umber of γH2AX foci , respectively , in control - and Molm - 14 - xenograft - derived : ( K ) KSL , n = 101 / 204 , and ( L ) LT - HSC , n = 33 / 53 . Images were processed similar to D - F . Scale bars are 3μm . Statistics : Student ' s t - test . ( * * P < 0 . 01 ) .

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

umber of γH2AX foci, respectively, in control- and Molm-14-xenograft-derived: (K) KSL, n=101 / 204, and (L) LT-HSC, n=33 / 53. Images were processed similar to D-F. Scale bars are 3μm. Statistics: Student's t-test. (** P< 0.01).


qRT - PCR analysis of the DNA - damage - genes panel in Molm - 14 - xenograft - derived : ( M ) KSL , and ( N ) LT - HSC , relative to matched controls . Data depicted as fold - change relative to matching controls , normalized to the endogenous control Gapdh and presented as mean ± SEM .

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

qRT-PCR analysis of the DNA-damage-genes panel in Molm-14-xenograft-derived: (M) KSL, and (N) LT-HSC, relative to matched controls. Data depicted as fold-change relative to matching controls, normalized to the endogenous control Gapdh and presented as mean ± SEM.


B ) , C ) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively , in CKO cells compared to WT ( q = Benjamini - Hochberg corrected p - value , n = 24 time points over 3 days ) . Statistically significant changes ( q ≤ 0 . 05 ) are shown in red . Some proteins are labelled as space allows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), C) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively, in CKO cells compared to WT (q = Benjamini-Hochberg corrected p-value, n= 24 time points over 3 days). Statistically significant changes (q ≤ 0.05) are shown in red. Some proteins are labelled as space allows.


A ) Fold - change in relative amplitude ( RA ) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis ( i . e . no RA cut - off ) . The mean fold change ( log ) in RA was an increase of 14 % in CKO cells compared to WT cells ( One sample t - test , * * * * p < 0 . 0001 ) . B ) For each genotype , all proteins were divided into 10 deciles of equal number , ranked by abundance . The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic . Linear regression lines are shown for each genotype , and the slopes were significantly non - zero ( F test , WT * * p = 0 . 0016 , CKO * * * * p < 0 . 0001 ) . The slopes were also significantly different to each other ( F test , p = 0 . 0001 ) . C ) Fold - change in relative amplitude ( RA ) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN . On average , the RA was increased in CKO cells compared to WT cells by 6 % ( One sample t - test , * p < 0 . 05 ) . D ) The same analysis in B ) was carried out for phosphopeptides . The slopes were significantly non - zero in CKO but not WT ( F test , WT p = 0 . 3 ( ns . : non significant ) , CKO * p = 0 . 04 ) . The slopes were also significantly different to each other ( F test , 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

A) Fold-change in relative amplitude (RA) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis (i.e. no RA cut-off). The mean fold change (log) in RA was an increase of 14% in CKO cells compared to WT cells (One sample t-test, ****p<0.0001). B) For each genotype, all proteins were divided into 10 deciles of equal number, ranked by abundance. The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic. Linear regression lines are shown for each genotype, and the slopes were significantly non-zero (F test, WT **p=0.0016, CKO ****p<0.0001). The slopes were also significantly different to each other (F test, p=0.0001). C) Fold-change in relative amplitude (RA) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN. On average, the RA was increased in CKO cells compared to WT cells by 6% (One sample t-test, *p<0.05). D) The same analysis in B) was carried out for phosphopeptides. The slopes were significantly non-zero in CKO but not WT (F test, WT p=0.3 (ns.: non significant), CKO *p=0.04). The slopes were also significantly different to each other (F test, p<0.0001).


B ) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two - way ANOVA correcting for multiple comparisons , controlling for false discovery rate by the Benjamini , Krieger and Yekutieli method ( q = 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two-way ANOVA correcting for multiple comparisons, controlling for false discovery rate by the Benjamini, Krieger and Yekutieli method (q=0.05).


D - E ) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells ( D , p = 0 . 47 ) , but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts ( E , * * p = 0 . 0013 ) ; Kolmogorov - Smirmov test ( n = 4 ) . Continuous lines = median , dotted lines = quartiles .

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

D-E) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells (D, p=0.47), but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts (E, **p=0.0013); Kolmogorov-Smirmov test (n=4). Continuous lines = median, dotted lines = quartiles.


A ) From the quantitative proteomics experiment , average abundance of catalytic proteasome subunits was calculated and normalised to WT means . Trypsin - like ( β2 ) , chymotrypsin - like ( β3 ) and caspase - like ( β1 ) catalytic subunits are shown . The average was calculated from all 24 time points of the proteomics experiment . Mean ± SD , 2 - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * 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

A) From the quantitative proteomics experiment, average abundance of catalytic proteasome subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


B ) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome , with anti - histone H3 as loading control . C ) Quantification of the blots in B ) , using all replicates ( n = 3 ) , normalised to WT mean . Mean ± SD , one - tailed Student ' s t test with Welch correction - prior prediction from experiment in A ) that CKO abundance would be lower , * p ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome, with anti-histone H3 as loading control. C) Quantification of the blots in B), using all replicates (n=3), normalised to WT mean. Mean±SD, one-tailed Student's t test with Welch correction - prior prediction from experiment in A) that CKO abundance would be lower, *p≤0.05.


D - E ) From one time - course experiment , ions , cytosolic proteins and total protein were extracted in parallel samples . The presented experiment is representative of 3 separate time - course experiments that were carried out ( N = 3 ) . Representative experiment shown ( n = 4 technical replicates for [ K + ] , n = 3 technical replicates for the other traces ) . Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration . Mean ± SEM , p - values from RAIN , red lines are fits by a damped cosine compared with a straight line ( null hypothesis ) . Parallel PER2 : : LUC recordings were also performed and plotted as a phase marker .

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

D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.


F ) Examples of key ion transporters are shown , as detected in the proteomics experiment . P values show the results of an F test comparing fits of damped cosine against straight line . All proteins except WT NKCC1 had RAIN p - values < 0 . 05 .

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

F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.


A ) WT and CKO cells were treated with 500 nM tunicamycin ( TUN ) and lysed in RIPA buffer at time points between 0 - 24 hours afterwards . Western blots were carried out , probing for phosphorylated eIF2α , total eIF2α and actin . Representative blots shown , n = 4 . B ) Quantification of all replicates represented in ( A ) , where phosphorylated eIF2α is normalised to total eIF2α . Mean ± SD . 2 - way ANOVA ( TWA ) with Holm - Sidak ' s multiple comparisons .

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

A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.


C ) Mice were housed in 12h : 12h light - dark cycles , with unrestricted feeding . Mouse lungs were collected 7 hours after transition to the light phase , 5 hours after intraperitoneal injection of mice with bortezomib ( BTZ , 2 . 5 mg / kg ) or vehicle ( Veh , 1 % DMSO in sterile PBS ) . Tissues were lysed in RIPA buffer and western blots were performed , probing for phosphorylated STAT3 , total STAT3 and actin . Male ( left ) and female ( right ) replicates shown , N = 2 mice for each condition . D ) Quantification of the blots shown in ( C ) , where phosphorylated STAT3 is normalised to total STAT3 . Mean ± SD . Multiple t - tests , corrected for multiple comparisons with the Holm - Sidak method , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.


A ) WT PER2 : : LUC cells were treated with epoxomicin or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . B ) Quantification of damping rate of the PER2 : : LUC recordings shown in A ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C ) WT PER2 : : LUC cells were treated with tunicamycin or vehicle control . Mean ( solid lines ) ± SD ( dotted lines ) . D ) Quantification of damping rate of the PER2 : : LUC recordings shown in C ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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

A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


E ) WT PER2 : : LUC cells were treated with radicicol or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . F ) Quantification of damping rate of the PER2 : : LUC recordings shown in E ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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

E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


I - J ) Representative double - plotted actograms of WT mice receiving drinking water with DMSO vehicle control ( N = 7 mice ) or 35 µg / mL ixazomib ( N = 8 mice ) . Two representative actograms are shown per condition . For the first 7 days the mice were in 12 : 12h light - dark cycles ( LD ) , and had drinking water supplemented with blackcurrant squash . Ixazomib / DMSO was added to this , and the lighting was changed to constant darkness ( DD ) . After 7 days the drug / vehicle was removed , and the mice remained in DD until the end of the experiment .

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

I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.


A . Bclaf1 wild type ( WT ) and two Bclaf1 knockout HeLa cell lines ( KO - 1 and - 2 ) were pre - treated with DMSO or cycloheximide ( CHX , 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) together with CHX for 12 hours . Afterwards , the cells were subjected to Annexin V / 7AAD staining followed by flow cytometry analysis , all Annexin V positive cells were counted for analysis , or protein extractions followed by western blotting analysis . B , C . HeLa ( B ) or primary MEFs ( C ) were transfected with control ( siCtrl ) or two siRNAs against Bclaf1 ( siBclaf1 - 1 and - 2 ) and then treated and analyzed as described in A . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


D , E . MEFs transfected with control ( siCtrl ) or siBclaf1 - 1 and / or siBclaf1 - 2 siRNAs were pre - treated with CHX ( 1 μg / ml ) / z - VAD ( 20 μM ) ( D ) or SM - 164 ( 100 nM ) / z - VAD ( 20 μM ) ( E ) for 30 min , and then added TNF ( 10 ng / ml ) for indicated times . The cells were subjected to Annexin V / 7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells ( up ) , or protein extractions and western blotting analysis at indicated time periods ( down ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


A , B . Cell lysates of HeLa Bclaf1 WT and KO cells ( A ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( B ) treated with TNF ( 10 ng / ml ) for indicated time periods were analyzed by western blotting with the indicated antibodies .

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

A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.


C , D . HeLa Bclaf1 WT and KO cells ( C ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( D ) were treated with TNF ( 10 ng / ml ) for the indicated time periods , and then subject to nucleus - cytoplasm fractionation as described in materials and methods followed by western blotting . Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions , respectively .

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

C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.


B . MEFs treated with TNF ( 10 ng / ml ) for 4 hours were subject to FADD immunoprecipitation , and then analyzed by western blotting .

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

B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.


G , H . HeLa cells transfected with siRNAs against Bclaf1 or c - FLIP were pretreated with CHX ( 1 μg / ml ) for 30 min and then stimulated with TNF ( 10 ng / ml ) in the presence of CHX for 12 hours followed by Annexin V / 7AAD staining and flow cytometry analysis ( G ) , or protein extractions followed by western blotting analysis ( H ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.


A - F . Bclaf1 WT , KO - 1 and - 2 HeLa cells ( A and D ) , as well as HeLa ( B and E ) and primary MEFs ( C and F ) which were transfected with siCtrl or siBclaf1 - 1 and - 2 were treated with TNF ( 10 ng / ml ) for 6 hours followed by western blotting analysis ( A - C ) , or for 4 hours followed by total RNA extraction and RT - PCR analysis ( D - F ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


H , I . CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa - Flag - Bclaf1 cells treated without ( H ) or with TNF ( I ) . The regions amplified by RT - PCR were delineated . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


B . CFLAR promoter were co - transfected with Bclaf1 or / and p50 into HeLa - Bclaf1 - KO cells , and the luciferase activity was measured as described before .

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B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.


E , HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre - treated with DMSO or CHX ( 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) for 12 hours . Cells were lysed and then analyzed by western

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E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blotting


G . CHIP analysis of the CFLAR promoter in HeLa - Flag - Bclaf1 cells . Immunoprecipitation was performed using anti - Flag and control IgG antibodies followed by RT - PCR analysis . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way or Two - way ANOVA test .

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G. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.


A . HeLa - Flag - Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF ( 10 ng / ml ) for 2 hours were separated into the cytoplasmic and the nuclear fraction . Endogenous Flag - Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting .

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A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.


B . HeLa - Flag - Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF ( 10 ng / ml ) for 2 hours . The nuclear fraction was isolated and subjected to immunoprecipitation with an anti - Flag or control IgG antibody , followed by western blotting analysis .

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B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.


D . CFLAR FL promoter were co - transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa - Bclaf1 - KO cells . The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant . One - way ANOVA test .

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D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for Bclaf1 ( B ) and Hematoxylin - and - eosin staining ( C ) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis ( B ) . Hematoxylin - and - eosin staining showing the morphological change ( C ) . Villus height and crypt depth were measured ( D ) . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μm in B , 100 μm in C .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 ( E ) , phospholated RIPK3 ( F ) CC3 + and p - RIPK3 + positive cells in five fields per intestine were quantified . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50

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

siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for western blotting analysis ( G ) .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G).


HIEC - 6 cells were transfected siCtrl or siBclaf1 - 1 and - 2 . Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis ( I and J )

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HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J)


K . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media ( PM ) after isolation . L . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media ( DM ) . hiPSC donor 1 .

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K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.


M - O . Quantification of the number of Pax7 ( M ) , MyoD ( N ) and Pax7 / MyoD double - positive ( O ) cells in hskMPs and eMPs in PM and after switching to DM . hiPSC donor 1 .

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M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM. hiPSC donor 1.


P . Representative immunostaining for the muscle fiber marker myosin heavy chain ( MyHC ) of eMPs maintained in DM . Q . Quantification of the fusion index of hskMPs and eMPs in PM and DM . hiPSC donor 1 .

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P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM. Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM. hiPSC donor 1.


U - Z , Representative immunostainings and quantification of eMPs in DM expressing Pax7 ( V ) , MyoD ( X ) , and MyoG ( Z ) , as well as the proliferation marker Ki67 . hiPSC donor 1 .

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U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67. hiPSC donor 1.


A . Representative immunostainings for dystrophin in cross - sections of muscles of mdx mice after transplantation of hskMPs or eMPs . Scale bars in the two left - hand side images = 300 μm . Scale bars in inserts 1 and 2 = 50 μm . B . Number of dystrophin positive fibers in mdx muscles injected with vehicle ( Ctrl ) , eMPs and hskMPs . C . Maximal distance of clusters of dystrophin positive fibers in cross - sections of mdx muscles transplanted with eMPs and hskMPs . A - C . 10 days post - transplantation . hiPSC donor 1 .

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A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm. B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs. C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs. A-C. 10 days post-transplantation. hiPSC donor 1.


( a - c ) Low - magnification cryo - EM projection images of pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Different regions of the flagellum are annotated as follows : green - neck , yellow - midpiece , coral - principal piece , pink - endpiece . ( d - f ) Tomographic slices through cryo - FIB milled lamellae of pig ( d ) , horse ( e ) , and mouse ( f ) sperm . Transverse slices ( d ' - f ' ) show complete triplets in the pig ( d ' ) and the horse ( e ' ) , but not in the mouse ( f ' ) . Complete triplets are indicated by green arrowheads with black outlines , while degenerated triplets are indicated by white arrowheads with green outlines . Note the electron - dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece ( asterisks in d and d ' ) .

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

(a-c) Low-magnification cryo-EM projection images of pig (a), horse (b), and mouse (c) sperm. Different regions of the flagellum are annotated as follows: green - neck, yellow - midpiece, coral - principal piece, pink - endpiece. (d-f) Tomographic slices through cryo-FIB milled lamellae of pig (d), horse (e), and mouse (f) sperm. Transverse slices (d'-f') show complete triplets in the pig (d') and the horse (e'), but not in the mouse (f'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in d and d').


( g - i ) Tomographic slices through cryo - FIB milled lamellae of the DC - to - axoneme transition in pig sperm show how the change in geometry ( g , white arrows and white dashed lines ) coincides with the appearance of axoneme accessory structures ( h , white arrows ) and with density in the A - tubule ( i , compare insets in white and black boxes ) . In ( i ) , the white and black arrows indicate where the cross - sections in white and black boxes were taken from .

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(g-i) Tomographic slices through cryo-FIB milled lamellae of the DC-to-axoneme transition in pig sperm show how the change in geometry (g, white arrows and white dashed lines) coincides with the appearance of axoneme accessory structures (h, white arrows) and with density in the A-tubule (i, compare insets in white and black boxes). In (i), the white and black arrows indicate where the cross-sections in white and black boxes were taken from.


( a - c ) Slices through defocus cryo - tomograms of the endpiece in pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Insets show digital zooms ( black boxes ) and subtomogram averages ( white boxes ) of microtubule tips . Note the presence of a plug ( arrowhead ) extending into the lumen .

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(a-c) Slices through defocus cryo-tomograms of the endpiece in pig (a), horse (b), and mouse (c) sperm. Insets show digital zooms (black boxes) and subtomogram averages (white boxes) of microtubule tips. Note the presence of a plug (arrowhead) extending into the lumen.


A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup / Cdt1 RNAi , cyclin E RNAi , Cdk2 RNAi , Dacapo overexpression ( + Dacapo ) , and 1 mM HU .

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A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.


B Analysis of Mcm2 ‐ 7 nuclear chromatin association at different points in the cell cycle . Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2 ‐ 7 by Western blot . A non ‐ specific band is indicated by an asterisk ( * ) . C Quantification of the ratio of chromatin ‐ bound Mcm2 ‐ 7 relative to Orc2 ( log10 scale ) for a minimum of 11 replicates ( mean ± SD ) .

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B Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).C Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).


Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h ( top ) . Western blot analysis of Mcm2 ‐ 7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2 ‐ 7 , Orc2 , and Dacapo .

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Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.


B FACS profiles of DNA content for each cell population assayed .

B FACS profiles of DNA content for each cell population assayed.


C Western blot analysis for Orc2 and Mcm2 ‐ 7 in the nuclear chromatin fraction and Orc2 , Mcm2 ‐ 7 , Dup / Cdt1 , and cyclin E in the cytoplasmic fraction for each condition assayed .

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C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.


A Genome ‐ wide analysis of ORC localization by ChIP ‐ chip . ORC enrichment from asynchronous cells is depicted for a 5 ‐ Mb section of chromosome 2L . B Genome ‐ wide analysis of Mcm2 ‐ 7 localization in early G1 by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from cyclin E RNAi ‐ depleted cells is depicted for a 5 ‐ Mb section of chromosome 2L . C Venn diagram depicting the overlap between ORC and Mcm2 ‐ 7 peaks .

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A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.


A Genome ‐ wide analysis of Mcm2 ‐ 7 localization at the G1 / S transition by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from HU ‐ arrested cells is depicted for a 5 ‐ Mb section of chromosome 2L . Inset : transcribed ( green ) and non ‐ transcribed ( red ) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom . B Bimodal distribution of Mcm2 ‐ 7 enrichment over transcribed and non ‐ transcribed genes . Histogram showing the distribution of probe scores found within transcribed ( green ) and non ‐ transcribed ( red ) genes . C " Meta " ‐ gene analysis of Mcm2 ‐ 7 enrichment for different deciles of gene expression and their aggregated probe intensities .

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A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C "Meta"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.


D Mcm2 ‐ 7 is displaced from chromatin by DNA replication . Genome ‐ wide analysis of Mcm2 ‐ 7 localization in late S ‐ phase ( 6 h post HU release ) by ChIP ‐ chip . Mcm2 ‐ 7 enrichment is depicted for a 10 ‐ Mb section of chromosome 2L ( filled gray ) , replication timing profile ( black line ) and early ( yellow ) and late ( purple ) replication timing domains . E Box ‐ plots representing late S ‐ phase Mcm2 ‐ 7 ChIP signal found within early ( 246 ) or late ( 167 ) replication domains .

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D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.


A . Ovaries from control heterozygous Rpp3018 . 2 flies ( left ) , homozygous Rpp3018 . 2 or transheterozygous Rpp3018 . 2 / Rpp30PE flies ( middle ) and homozygous Rpp3018 . 2 carrying a copy of ubiRpp30GFP transgene ( right ) . Scale bar , 100µm .

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A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm.


D . Ovaries were dissected , lysed and protein extracts analyzed by Western Blot using anti - Rpp30 , anti - GFP or anti - tubulin antibodies . Orange arrows indicate overexpressed and endogenous Rpp30 signals . Genotypes are numbered as 1 , 2 , 3 .

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D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3.


E . Rpp30 - dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds : Left . Homozygous Rpp3018 . 2 ovariole expressing ubiquitous ubiRpp30GFP ( st , stage ) . Right . Transheterozygous Rpp3018 . 2 / Rpp30PE ovarioles overexpressing germline specific nanosGal4 , UASRpp30GFP . Note follicular somatic cells with no Rpp30GFP expression ( arrowhead ) . Magnifications show nuclear Rpp30 localization , Rpp30 perinuclear foci ( arrows ) , and Orb ( a specific oocyte marker ) . Scale bar , 10µm .

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E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm.


F . flp / FRT clones mutant for Rpp3018 . 2specifically in the germline ( star ) and / or the follicular cells ( arrows ) are detected by the absence of GFP . Magnifications show mutant oocytes with Orb mislocalization next to the karyosome ( dotted circle ) instead of being localized to the posterior , as seen in stage 3 ( st . 3 ) wild - type clones . Scale bar , 10µm .

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F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.


Figure 2 . Rpp30 is required for pre - tRNA processing . Whole fly RNA extracts were used to study tRNA processing by Northern Blot . Top box : the three different probes used are depicted : 5 ' and 3 ' probes ( red and green , respectively ) can detect full pre - tRNAs and tRNAs intermediates , but will not detect mature tRNAs . An Internal Probe ( IP , blue ) corresponding to the anti - codon region was used to detect all non - mature and mature tRNAs . The genotypes used in this experiment are numbered at the bottom ( 1 - 6 ) . Northern Blot panels : tRNA - his 5 ' probe ( top ) , tRNA - his 3 ' probe ( middle ) and tRNA - his IP ( bottom ) . U6 probe was used as loading control .

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Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.


A . Rpp3018 . 2 homozygous early oogenesis arrest is partially rescued by p53 mutation . Scale bar , 100μm .

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A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,100μm.


B . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is rescued by chk2 mutation ( Rpp3018 . 2 , mnkp6 and Rpp3018 . 2 / Rpp30PE , mnkp6 ) . Scale bar , 100μm . Star : example of one rescued stage 9 egg chamber . Arrow : example of non - rescued egg chambers .

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B. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers.


C . Germline clones mutant for Rpp3018 . 2 were immunostained for PCNA ( red ) . DAPI is in blue . Magnifications : PCNA signal in a control or mutant nurse cell . Scale bar , 10μm .

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C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm.


D . Germline clones mutant for Rpp3018 . 2 were immunostained for Brf ( pol - III ) ( red ) . DAPI is in blue . A Z - projection of Brf staining is shown . The arrow points Brf aggregates in a mutant chamber . Scale bar , 10μm .

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D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm.


E . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is partially rescued by claspin mutation ( Rpp3018 . 2 ; claspinEP / TM3 ) . Scale bar , 100μm .

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E. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm.


G . Top panel . Early oogenesis arrest found in Rpp30182 . / PEtransheterozygous ovaries is rescued by sqd - S - HAoverexpression . Scale bars : 100µm . Low panel . Rpp3018 . 2 / PE ; sqd - S - HA / sqd - S - HA ovaries were dissected and stained for Orb ( green ) , HA ( squid , red ) and DAPI ( blue ) . Scale bar , 10µm .

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G. Top panel. Early oogenesis arrest found in Rpp30182./PEtransheterozygous ovaries is rescued by sqd-S-HAoverexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.


. D . Nuage specific markers ( Aub , Ago3 and Mael ) are shown in stage 3 wild - type and germline mutant clones for Rpp3018 . 2 . Scale bar , 10µm .

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. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.


E - H . Double mutant ovaries for Rpp30 and chk2 ( Rpp3018 . 2 , mnkp6 ) rescued the specific and general piRNA populations , nuage morphology and the characteristic ping - pong signal . Scale bar , 10µm .

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E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.


D . Testes from control heterozygous , transheterozygous Rpp3018 . 2 / Rpp30PE or aubHN / QC42 were fixed and stained for Stellate crystal formation . Z - projections are shown .

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D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.


( A ) 3D time ‐ lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron . Neurons were transfected with Mito ‐ DsRed2 , pretreated with the pan ‐ caspase inhibitor zVAD ‐ fmk methyl ester ( 100 μM ) , and exposed to SNOC ( 200 μM ) . Images were 3D iso ‐ surface rendered . Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure ( upper panels ; scale bar , 15 μm ) and closeup views ( lower panels ; scale bar , 3 μm ) . See Supplementary Video 1 .

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(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.


( B ) Mitochondrial fragmentation within the field of view shown in ( A ) results in increased mitochondrial numbers .

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(B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers.


( C ) NO induces dose ‐ dependent mitochondrial fission . Neurons expressing Mito ‐ DsRed2 were exposed to SNOC and fixed at 60 min . The fraction of neurons displaying fragmented mitochondria is shown as the mean ± s . e . m .

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(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.


( D ) Mito ‐ DsRed2 ‐ expressing corticalneurons were pretreated with 1 mM nitro ‐ L ‐ arginine ( 20 min ) and exposed to 25 μM NMDA . The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean ± s . e . m . of quintuplicate samples from a representative experiment ( † † † significance at P0 . 001 or n . s . † , not significant compared to control ; * * * significance at P0 . 001 as compared to NMDA treatment ) .

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(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).


Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria . Neurons were co ‐ transfected with vectors encoding ( A ) Mito ‐ DsRed2 and ( B ) MyrPalm ‐ mCFP plus ( C ) AIF ‐ GFP and the effects of SNOC were recorded using 3D time ‐ lapse imaging . Projection images zoomed in onto the dendritic arbor of one neuron ( representative of n = 42 ) . Arrows indicate dendritic spines . The inset shows the soma at appropriate grayscale levels . The apparent brightening of AIF ‐ GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF . Scale bars , 10 μm . See also Supplementary Video 2 .

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Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.


( D ) 3D time ‐ lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition . Scale bar , 10 μm . Five mitochondria were selected and motion tracked throughout the imaging series .

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(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.


( E ) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion . Each trace color corresponds to the same colored mitochondrion . ( representative of n = 9 )

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(E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9)


( F - G ) Immunocytochemistry for staining anti ‐ AIF antibodies ( red ) anti ‐ NeuN antibodies ( green ) and nuclei labeled with Hoechst 33342 dye ( blue ) after ( F ) aged SNOC or ( G ) SNOC ( 150 μM ; 4 h ) treatment . Volume rendered 3D reconstruction of confocal image stacks are shown above , and a single confocal plane in grayscale below . Grid , 3 . 3 μm for ( F ) and 1 . 9 μm for ( G ) , scale bars , 5 μm .

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(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.


( H ) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body , outlined by the NeuN staining ( excluding the nucleus ) . The bar diagram summarizes the mean ± s . e . m . of 282 and 262 neurons measured in n = 19 and 20 image stacks in three independent experiments for aged SNOC and SNOC , respectively ( P ∼ 0 . 99 not significant by Student ' s t ‐ test ) .

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(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).


( I ) DEVDase activity . Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine , as positive control . The rate of zDEVD ‐ AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units . Data show mean ± s . e . m . for four independent experiments ( * significance at P0 . 01 by ANOVA ) .

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(I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).


( A ) Elongated mitochondrion ( arrow ) in neuronal process of the unaffected hemisphere ( top ) . Small mitochondrial segments ( arrows ) are in close proximity to each other in a neuronal process of the ischemic brain region ( bottom ) . Images are representative of three analyzed mice . Scale bars , 500 nm .

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(A) Elongated mitochondrion (arrow) in neuronal process of the unaffected hemisphere (top). Small mitochondrial segments (arrows) are in close proximity to each other in a neuronal process of the ischemic brain region (bottom). Images are representative of three analyzed mice. Scale bars, 500 nm.


( B ) Histogram comparing mitochondrial cross ‐ section length of contralateral , control ( red ) and ischemic ( black ) hemispheres . The lower bins of the histogram ( 2000 nm ) were compared with χ2 ‐ test and were not different . The table shows the presence of long mitochondrial cross ‐ sections ( > 2000 nm ) in the control hemisphere and their absence in the ischemic hemisphere ( P0 . 01 ; Fisher ' s exact test ) .

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(B) Histogram comparing mitochondrial cross‐section length of contralateral, control (red) and ischemic (black) hemispheres. The lower bins of the histogram (2000 nm) were compared with χ2‐test and were not different. The table shows the presence of long mitochondrial cross‐sections (>2000 nm) in the control hemisphere and their absence in the ischemic hemisphere (P0.01; Fisher's exact test).


( C ) Proximities of mitochondria comparing the contralateral ( red ) and ischemic ( black ) hemispheres . Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm . The same data set is summarized as a contingency table on the right , ( P0 . 001 ; two ‐ way Fisher ' s exact test ) .

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(C) Proximities of mitochondria comparing the contralateral (red) and ischemic (black) hemispheres. Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm. The same data set is summarized as a contingency table on the right, (P0.001; two‐way Fisher's exact test).


Fission is associated with ultrastructural changes in mitochondria . EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission . Cortical cultures were exposed to aged SNOC control ( A - D ) or 200 μM SNOC ( E - L ) for 6 h in the presence of 80 μM zVAD ‐ fmk methyl ester . A slice from each tilt series is shown ( A , E and I ) , followed by orthogonal views of 3D reconstructions after segmentation of the OMM ( blue ) and IMM ( gray ) , with cristae ( various arbitrary colors ) ( B - D , F - H and J - L ) . For clarity , 10 representative cristae are shown in ( B , D , F , G , J and K ) , while all cristae are displayed in ( C , H and L ) . The control mitochondrion ( A - D ) is elongated with a dark matrix , indicating no swelling , and 34 cristae . The NO ‐ exposed mitochondrion in ( E - H ) is dividing , and the lower mitochondrion of the couplet shows severe damage , recognized by an outer membrane rupture , a regionally confined inner membrane blowout , slight matrix swelling and cristae fragmentation . Each of the daughter mitochondria is shorter and rounder than controls . Unlike the upper daughter , few of the cristae extend throughout the lower mitochondrion . Additionally , cristae are considerably smaller and regionally confined . The lower mitochondrion of the couplet contains 82 cristae , while the upper contains only 39 , indicating cristae fragmentation in the former . The NO ‐ exposed mitochondrial triplet in ( I - L ) displays further damage , including membrane degradation , accumulation of an electron dense mass , greater cristae fragmentation , and apparent mitochondrial fragmentation . All cristae are smaller than controls , with 73 in the top mitochondrion , 112 in the middle , and 47 in the bottom . Scale bars , 400 nm . See Supplementary Video 3 , 4 and 5 of tomographic reconstruction .

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Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.


( A ) Projection image of two neurons expressing Mito ‐ DsRed2 ( red ) and MyrPalm ‐ CFP ( blue ) . Images were high ‐ pass filtered and z ‐ stacks were projected over maximum intensity .

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(A) Projection image of two neurons expressing Mito‐DsRed2 (red) and MyrPalm‐CFP (blue). Images were high‐pass filtered and z‐stacks were projected over maximum intensity.


( B ) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or ( C ) as mitochondrial numbers .

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(B) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or (C) as mitochondrial numbers.


( D ) Magnified representative frames of the time ‐ lapse recording of the area within the white rectangle of ( A ) indicate mitochondrial morphology at time 0 , 20 min and 1 h . These time points are depicted in ( B ) and ( C ) by red arrows . Data are representative of 26 neurons , which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons ( six experiments ) . Scale bar , 10 μm . See Supplementary Video 6 .

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(D) Magnified representative frames of the time‐lapse recording of the area within the white rectangle of (A) indicate mitochondrial morphology at time 0, 20 min and 1 h. These time points are depicted in (B) and (C) by red arrows. Data are representative of 26 neurons, which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons (six experiments). Scale bar, 10 μm. See Supplementary Video 6.


( E ) Autophagy in neurites at 6 h after SNOC exposure . Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image . The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes . White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome . Scale bars , 500 nm .

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(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.


( A ) Mitochondrial fission is linked to a drop in ATP . Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors ( 2 μM rotenone plus 2 μg / ml oligomycin ; Rot / Olig ) . ATP concentrations are shown as the mean ± s . e . m . normalized to the plating density of neurons ( n = 6 )

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(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)


( B ) [ ATP ] / [ ADP ] ratios in purified corticalneurons exposed to increasing SNOC concentrations ( n = 4 ) .

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(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).


( C ) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2 . 5 h .

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(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.


( D ) Cortical neurons expressing Mito ‐ GFP ( green ) were exposed to aged or fresh 200 μM SNOC solution . After 2 . 5 h cultures were loaded with hydroethidine . Images are representative of more that 20 neurons analyzed ( in D ) for each condition from at least three independent experiments . Scale bar , 10 μm .

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(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.


( E ) Reduced glutathione ( GSH ) partially blocks mitochondrial fission . Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester , washed once and then exposed to 35 μM SNOC . Mitochondrial fission was scored after 1 h . Data are means ± s . e . m . of quintuplicate samples from one representative experiment of a total of three ( † † † significance at P0 . 001 or n . s . , † not significant compared to control ; * significance at P0 . 05 ) .

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(E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).


( A ) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC ( 175 μM ; 7 h ) or rotenone ( 30 nM ; 2 h ) treatments and a comparison of the effects of Aβ25 - 35 versus Aβ35 - 25 ( 10 μM , 6 h ) , as indicated . Scale bar , 20 μm .

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(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.


( B ) Percentage of mitochondrial fission and cell death at 18 h ( † and † † † significance at P0 . 05 and 0 . 001 as compared to control pcDNA3 transfection ; * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to SNOC treated , pcDNA3 transfected neurons ; n = 3 independent experiments ) . Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining .

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(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.


( C ) Enforced Drp1 or Fis1 expression ( 60 h ) evokes fission and neuronal cell death ( * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to pcDNA3 transfected neurons ; n = 3 independent experiments ) .

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(C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments).


( D ) Rotenone induces dose ‐ dependent mitochondrial fission . The fraction of neurons displaying fissioned mitochondria is shown as the mean ± s . e . m . ( * significance at P0 . 05 , n = 4 ) .

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(D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).


( E ) Mfn1 or Drp1K38A inhibits 100 nM rotenone ‐ induced mitochondrial fission ( 4 h ) and cell death ( 48 h ) . Data indicate means ± s . e . m . of triplicate measurements ( † † and † † † significance at P0 . 01 and 0 . 001 as compared to untreated control ; * * * significance at P0 . 001 as compared to rotenone treatment with pcDNA3 transfection ; n = 3 ) .

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(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).


( F ) Mitochondrial fission by Aβ35 - 25 or Aβ25 - 35 exposure . The fraction of neurons exhibiting fragmented mitochondria is shown as the mean ± s . e . m . ( † † significance at P0 . 01 compared to pcDNA3 transfected , Aβ35 − 25 treated control ; * and * * significance at P0 . 05 and 0 . 001 , respectively , compared to pcDNA3 transfected , Aβ25 - 35 treated neurons ; n = 3 ) .

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(F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).


A , B Co - immunoprecipitation ( IP ) of EBNA2 ( A ) and PLK1 ( B ) using EBNA2 - , PLK1 - specific antibodies , or isotype - matched control antibodies performed on total cell lysates ( L ) of lymphoblastoid long - term B cell cultures ( LCLs ) infected with recombinant EBV strain 2089 , which is based on EBV strain B95 -

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A, B Co-immunoprecipitation (IP) of EBNA2 (A) and PLK1 (B) using EBNA2-, PLK1-specific antibodies, or isotype-matched control antibodies performed on total cell lysates (L) of lymphoblastoid long-term B cell cultures (LCLs) infected with recombinant EBV strain 2089, which is based on EBV strain B95-8


GFP - tagged EBNA2 fragments to co - precipitate endogenous PLK1 . Total protein lysates and immunoprecipitates ( IP ) were analyzed by Western blotting ( WB ) .

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GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB).


( H ) Co - immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1 .

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(H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.


( D ) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co - immunoprecipitations followed by Western blotting .

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(D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting.


( F ) GST EBNA2 fragment 246 - 487 wt and mutant S457A / T465V were treated with recombinant active PLK1 ( + ) in the presence of [ γ - 32P ] ATP in vitro or left untreated ( - ) . CRS , an artificial PLK1 test substrate was used as a positive control .

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(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.


( H ) HA - tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2 / CBF1 responsive promoter reporter luciferase plasmid . Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity .

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(H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity.


( A ) Flag - tagged active ( PLK1 ) and kinase inactive PLK1 ( K82M ) were co - expressed with HA - EBNA2 wt , docking site mutant ( S379A ) and phosphorylation mutant ( S457A / T465V ) and tested for activation of an EBNA2 / CBF1 responsive promoter reporter in DG75 cells . Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity .

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(A) Flag-tagged active (PLK1) and kinase inactive PLK1 (K82M) were co-expressed with HA-EBNA2 wt, docking site mutant (S379A) and phosphorylation mutant (S457A/T465V) and tested for activation of an EBNA2/CBF1 responsive promoter reporter in DG75 cells. Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity.


( B ) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag - , HA - and GAPDH - specific antibodies .

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(B) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag-, HA- and GAPDH-specific antibodies.


( C ) Expression of EBNA2 , LMP1 , PLK1 , MYC , and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors .

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(C) Expression of EBNA2, LMP1, PLK1, MYC, and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors.


( B ) Survival curve of humanized mice infected with 105 GRU EBV wt ( n = 13 ) , EBV S379A ( n = 11 ) or EBV S457A / T465V EBV ( n = 9 ) .

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(B) Survival curve of humanized mice infected with 105 GRU EBV wt (n = 13), EBV S379A (n = 11) or EBV S457A/T465V EBV (n = 9).


( E ) Development of viral loads in blood of infected mice over a period of five weeks . Error bars indicate mean ± SEM . Number of biological replicates per group : EBNA2 WT EBV week 2 - 5 : n = 8 ; EBNA2 S379A EBV week 2 - 5 : n = 7 ; EBNA2 S457A T465V EBV week 2 : n = 9 , week 3 : n = 8 , week 4 and week 5 : n =

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(E) Development of viral loads in blood of infected mice over a period of five weeks. Error bars indicate mean ± SEM. Number of biological replicates per group: EBNA2 WT EBV week 2 - 5: n = 8; EBNA2 S379A EBV week 2 - 5: n = 7; EBNA2 S457A T465V EBV week 2: n = 9, week 3: n = 8, week 4 and week 5: n = 7


( F ) Flow - cytometric analyses of CD8 + / CD4 + T cell ratios in the blood of infected mice over a period of five weeks . : EBNA2 WT EBV week 0 , 2 , 4 , 5 : n = 13 , week 3 : n = 8 ; EBNA2 S379A week 0 , 2 , 4 , 5 : n = 11 , week 3 : n = 7 ; EBNA2 S457A T465V week 0 , 2 : n = 12 , week 3 : n = 7 , week 4 : n = 10 , week 5 : n = 9 .

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(F) Flow-cytometric analyses of CD8+/CD4+ T cell ratios in the blood of infected mice over a period of five weeks. : EBNA2 WT EBV week 0, 2, 4, 5: n = 13, week 3: n = 8 ; EBNA2 S379A week 0, 2, 4, 5: n = 11, week 3: n = 7 ; EBNA2 S457A T465V week 0, 2: n = 12, week 3: n = 7, week 4: n = 10, week 5: n = 9.


Gel shift assay of biotin - labeled mascRNA and 5S rRNA with the cytosol ( Cyto ) and the postnuclear membrane lysate ( Mem ) .

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Gel shift assay of biotin-labeled mascRNA and 5S rRNA with the cytosol (Cyto) and the postnuclear membrane lysate (Mem).


Northern blotting of mascRNA ( top panel ) and western blotting of β - Tubulin and Mortalin ( bottom two panels ) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 cells .

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Northern blotting of mascRNA (top panel) and western blotting of β-Tubulin and Mortalin (bottom two panels) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 cells.


SDS - PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples . Biotin - labeled mascRNA ( S ) and the antisense RNA ( AS ) were used to pull down the RNA - protein complexes . Representive data of mass spectrometry analysis of the RNA pulldown samples . Proteins most enriched in mascRNA ( S ) RNA pulldown sample are listed .

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SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.   Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.


Western blots of the proteins in mascRNA S and AS pulldown samples ( top 4 panels ) , and mascRNA and menRNA pulldown samples ( Bottom 2 panels ) . β - tubulin was used as a loading control .

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Western blots of the proteins in mascRNA S and AS pulldown samples (top 4 panels), and mascRNA and menRNA pulldown samples (Bottom 2 panels). β-tubulin was used as a loading control.


RT - PCR analysis of RNAs extracted from ARS immunoprecipitation ( RIP ) samples ( erro bars : mean ± SD ; biological replicates n = 3 ) . Eight complex - forming ARSs ( DARS , EPRS , IARS , KARS , LARS , MARS , QARS and RARS ) and GARS tagged with FLAG were overexpressed in HEK cells . ARS - RNA complexes were imminoprecipitated with anti - FLAG Magnetic Beads . The cell line constructed with PQCXIP vector ( PQ ) was used as a negative control . GAPDH was used as an internal control for qRT - PCR .

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RT-PCR analysis of RNAs extracted from ARS immunoprecipitation (RIP) samples (erro bars: mean ± SD; biological replicates n = 3). Eight complex-forming ARSs (DARS, EPRS, IARS, KARS, LARS, MARS, QARS and RARS) and GARS tagged with FLAG were overexpressed in HEK cells. ARS-RNA complexes were imminoprecipitated with anti-FLAG Magnetic Beads. The cell line constructed with PQCXIP vector (PQ) was used as a negative control. GAPDH was used as an internal control for qRT-PCR.


Northern blot validation of mascRNA in the RIP samples . 5S rRNA from the RIP samples was used as an internal control .

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Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control.


Northern blot ( top 2 panels ) and western blot ( bottom 3 panels ) analysis of ARS immunoprecipitation samples ( RIP ) . RIP was performed using EPRS , QARS and GARS antibodies .

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Northern blot (top 2 panels) and western blot (bottom 3 panels) analysis of ARS immunoprecipitation samples (RIP). RIP was performed using EPRS, QARS and GARS antibodies.


Gel shift assay of biotin - labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody .

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Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.