in ( A ) were analyzed . The mean of 19 - 23 FRAP curves and the fitting curves are shown in black and red respectively . In gray , the SD is shown . Intranuclear distribution of 20S proteasomes , HSP70 , VCP and DRiPs in HeLa cells treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h and allowed to recover for 5 h in drug - free medium ( control ) , with VER ( 40 µM ) , Eeyarestatin I ( 5 µM ) or MG132 ( 10 µM ) . Scale bars : 5 µm . GFP - PSMA7 HeLa Kyoto cells were left untreated or treated as described in A . Cells were subjected to FRAP to analyze GFP - PSMA7 mobility . Quantitation of three populations of GFP - PSMA7 molecules is shown : fast moving , slow moving and immobile ; statistical significance via One - way ANOVA ; n = 12 - 14 ± SEM .

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 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

Figure 6PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm. Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.GFP-PSMA7 HeLa Kyoto cells were left untreated or treated as described in A. Cells were subjected to FRAP to analyze GFP-PSMA7 mobility. Quantitation of three populations of GFP-PSMA7 molecules is shown: fast moving, slow moving and immobile; statistical significance via One-way ANOVA; n = 12 - 14 ± SEM.


Figure 7Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) . Where indicated , cells were treated with MG132 , VER or EeyI alone for 5 h . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( A ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 1112 - 1537 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 . HeLa cells were lipofected with cDNAs encoding for HA - Ubiquitin , Flag - Ubiquitin or Flag - VCP 24 h post - transfection , cells were either left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) . Cells were processed for staining of HA , Flag and endogenous 53BP1 . * indicates cells that do not express HA - or Flag - Ubiquitin and show no rescue of 53BP1 foci formation after stress . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( C ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 710 - 938 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 . Colony formation assay . The number of colonies surviving 10 days after plating is shown . Control cells or cells treated with MG132 ( 10 µM ) and puromycin ( 5 µg / ml ) , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) are shown . Where indicated , cells were treated with VER or EeyI alone for 5 h . Number of colonies counted / condition : up to 526 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 03 , ± s . e . m .

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 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

Figure 7Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.


Figure 1A , B Functional freeMitos association with PMN . Overlay dot plot population , representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN ( anti - CD11b - PE ) and freeMitos . Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed . CD11b - labeled PMN were incubated in absence ( A ) or presence ( B ) of freeMitos at a 1 : 5 ratio ( PMN : mitochondria ) . C Confocal microscopy imaging of PMN cell membrane ( red ) labeled with CellMask Orange in absence ( upper panel ) or presence ( lower panel ) of MitoTrackerTM Deep Red - labeled freeMitos ( magenta ) . PMN nuclei were stained with DAPI ( cyan ) . The white scale bars shown in the lower right of the images represent 10 μm in size . D , E FreeMitos associate with PMN and modulate gene expression . Volcano plot , of 4 biological replicates , representing the gene expression modulation induced by mitochondrial DAMPs preparation ( D ) and freshly isolated functional freeMitos ( E ) in PMN . FreeMitos effect on the respiratory state of PMN . O2 consumption rates of resting or activated ( incubated with TNF - a and GM - CSF ) PMN were measured using high - resolution O2 respirometry ( Oroboros Oxygraph ) and revealed no significant differences between the basal state ( left panel ) , leak state ( middle panel ) and the maximum electron transport system capacity ( ETS , right panel ) of cellular respiration ( F ) . Data information : For panels F data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different ( P < 0 . 05 ) . Immunoblot of mitochondrial cytochrome c oxidase ( COX IV ) and histone H3 as a loading control of PMN after co - incubations with freeMitos ( G ) with subsequent densitometry analysis ( H ) . Data information : For panels H , data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels H . Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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

Figure 1A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN (anti-CD11b-PE) and freeMitos. Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed. CD11b-labeled PMN were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).C Confocal microscopy imaging of PMN cell membrane (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei were stained with DAPI (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.D, E FreeMitos associate with PMN and modulate gene expression. Volcano plot, of 4 biological replicates, representing the gene expression modulation induced by mitochondrial DAMPs preparation (D) and freshly isolated functional freeMitos (E) in PMN.FreeMitos effect on the respiratory state of PMN. O2 consumption rates of resting or activated (incubated with TNF-a and GM-CSF) PMN were measured using high-resolution O2 respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Data information: For panels F data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different (P < 0.05).Immunoblot of mitochondrial cytochrome c oxidase (COX IV) and histone H3 as a loading control of PMN after co-incubations with freeMitos (G) with subsequent densitometry analysis (H). Data information: For panels H, data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels H. Values without at least one common superscript letter are different (P < 0.05).


Figure 2A , B FreeMitos induce PMN intracellular calcium release in PMN . Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co - incubated with 5 x 106 mitochondria ( A ) . Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo - 3 AM . The gray line represents PMN with the vehicle ( HBSS ) while the dark line represents PMN incubated with mitochondria . PMN intracellular calcium mobilization induced by either the vehicle ( HBSS ) , freeMitos or mitochondrial DAMPs and quantified by flow cytometry ( B ) . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel B , One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels B , Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Functional freeMitos induce the release of PMN - derived microvesicles . A dose - response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles . PMN microvesicles labeling was performed with either anti - CD66b - FITC labelling ( C , left panel ) or anti - CD11b - PE ( C , right panel ) . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel C two - way ( panel C ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . PMN - derived microparticle gating strategy is shown in panel D . Flow cytometry panel established to distinguish the PMNL - MVs from the background using the leucocyte specific integrin alpha - M beta - 2 ( CD11b ) , the granulocyte specific carcinoembryonic antigen - related cell adhesion molecule 8 ( CD66b ) , and size reference beads . PMN were either incubated with the vehicle ( Tyrode ' s Buffer , upper panels ) or freeMitos ( lower panels ) . Following co - incubation , PMN were washed twice and resuspended in HBSS before processing on flow cytometer . Only single events , identified by the singlets gate ( left upper and lower panels ) using side scatter area ( SSC - A ) vs side scatter heights ( SSC - H ) were analyzed . Light scatter ( side scatter ( SSC ) and forward scatter ( FSC ) ) approach for determining the background noise in HBSS . Histogram displaying the fluorescence intensity of samples labelled with either CD11b - FITC or CD66b - PE . Overlay dot plot of PMNL - MVs population and size reference beads , representative of 4 biological replicates , each performed in 2 technical replicates . E Visual confirmation of PMN and PMN - derived microvesicles . Transmission electron microscopy ( TEM ) imaging of PMN - derived microvesicles ( black arrowhead ) , representative of 3 biological replicates , from resting or activated PMN ( 106 cells ) incubated with either mitochondria , mitochondrial DAMPs or fMLP as positive control . The black scale bars are shown in the lower right of the images for the vehicles ( 400 nm ) , mitochondria ( 600 nm ) , DAMPs ( 600 nm ) and fMLP ( 500 nm ) conditions . F Quantification of resting PMN microvesicles produced by PMN ( 106 cells ) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . G Quantification of calpain activity , by fluorescence assay , in cell lysate of either resting ( left panel ) or activated ( right panel ) PMN ( 2 x 105 cells ) incubated in presence of freeMitos for 2 hr . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel G ) . One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels G . Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . H ELISA quantification of IL - 8 concentrations present in the supernatant following the incubation of PMN ( 106 cells ) with freeMitos ( 107 ) , DAMPs ( 25 µg ) or fMLP ( 1 µM ) for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel H ) One - way ( panel H ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2A, B FreeMitos induce PMN intracellular calcium release in PMN. Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co-incubated with 5 x 106 mitochondria (A). Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN with the vehicle (HBSS) while the dark line represents PMN incubated with mitochondria. PMN intracellular calcium mobilization induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles. PMN microvesicles labeling was performed with either anti-CD66b-FITC labelling (C, left panel) or anti-CD11b-PE (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry panel established to distinguish the PMNL-MVs from the background using the leucocyte specific integrin alpha-M beta-2 (CD11b), the granulocyte specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN were washed twice and resuspended in HBSS before processing on flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC or CD66b-PE. Overlay dot plot of PMNL-MVs population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.E Visual confirmation of PMN and PMN-derived microvesicles. Transmission electron microscopy (TEM) imaging of PMN-derived microvesicles (black arrowhead), representative of 3 biological replicates, from resting or activated PMN (106 cells) incubated with either mitochondria, mitochondrial DAMPs or fMLP as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria (600 nm), DAMPs (600 nm) and fMLP (500 nm) conditions.F Quantification of resting PMN microvesicles produced by PMN (106 cells) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.G Quantification of calpain activity, by fluorescence assay, in cell lysate of either resting (left panel) or activated (right panel) PMN (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.H ELISA quantification of IL-8 concentrations present in the supernatant following the incubation of PMN (106 cells) with freeMitos (107), DAMPs (25 µg) or fMLP (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


Figure 1 ( C ) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA . The dCas9 - ω was verified in the ∆ rpoZ strain background , and the rest of candidates were verified in the wild - type strain ( BW25113 ) . ( D ) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9 - AsiA with gRNA - H3 under kanamycin selection ( 2 . 5 μg / ml ) . ( E ) Different gRNAs were paired with dCas9 - AsiA and dCas9 - ω to profile the optimal gRNA binding distance . The dCas9 - AsiA was verified in the wild - type strain ( BW25113 ) , and dCas9 - ω was examined in the ∆ rpoZ strain background . ( F ) Comparing single and multiple gRNA with dCas9 - AsiA .

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 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

Figure 1(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml). (E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background. (F) Comparing single and multiple gRNA with dCas9-AsiA.


Figure 2 ( C ) Distribution of fluorescence signal of the GFP reporter induced by different dCas9 - AsiA variants ( top ) . Fold induction by different dCas9 - AsiA variants is shown ( bottom ) . ( D ) CRISPRa induction of promoters with varying basal expression levels . CRISPRa - , basal expression of the promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 and associated gRNA - H4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).  (D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.


Figure 3 ( A ) CasTA2 . 1 upregulates a genomically inserted GFP reporter . ( B ) Chromosomal gene targets activated by CasTA2 . 1 with bars showing the activation fold change and dots showing basal expression of each gene . Expression was quantified using RT - qPCR . ( C ) CasTA2 . 1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene . A non - specific gRNA is used as the negative control . ( D ) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2 . 1 on a reporter containing GFP and mScarlet . Parental cells had low basal GFP and high basal mScarlet expression .

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Figure 3(A) CasTA2.1 upregulates a genomically inserted GFP reporter. (B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR. (C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control. (D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.