Figure 5A . Glutamate content in the culture medium ( [ Glu ] e ) from either hTauAT ( n = 10 culture inserts containing 6 slices each ) or control slices ( n = 5 - 12 culture inserts ) analyzed at DIV 5 , 10 , 20 and 25 . Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point . Increased levels were observed between DIV 5 and DIV 20 . At DIV 25 glutamate levels already declined to control levels . One - way ANOVA followed by Tukey ' s post - hoc test * p < 0 . 05 ; * * p < 0 . 01 * * * p < 0 . 001 . B . Lactate dehydrogenase ( LDH ) release ( a measure of cytotoxicity ) analyzed in hTauAT ( n = 17 - 19 culture inserts containing 6 slices each ) or control slices ( n = 15 - 16 culture inserts ) at DIV 5 , 10 , 20 and 25 . LDH release of hTauAT slices is shown in percent of control at each time point . A pronounced increase ( ~ 25 % ) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One - way ANOVA : * p < 0 . 05 ; * * p < 0 . 01 and * * * p < 0 . 001 . C . Slice cultures from control ( left ) , hTauAT ( middle ) or hTauAT treated with ceftriaxone ( CEF ; 100 µM ; right ) stained against neuronal nuclear protein ( NeuN ) at DIV 30 . Representative neuronal cell layers are shown . hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 ( middle panel , red arrows ) . Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3 . D . NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest ( ROI ) . The number of neurons was reduced in hTauAT slices , both within area DG and CA3 , whereas the number of neurons in the CA1 was only slightly affected . Neuronal loss was largely prevented in cultures treated with ceftriaxone ( CEF ) in dentate gyrus and area CA3 ( n = 14 - 19 slices per group and area ; prepared from at least 6 animals ) . One - way ANOVA followed by Tukey ' s post - hoc test * p < 0 . 05 ; * * p < 0 .

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

Figure 5A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01


Figure 6A . Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months . Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining . The contrast of photomicrographs was inverted . B . Dendritic spines from heterozygous hTauATmice , seen by Golgi staining . C . Quantification of dendritic spine densities in control littermate and hTauAT mice . Note that the spine density in heterozygous hTauAT is increased ( control littermates : n = 2 animals ; heterozygous hTauATn = 4 animals ; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal ; unpaired t - test ) . D . Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12 ± 1 month old mice demonstrating significantly enhanced basal synaptic transmission ( Multiple repeat ANOVA , F ( 9 , 126 ) = 2 . 00 , p = 0 . 044 ) in area CA3 in mice expressing hTauAT . Scale bars : 500 µV vertical bar and 20 msec horizontal bar . E . Long - term potentiation in the mossy fiber pathway demonstrates no differences in long - term plasticity between control ( black ) and hTauAT expressing mice ( red ) . F . Input / output curve of field potential recording in stratum pyramidale ( s . p . ) of area CA3 in organotypic hippocampal slices at DIV 30 . Somatic field potentials are increased in hTauAT compared to control littermates . Scale bars : 800 µV vertical bar and 20 msec horizontal bar .

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

Figure 6A. Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months. Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining. The contrast of photomicrographs was inverted.B. Dendritic spines from heterozygous hTauATmice, seen by Golgi staining.C. Quantification of dendritic spine densities in control littermate and hTauAT mice. Note that the spine density in heterozygous hTauAT is increased (control littermates: n = 2 animals; heterozygous hTauATn = 4 animals; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal; unpaired t-test).D. Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12±1 month old mice demonstrating significantly enhanced basal synaptic transmission (Multiple repeat ANOVA,F(9, 126)=2.00, p=0.044) in area CA3 in mice expressing hTauAT. Scale bars: 500 µV vertical bar and 20 msec horizontal bar.E. Long-term potentiation in the mossy fiber pathway demonstrates no differences in long-term plasticity between control (black) and hTauAT expressing mice (red).F. Input / output curve of field potential recording in stratum pyramidale (s.p.) of area CA3 in organotypic hippocampal slices at DIV 30. Somatic field potentials are increased in hTauAT compared to control littermates. Scale bars: 800 µV vertical bar and 20 msec horizontal bar.


Figure 7A . Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice ( DIV30 ) during wash - in of 50 µm picrotoxin . This leads to blockage of GABAergic transmission followed by epileptiform discharges ( burst ) occurring approximately after 3 min . The zoom - in reveals that such an epileptiform discharge ( burst ) consists of repetitive firings . B . Similar example trace as in ( A ) recorded in a hTauAT expressing slice ( red ) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression . C . Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~ 2 - fold increase in hTauAT slices ( n = 8 slices per group ) . D . Firings per burst are increased ~ 1 . 5 - fold already at the early time point DIV10 due to hTauAT expression . E . Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10 . Burst frequency is enhanced in hTauAT expressing slices ( n = 10 slices ) . If Ceftriaxone ( CEF ) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented ( n = 5 slices ) . F . Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30 . Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation ( n = 6 slices ) . G . Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN ( red ) and pan - Tau ( K9JA antibody , green ) . Axonal projections ( green ) of granule cells ( red ) in the dentate gyrus are depicted . H . Hippocampal slice culture expressing hTauAT stained against NeuN ( red ) and pan - Tau ( green ) showing mossy fiber sprouting in the hilar region , CA3 and dentate gyrus ( white arrows ) .

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

Figure 7A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).


Figure 8A . Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin ( TeNT ) or 1 µM tetrodotoxin ( TTX ) at DIV5 . Under both conditions , glutamate levels in the culture medium of hTauAT slices decreased to control levels ( n = 6 culture inserts containing 6 slices each / group and condition ) . B . Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV1 - DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices ( n = 8 - 9 culture inserts containing 6 slices each / group and condition ) . One - Way ANOVA F ( 3 , 30 ) = 14 , 89 ; p < 0 . 001C . Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV 1 to DIV 20 ( n = 6 - 12 culture inserts containing 6 slices each / group and condition ) . One - Way ANOVA followed by Tukey ' s post - hoc test . D . Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated ( n = 7 - 8 culture inserts containing 6 slices each / group and condition ) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices . One - Way ANOVA F ( 3 , 25 ) = 9 . 075 ; p = 0 . 0003 ) . E . LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures ( n = 8 - 10 culture inserts containing 6 slices each / group and condition ) . One Way ANOVA F ( 3 , 32 ) = 7 . 685 ; p = 0 . 0005 ) . F . Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days ( n = 5 - 9 slices ; prepared from at least three animals ) . One way ANOVA F ( ( 3 , 25 ) = 7 . 317 ; p = 0 . 0011 ) .

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 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 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 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

Figure 8A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey's post-hoc test.D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).


Figure 1A . Genes involved in innate immunity are significantly upregulated in nuo - 6 and isp - 1 worms . Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild - type N2 worms . Results represent counts per million ( CPM ) expressed as a percentage of wild - type of six biological replicates per strain . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison testB . Upregulation of innate immunity genes is dependent on the p38 - mediated innate immune signaling pathway including NSY - 1 , SEK - 1 , PMK - 1 and ATF - 7 . Gene expression changes were examined by quantitative RT - PCR of three biological replicates per strain . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison testC . Using a fluorescent reporter strain for the innate immunity gene T24B8 . 5 confirms that innate immunity genes are upregulated in the long - lived mitochondrial mutants and that components of the p38 - mediated innate immune signaling pathway are required for their upregulation . Scale bar indicates 250 µM . Three biological replicates per strain were quantified . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a two - way ANOVA with Bonferroni post -

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

Figure 1A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testB. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testC. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test


Figure 2A - D . Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay . Both nuo - 6 and isp - 1 long - lived mitochondrial mutants show increased resistance compared to wild - type worms ( A ) . Disruption of components of the p38 - mediated innate immune signaling pathway ( nsy - 1 , sek - 1 , pmk - 1 , atf - 7 ) markedly decreases resistance to PA14 in wild - type ( B ) , nuo - 6 ( C ) and isp - 1 ( D ) worms . All strains were tested in a single parallel experiment . Two biological replicates per strain were performed with a total of at least 175 animals per strain . Data information : * * * p < 0 . 001 . Statistical significance for survival plots was determined with the log - rank test . Significance is indicated between the strain listed on top and all other strains . Data from panel A is repeated in panels B , C , and D for direct comparison .

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

Figure 2A-D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared to wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C) and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. Data information: ***p<0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A is repeated in panels B, C, and D for direct comparison.


Figure 3A - H . To examine the role of the p38 - mediated innate immune signaling pathway in the long - lifespan of the nuo - 6 and isp - 1 mitochondrial mutants , we crossed the long - lived mitochondrial mutants to worms with mutations in nsy - 1 , sek - 1 , pmk - 1 and atf - 7 ( gof ) . In each case , mutation of genes involved in p38 - mediated innate immunity signaling markedly reduces the lifespan of the long - lived mitochondrial mutant but have little or no impact on wild - type lifespan . In panel G , we utilized pmk - 1 RNAi instead of the pmk - 1 mutation because of the close proximity of isp - 1 and pmk - 1 on the same chromosome . EV indicates empty vector . The lifespan results for panels A , B , D , E , F , and H were performed in a single parallel experiment . Results are from a minimum of three biological replicates per strain . Data information : Statistical significance for survival plots was determined with the log - rank test . p - values indicate significance between red and purple lines . Control strains are shown in multiple panels for direct comparison .

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

Figure 3A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain. Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.


Figure 4A , B . To determine the extent to which the p38 - mediated innate immunity pathway is activated in nuo - 6 and isp - 1 mutants , we measured the ratio of phosphorylated PMK - 1 / p38 to total PMK - 1 / p38 by Western blotting . Representative blots are shown ( A ) . There is no difference in the ratio of phosphorylated to total PMK - 1 / p38 in the long - lived mitochondrial mutants compared to wild - type ( B ) . Eight biological replicates of wild - type worms and nine of mitochondrial mutants were quantified . Data information : Error bars indicate SEM . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison testF . Constitutive activation of atfs - 1 is sufficient to decrease food consumption to the same level as in long - lived mitochondrial mutants . Three biological replicates per strain were measured , each including at least three technical replicates . atfs - 1 ( lof ) allele is gk3094 , atfs - 1 ( gof ) allele is et17 . Data information : Error bars indicate SEM . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison test

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 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

Figure 4A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison testF. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test


Figure 5A . A deletion of daf - 16 completely abolishes the increased resistance to bacterial pathogens in isp - 1 mutants . p - value indicates the significance of difference between red and purple lines . Three biological replicates per strain were performed . Data information : Statistical significance was assessed using log - rank test . B , C . While daf - 16 RNAi effectively decreases the expression of DAF - 16 target genes ( B , sod - 3 , dod - 3 , mtl - 1 , ftn - 1 , icl - 1 , sodh - 1 ) , it does not affect the expression of any of the innate immunity genes ( C , T24B8 . 5 , K08D8 . 5 , F55G11 . 8 , clec - 65 , clec - 67 , dod - 22 , Y9C9A . 8 and C32H11 . 4 ) . This indicates that DAF - 16 is not required for expression of innate immune signaling pathway target genes in isp - 1 mutants . daf - 16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation . RNA was isolated from six biological replicates at the young adult stage of the experimental generation . RNA from the six biological replicates was pooled for RNA sequencing .

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 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

Figure 5A. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed. Data information: Statistical significance was assessed using log-rank test.B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not affect the expression of any of the innate immunity genes (C, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.


Figure 6A . To examine the role of the ATFS - 1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo - 6 mutants , we examined the effect of disrupting atfs - 1 in nuo - 6 mutants . Loss of atfs - 1 completely abolishes the increased resistance to bacterial pathogens in nuo - 6 worms . p - value indicates significance of difference between red and purple lines . nuo - 6 ; atfs - 1 contains the gk3094 deletion allele . Three biological replicates per strain were measured . B . To examine the role of ATFS - 1 in the upregulation of innate immunity genes in nuo - 6 mutants , we compared gene expression between nuo - 6 and nuo - 6 ; atfs - 1 mutants with wild - type worms and atfs - 1 mutants as controls . Deletion of atfs - 1 significantly decreases the expression of genes involved in innate immunity in nuo - 6 worms but does not decrease the expression of these genes in wild - type worms . Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype . Results represent counts per million ( CPM ) expressed as a percentage of wild - type . Worms in a wild - type background ( control ) are shown with white bars , while worms with atfs - 1 deletion are shown with blue bars . Data information : Error bars indicate SEM . * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance in panel A was assessed using log - rank test . Statistical significance in panel B was assessed using a two - way ANOVA with Bonferroni posttest .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. To examine the role of the ATFS-1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo-6 mutants, we examined the effect of disrupting atfs-1 in nuo-6 mutants. Loss of atfs-1 completely abolishes the increased resistance to bacterial pathogens in nuo-6 worms. p-value indicates significance of difference between red and purple lines. nuo-6; atfs-1 contains the gk3094 deletion allele. Three biological replicates per strain were measured. B. To examine the role of ATFS-1 in the upregulation of innate immunity genes in nuo-6 mutants, we compared gene expression between nuo-6 and nuo-6; atfs-1 mutants with wild-type worms and atfs-1 mutants as controls. Deletion of atfs-1 significantly decreases the expression of genes involved in innate immunity in nuo-6 worms but does not decrease the expression of these genes in wild-type worms. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Worms in a wild-type background (control) are shown with white bars, while worms with atfs-1 deletion are shown with blue bars. Data information: Error bars indicate SEM. **p<0.01, ***p<0.001. Statistical significance in panel A was assessed using log-rank test. Statistical significance in panel B was assessed using a two-way ANOVA with Bonferroni posttest.


Figure 7A . Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs - 1 mutants ( et15 and et17 ) revealed that innate immunity genes are upregulated when ATFS - 1 is activated . This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38 - mediated innate immune pathway . Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype . Results represent counts per million ( CPM ) expressed as a percentage of wild - type . Data information : Error bars indicate SEM . * p < 0 . 05 * * p < 0 . 01 , * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison test .

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

Figure 7A. Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs-1 mutants (et15 and et17) revealed that innate immunity genes are upregulated when ATFS-1 is activated. This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38-mediated innate immune pathway. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test.


Figure 1Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum . Eluates were analysed by SDS - PAGE and western botting . Total 1 . 5 % , Eluate , 100 % . Wild type mitochondria were solubilized and subjected to 2D - BN / SDS - PAGE and western blotting with indicated antibodies . Solubilized mitochondria from MITRAC12FLAG - or ACAD9FLAG - expressing cells were subjected to anti - FLAG immunoisolation and native elution . Eluates were analysed by 2D - BN / SDS - PAGE and western blot analysis . Total 0 . 8 % ( MITRAC12FLAG isolation ) , 0 . 35 % ( ACAD9FLAG isolation ) , Eluate 100 % . Solubilized mitochondria from MITRAC15FLAG - expressing cells were first subjected to native anti - FLAG immunoisolation ( Total 0 . 1 % , Eluate 10 % , lane 4 ) . Purified complexes were applied to anti - MITRAC12 immunoisolation ( Eluates 100 % , lane 5 ) . All samples were analyzed by SDS - PAGE and immunoblotting .

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

Figure 1Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum. Eluates were analysed by SDS-PAGE and western botting. Total 1.5 %, Eluate, 100 %.Wild type mitochondria were solubilized and subjected to 2D-BN/SDS-PAGE and western blotting with indicated antibodies.Solubilized mitochondria from MITRAC12FLAG- or ACAD9FLAG-expressing cells were subjected to anti-FLAG immunoisolation and native elution. Eluates were analysed by 2D-BN/SDS-PAGE and western blot analysis. Total 0.8 % (MITRAC12FLAG isolation), 0.35 % (ACAD9FLAG isolation), Eluate 100 %.Solubilized mitochondria from MITRAC15FLAG-expressing cells were first subjected to native anti-FLAG immunoisolation (Total 0.1 %, Eluate 10 %, lane 4). Purified complexes were applied to anti-MITRAC12 immunoisolation (Eluates 100 %, lane 5). All samples were analyzed by SDS-PAGE and immunoblotting.


Figure 2Mitochondrial translational products were labelled with [ 35S ] methionine and anti - FLAG immunoisolation was performed as described in Fig . 1D . Eluates were analyzed by SDS - PAGE and digital autoradiography . Total 1 . 5 % , Eluate 100 % . Solubilized mitochondria from MITRAC15FLAG - or TIM21FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by 2D - BN / SDS - PAGE and western blotting . MITRAC15FLAG isolation : Total 0 . 1 % , Eluate 100 % . TIM21FLAG isolation : Total 0 . 2 % , Eluate 66 % ( TIM21FLAG decoration ) , 33 % ( MITRAC15 , ACAD9 , MITRAC12 , COX1 decoration ) . Solubilized mitochondria from MITRAC15FLAG - or C12ORF62FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by SDS - PAGE and immunoblotting . Total 0 . 4 % , Eluate 100 % ( MITRAC15FLAG isolation ) , 70 % ( C12ORF62FLAG isolation ) . Solubilized mitochondria from ACAD9FLAG - or MITRAC15FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by SDS - PAGE and immunoblotting . Total 0 . 5 % ( ACAD9FLAG isolation ) , 0 . 4 % ( MITRAC15FLAG isolation ) Eluate 100 % . Asterisk ( * ) indicates TIM21 signal . ACAD9 / ACAD9FLAG could not be separated with the used gel system , both proteins are detected with ACAD9 antiserum .

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 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

Figure 2Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %.Solubilized mitochondria from MITRAC15FLAG- or TIM21FLAG-expressing cells were subjected to anti-FLAG immunoisolation and analyzed by 2D-BN/SDS-PAGE and western blotting. MITRAC15FLAG isolation: Total 0.1 %, Eluate 100 %. TIM21FLAG isolation: Total 0.2 %, Eluate 66 % (TIM21FLAG decoration), 33 % (MITRAC15, ACAD9, MITRAC12, COX1 decoration).Solubilized mitochondria from MITRAC15FLAG- or C12ORF62FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.4 %, Eluate 100 % (MITRAC15FLAG isolation), 70 % (C12ORF62FLAG isolation).Solubilized mitochondria from ACAD9FLAG-or MITRAC15FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.5 % (ACAD9FLAG isolation), 0.4 % (MITRAC15FLAG isolation) Eluate 100 %. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.


Figure 3Mitochondria from wild type and MITRAC15 - / - cells were isolated and subjected to steady - state protein analyses by western blotting . Mitochondria from wild type and MITRAC15 - / - cells were isolated and analyzed by BN - PAGE . Triton X - 100 ( left site ) or digitonin ( right site ) were used for solubilization . The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels ( error bars indicate SEM from three biological replicates ) . Complex I and citrate synthase activity were measured in WT and MITRAC15 - / - cells ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) . Pulse - chase [ 35S ] methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) . Pulse radiolabeling after re - expressing MITRAC15FLAG in MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Relative pulse signal intensity of ND2 was normalized to ATP6 ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 ; unpaired t - test ) .

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

Figure 3Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting.Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).


Figure 4Mitochondrial translation products were radiolabelled with [ 35S ] methionine after siRNA - mediated depletion of ACAD9 in wild type and MITRAC15 - / - cells . Cell lysates were analyzed by SDS - PAGE and digital autoradiography . For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal . ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 * * * p < 0 . 001 ; unpaired t - test ) . FLAG - immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG - expressing cells after siRNA - mediated down regulation of ACAD9 and pulse radiolabeling . SDS - PAGE followed by autoradiography was used to analyze eluates . Relative signal intensity of ND2 , ND3 and ND4L were normalized to ATP6 ( total ) or isolation efficiency ( eluate ) . Total 2 % , Eluate 100 % . Error bars indicate SEM from three biological replicates ; * * p < 0 . 01 ; unpaired t - test . After C12orf62 down regulation , [ 35S ] methionine labelling was performed . During labeling mitochondrial translation was inhibited with puromycin ( pur ) ( 2 µg / ml ) . MITRAC15FLAG interacting nascent chains were isolated by FLAG - immunoprecipitation from cell lysates . Eluates were subjected to SDS - PAGE , analyzed by digital autoradiography . Total 2 % , MITRAC15FLAG eluates 100 % ( in case of C12ORF62FLAG , 25 % of eluate was loaded as a standard ) . Red asterisks , accumulating nascent chains of ND2 . The accumulating nascent chains of ND2 of lane 12 is presented in a magnification . F1 - F3 mark nascent chains of COX1 [ 18 ] . HEK293T cells were pulse labelled with [ 35S ] methionine in the presence of puromycin ( 2 µg / ml ) . During puromycin treatment , cells were incubated for 5 min and 10 min in medium lacking [ 35S ] methionine . Anti - FLAG immunoisolations were performed as described and eluates analyzed as described in Fig . 4C . Total 1 % , Eluates 100 % . The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; paired t - test ) . Red asterisks , accumulating nascent chains of ND2 .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).FLAG-immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG-expressing cells after siRNA-mediated down regulation of ACAD9 and pulse radiolabeling. SDS-PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3 and ND4L were normalized to ATP6 (total) or isolation efficiency (eluate). Total 2 %, Eluate 100 %. Error bars indicate SEM from three biological replicates; **p < 0.01; unpaired t-test.After C12orf62 down regulation, [35S]methionine labelling was performed. During labeling mitochondrial translation was inhibited with puromycin (pur) (2 µg/ml). MITRAC15FLAG interacting nascent chains were isolated by FLAG-immunoprecipitation from cell lysates. Eluates were subjected to SDS-PAGE, analyzed by digital autoradiography. Total 2 %, MITRAC15FLAG eluates 100 % (in case of C12ORF62FLAG, 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 is presented in a magnification. F1-F3 mark nascent chains of COX1 [18].HEK293T cells were pulse labelled with [35S]methionine in the presence of puromycin (2 µg/ml). During puromycin treatment, cells were incubated for 5 min and 10 min in medium lacking [35S]methionine. Anti-FLAG immunoisolations were performed as described and eluates analyzed as described in Fig. 4C. Total 1 %, Eluates 100 %. The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % (error bars indicate SEM from three biological replicates; *p < 0,05; paired t-test). Red asterisks, accumulating nascent chains of ND2.


Figure 5Radiolabeling with [ 35S ] methionine , anti - FLAG immunoisolation and sample analyses were performed Total 1 % , Eluates 100 % . Red asterisks , accumulating nascent chains of ND2 . Depletion of ACAD9 by siRNA was performed Total 1 % , Eluates 100 % . Red asterisks , accumulating nascent chains of ND2 .

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

Figure 5Radiolabeling with [35S]methionine, anti-FLAG immunoisolation and sample analyses were performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.Depletion of ACAD9 by siRNA was performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.


Figure 1 ( A ) The relative fold change of carbon metabolites with or without HU treatment for 60 min . Error bars represent standard deviations ( SD ) from six independent experiments . ( D , E ) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT , snf1Δ or snf1Δmig1Δ cells ( D ) . Protein extracts were probed with an anti - GFP antibody . Mcm2 was applied as a loading control . Three independent experiments were carried out and the relative fold changes were measured by Image J ( E ) . Error bars represent standard deviations ( SD ) from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 0 . 05 ; * * 0 . 001 < P - values < 0 . 01 ) .

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 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

Figure 1(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments.(D, E) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT, snf1Δ or snf1Δmig1Δ cells (D). Protein extracts were probed with an anti-GFP antibody. Mcm2 was applied as a loading control. Three independent experiments were carried out and the relative fold changes were measured by Image J (E). Error bars represent standard deviations (SD) from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05; **0.001< P-values <0.01).


Figure 2 ( C ) Rad53 phosphorylation in various snf1 alleles in response to HU . The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ . Rad53 phosphorylation was detected as above . ( D ) The snf1 kinase - defective mutants are sensitive to HU . Yeast spot assays were performed as in Fig 2A . The phospho - mimetic snf1T210D ( a constitutive active mutant ) was applied as a control for nonphosphrylable snf1T210A . ( F , G ) HU induces a rapid Snf1 T210 phosphorylation . Exponential cells were treated by 200 mM HU for the indicated time . Protein extracts were prepared as described in the Methods and Materials . Three independent experiments were done . Quantitation of Snf1 T210 and Snf1 signals was performed by Image J . The relative ratio of Snf1 T210 / Snf1 was shown in ( H ) . Error bars represent standard deviations ( SD ) from three biological repeats .

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

Figure 2(C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above.(D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A.(F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.


Figure 3 ( C ) Snap shot of Rfa1 ChIP - Seq at Chromosome VI . Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min . The sequencing reads were mapped to the yeast reference genome . The red dashed box indicated the relative read intensity of ARS607 . ( E , F ) SNF1 is required for efficient recruitment of Rfa1 , but not Psf2 , to HU - stalled replication forks . Cells were grown and synchronized in G1 by α - factor before release into the fresh medium supplemented with 200 mM HU for the indicated time . Cell extracts were prepared and subjected to MYC - ChIP of Rfa1 - 13MYC ( E ) or Psf2 - 13MYC ( F ) . The amounts of DNA in the precipitates were quantified by qPCR . Error bars represent SD from three biological repeats .

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 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

Figure 3(C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607.(E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.


Figure 4mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU . The experiments were done as in Fig 2 . ( E , F ) snf1Δ shows less RPA on chromatin , which can be restored by mig1Δ . Cells were collected and fractionated into chromatin - bound ( Chromatin ) and non - chromatin - bound fractions ( non - Chromatin ) followed by immunoblotting . Tubulin and Orc2 were applied as controls for non - chromatin and chromatin fractions , respectively . The signals were quantified by Image J and quantifications were shown in ( F ) . Error bars represent SD from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 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 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

Figure 4mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU. The experiments were done as in Fig 2.(E, F) snf1Δ shows less RPA on chromatin, which can be restored by mig1Δ. Cells were collected and fractionated into chromatin-bound (Chromatin) and non-chromatin-bound fractions (non-Chromatin) followed by immunoblotting. Tubulin and Orc2 were applied as controls for non-chromatin and chromatin fractions, respectively. The signals were quantified by Image J and quantifications were shown in (F). Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).


Figure 5Phospho - mimetic mig1 - 2D bypasses the role of SNF1 in HU resistance ( C ) Phospho - mimetic mig1 - 2D bypasses the role of SNF1 in RPA recruitment ( D ) . ( E ) The diagram of the strategy to reinforce the Mig1 - Ssn6 interaction via a GFP and GBP pair ( left panel ) . The persistent Mig1 - Ssn6 association abolishes the phospho - mimicking effect of mig1 - 2D ( right panel ) .

O B-PANEL_START 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

Figure 5Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C)Phospho-mimetic mig1-2D bypasses the role of SNF1 in RPA recruitment (D). (E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel).


Figure 6 ( C ) Rfa1 - 13MYC ChIP assays after HU treatment . Strains were synchronized in G1 phase by α - factor prior to release into fresh media containing 200 mM HU for the indicated time points . Cell extracts were prepared as in Fig 3 . Error bars represent SD from three biological repeats . ( D ) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ . The experiments were done as in Fig 1 . Error bars represent SD from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 0 . 05 ; * * 0 . 001 < P - values < 0 . 01 ) .

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 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

Figure 6(C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats.(D) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ. The experiments were done as in Fig 1. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).


Figure 7 ( B ) The effects of α - KG on the HU sensitivity of snf1Δ and other checkpoint mutants . Yeast spot assays were performed as in Fig 2A . ( D ) α - KG restores Rad53 phosphorylation in snf1Δ . CBB indicates coomassie bright blue ( CBB ) staining of the membrane as the loading control . ( E ) The effects of α - KG on the HU sensitivity of Rfa1 zinc finger mutants ( C486S , C491S , C505S , C508S ) . Yeast spot assays were performed as in Fig 2A .

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

Figure 7(B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A.(D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control.(E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A.


Figure 1a , b . The amount of FDP - lysine , which was major protein - bound acrolein , in the sera of healthy controls and PD patients was determined by an ELISA ( a . all patients , ( control n = 22 ; PD n = 94 ) b . staged by the H & Y scale , a five - steps index of PD severity . Larger number indicates more severe condition . n = 94 ; H & Y stage I ( n = 18 ) , stage II ( n = 42 ) stage III ( n = 23 ) , stage IV ( n = 7 ) , and stage V ( n = 4 ) .

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

Figure 1a, b. The amount of FDP-lysine, which was major protein-bound acrolein, in the sera of healthy controls and PD patients was determined by an ELISA (a. all patients, (control n= 22; PD n=94) b. staged by the H&Y scale, a five-steps index of PD severity. Larger number indicates more severe condition. n=94; H&Y stage I (n=18), stage II (n=42) stage III (n=23), stage IV (n=7), and stage V (n=4).


Figure 2SH - SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h ( a ) Cells were fixed and stained with an anti - LAMP2 ( green ) antibody as a lysosomal marker . Nuclei were stained with DAPI ( blue ) . Cell membrane is indicated by the white line . Clustered relative to whole lysosomes were quantified as lysosomal clustering value ( a , right ) . e . SH - SY5Y cells were treated with acrolein for 2 h . Cell lysates were immunoblotted with the indicated antibodies . h . SH - SY5Y cells were treated with acrolein for 2 h . Cell lysates were immunoblotted with the indicated antibodies .

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

Figure 2SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right).e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.h. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.


Figure 3d . SH - SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h . ( upper ) Cells were fixed and stained with an anti - LAMP2 antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 µm . ( lower ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value .

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

Figure 3d. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. (upper) Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.


Figure 4a . Cells were transfected with the TMEM55B - myc - DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h . Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies . b . SH - SY5Y cells were treated with 40 μM acrolein for 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . c . SH - SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed , subjected to Phos - tag PAGE , and immunoblotted with an anti - JIP4 antibody . d . SH - SY5Y cells were pre - treated with 10 µM Jak3 inhibitor VI ( Jak3 i VI ) for 1 h and then treated with 40 µM acrolein for an additional 2 h . Cells lysates were subjected to Phos - tag PAGE and immunoblotted with an anti - JIP4 antibody . e . SH - SY5Y cells were pre - treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . SH - SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h . Cell lysates were subjected to Phos - tag PAGE and immunoblotted with anti - JIP4 antibody ( f , left ) . CaMK2G knockdown efficiency was confirmed by qRT - PCR . ; * * * p < 0 . 001 , n = 3 technical replicates . Values are mean ± SD . At least three experiments were replicated ( f , right ) . j . His6 - tagged recombinant truncated JIP4 ( 100 - 300 aa ) was purified from E . coli and incubated with recombinant CaMK2G with / without Ca2 + and calmodulin at 30 ° C for 1 h . The reactant was subjected to Phos - tag PAGE and immunoblotted with anti - His antibody . Jak3 inhibitor VI was pre - reacted with CaMK2G for 30 min .

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 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 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 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

Figure 4a. Cells were transfected with the TMEM55B-myc-DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h. Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies.b. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).c. SH-SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed, subjected to Phos-tag PAGE, and immunoblotted with an anti-JIP4 antibody. d. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI (Jak3 i VI) for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells lysates were subjected to Phos-tag PAGE and immunoblotted with an anti-JIP4 antibody.e. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).SH-SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h. Cell lysates were subjected to Phos-tag PAGE and immunoblotted with anti-JIP4 antibody (f, left). CaMK2G knockdown efficiency was confirmed by qRT-PCR.; ***p <0.001, n=3 technical replicates. Values are mean ± SD. At least three experiments were replicated (f, right).j. His6-tagged recombinant truncated JIP4 (100-300 aa) was purified from E. coli and incubated with recombinant CaMK2G with/without Ca2+ and calmodulin at 30°C　for 1 h. The reactant was subjected to Phos-tag PAGE and immunoblotted with anti-His antibody. Jak3 inhibitor VI was pre-reacted with CaMK2G for 30 min.


Figure 5c . GFP or GFP - ALG2 transfected TRPML1 - mCherry stably expressing cells were treated with acrolein for 2 h . Cell lysates were immunoprecipitated with anti - RFP magnetic beads and immunoblotted with the indicated antibodies . f . SH - SY5Y cells transfected with GFP or GFP - ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies .

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

Figure 5c. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2 h. Cell lysates were immunoprecipitated with anti-RFP magnetic beads and immunoblotted with the indicated antibodies.f. SH-SY5Y cells transfected with GFP or GFP-ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies.


Figure 6Parental and JIP4 KO SH - SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h ( a ) . Cell lysates were immunoblotted with the indicated antibodies . Autophagic flux was determined as the LC3 - II production rate calculated as the level of LC3 - II with bafilomycin A1 treatment . SH - SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI ( Jak3 i VI ) for 2 h ( b ) . Cell lysates were immunoblotted with the indicated antibodies . Autophagic flux was determined as the LC3 - II production rate calculated as the level of LC3 - II with bafilomycin A1 treatment . d . ( upper ) SH - SY5Y cells were pretreated with 10 µM BAPTA - AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 μm . ( lower ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value . e . SH - SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h . Cells were lysed , subjected to Phos - tag PAGE , and immunoblotted with anti - JIP4 antibody . f . ( left ) SH - SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h . Cells were fixed and stained with anti - γ - tubulin ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 μm . ( right ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value . * * *

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

Figure 6Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h (a). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI (Jak3 i VI) for 2 h (b). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.d. (upper) SH-SY5Y cells were pretreated with 10 µM BAPTA-AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.e. SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were lysed, subjected to Phos-tag PAGE, and immunoblotted with anti-JIP4 antibody.f. (left) SH-SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were fixed and stained with anti-γ-tubulin (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (right) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ***


Figure 7b . Parental and JIP4 KO SH - SY5Y cells were treated with various concentrations of acrolein for 24 h . Cytotoxicity was measured by an LDH assay . c . SH - SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h . Cytotoxicity was measured by an LDH assay .

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

Figure 7b. Parental and JIP4 KO SH-SY5Y cells were treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. c. SH-SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay.


Figure 1Expression of ERα in control ( shCtrl ) and knockdown ( shERα ) BM67 cells was determined by western blotting ( β - actin served as a loading control ) . Gene expression was determined using polysome - profiling which quantifies both total mRNA and efficiently translated polysome - associated mRNA ( n = 3 ) . Distributions ( quantified by kernel density estimation ) of p - values for differential expression ( shERα vs . shCtrl BM67 cells ) using data from polysome - associated mRNA ( orange ) , cytosolic mRNA ( purple ) or from analysis of changes in translational efficiency ( i . e . changes in polysome - associated mRNA not paralleled by changes in cytosolic mRNA ) leading to altered protein levels ( red ; a higher density of low p - values indicates a higher frequency of changes in gene expression ) . Scatterplot of polysome - associated mRNA vs . cytosolic mRNA log2 fold - changes ( shERα vs . shCtrl ) . Areas of the plot are colored according to the density of data points ( genes ; dark blue corresponds to areas with many genesSame plot as in ( B ) but including a blue density of p - values from the analysis of translational offsetting . A scatter plot similar to ( C ) but where genes are colored according to their mode of regulation derived from anota2seq analysis . A relaxed threshold ( p < 0 . 05 ) was used to identify a set of transcripts regulated via translation , which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting ( FDR < 0 . 1 ) . Confidence ellipses ( level 0 . 7 ) are overlaid for each mode of regulation . A bar graph indicates the number of mRNAs regulated via each mode .

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

Figure 1Expression of ERα in control (shCtrl) and knockdown (shERα) BM67 cells was determined by western blotting (β-actin served as a loading control). Gene expression was determined using polysome-profiling which quantifies both total mRNA and efficiently translated polysome-associated mRNA (n = 3).Distributions (quantified by kernel density estimation) of p-values for differential expression (shERα vs. shCtrl BM67 cells) using data from polysome-associated mRNA (orange), cytosolic mRNA (purple) or from analysis of changes in translational efficiency (i.e. changes in polysome-associated mRNA not paralleled by changes in cytosolic mRNA) leading to altered protein levels (red; a higher density of low p-values indicates a higher frequency of changes in gene expression).Scatterplot of polysome-associated mRNA vs. cytosolic mRNA log2 fold-changes (shERα vs. shCtrl). Areas of the plot are colored according to the density of data points (genes; dark blue corresponds to areas with many genesSame plot as in (B) but including a blue density of p-values from the analysis of translational offsetting.A scatter plot similar to (C) but where genes are colored according to their mode of regulation derived from anota2seq analysis. A relaxed threshold (p<0.05) was used to identify a set of transcripts regulated via translation, which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting (FDR<0.1). Confidence ellipses (level 0.7) are overlaid for each mode of regulation. A bar graph indicates the number of mRNAs regulated via each mode.


Figure 2Targets from each mode of regulation ( cf . Fig 1E ) were selected for validation by Nanostring . Shown is a scatterplot of Nanostring quantification of polysome - associated mRNA vs . cytosolic mRNA log2 fold - change ( shERα vs . shCtrl ; n = 3 ) . Genes are colored according to their identified mode of regulation ( i . e . from Fig 1E ) . Confidence ellipses ( based on Nanostring data , level 0 . 7 ) are overlaid for each mode of regulation . Selected genes from each mode of regulation are indicated . Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications . β - actin served as loading control . Graphs represent mean ± standard deviation ( n = 3 - 5 as indicated ) . Protein expression between groups were compared using two - sided paired Student ' s t tests . Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg / mL cycloheximide as indicated . Graphs represent mean ± standard deviation ( n = 5 ) .

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

Figure 2Targets from each mode of regulation (cf. Fig 1E) were selected for validation by Nanostring. Shown is a scatterplot of Nanostring quantification of polysome-associated mRNA vs. cytosolic mRNA log2 fold-change (shERα vs. shCtrl; n = 3). Genes are colored according to their identified mode of regulation (i.e. from Fig 1E). Confidence ellipses (based on Nanostring data, level 0.7) are overlaid for each mode of regulation. Selected genes from each mode of regulation are indicated.Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications. β-actin served as loading control. Graphs represent mean ± standard deviation (n = 3-5 as indicated). Protein expression between groups were compared using two-sided paired Student's t tests.Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).


Figure 3nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells . The number of detected transcription start sites ( peaks ) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries . Boxplots for offset and non - offset mRNAs comparing 5 ' UTR weighted lengths ( log2 scale ) , GC content ( % ) , free energy ( log10 scale , kcal / mole ) . In boxplots , the solid horizontal middle line is the median ; the lower and upper box limits correspond to the first and third quartiles ; the whiskers extend from the box limits to the most extreme value but not further than + / - 1 . 5 x the inter - quartile range ( IQR ) from the box limits . The notches extend to 1 . 58 x the IQR / √ n where n is the number of data points . Data beyond the end of the whiskers are plotted as points . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to assess differences between translationally offset and non - offset mRNAs . Number of transcripts harboring at least one ( dark gray ) or no ( light gray ) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one - sided Fisher ' s exact tests . A global Bonferroni adjustment was applied on p - values . Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells ; the numbers of transcripts in each group are indicated in the figure .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries.Boxplots for offset and non-offset mRNAs comparing 5' UTR weighted lengths (log2 scale), GC content (%), free energy (log10 scale, kcal/mole). In boxplots, the solid horizontal middle line is the median; the lower and upper box limits correspond to the first and third quartiles; the whiskers extend from the box limits to the most extreme value but not further than +/- 1.5 x the inter-quartile range (IQR) from the box limits. The notches extend to 1.58 x the IQR / √n where n is the number of data points. Data beyond the end of the whiskers are plotted as points. Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between translationally offset and non-offset mRNAs. Number of transcripts harboring at least one (dark gray) or no (light gray) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one-sided Fisher's exact tests. A global Bonferroni adjustment was applied on p-values. Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells; the numbers of transcripts in each group are indicated in the figure.


Figure 4Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq . Down - and up - regulated miRNAs ( FDR < 0 . 2 and fold - change > 1 . 25 ) are colored in blue and red , respectively . Validation of miRNA expression using qPCR . Normalized expression ( mean ± standard deviation ; n = 3 ) ( B ) and expression fold change ( shERα vs . shCtrl ) obtained from small RNAseq vs . qPCR ( C ) . Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells ( mean ± standard deviation ; n = 3 ) . Assessment of the relationship between presence of miRNA target sites and translational offsetting . ( E ) For visualization , mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome - associated mRNA obtain an angle of ~ 45 degrees , whereas offset mRNAs are assigned an angle of ~ 0 degrees . ( F ) The distributions ( quantified by kernel density estimation ) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion , which were also targets of each suppressed miRNAs separately ( blue lines ) ; and the set of background transcripts ( mRNAs induced upon ERα depletion but not targets of suppressed miRNAs ; black ) . ( G ) For such subsets of miRNA targets ( i . e . from F ) , the relative number of offset and non - offset mRNAs ( as determined by anota2seq analysis ) was also compared to the background ( same as in F ) using Fisher ' s exact test ; and the resulting adjusted p - values ( FDRs ; across all assessed miRNAs ) were visualized as a histogram . ( H - I ) A similar assessment as in ( F - G ) but for suppressed mRNAs and induced miRNAs . ( J ) A similar assessment as in ( H ) but for randomly selected groups of 10 miRNAs ( gray lines ) compared to background ( black ) and targets of all induced miRNA ( red ; i . e . from H ) .

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

Figure 4Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq. Down- and up-regulated miRNAs (FDR<0.2 and fold-change > 1.25) are colored in blue and red, respectively.Validation of miRNA expression using qPCR. Normalized expression (mean ± standard deviation; n = 3) (B) and expression fold change (shERα vs. shCtrl) obtained from small RNAseq vs. qPCR (C).Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells (mean ± standard deviation; n = 3).Assessment of the relationship between presence of miRNA target sites and translational offsetting. (E) For visualization, mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome-associated mRNA obtain an angle of ~45 degrees, whereas offset mRNAs are assigned an angle of ~0 degrees. (F) The distributions (quantified by kernel density estimation) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion, which were also targets of each suppressed miRNAs separately (blue lines); and the set of background transcripts (mRNAs induced upon ERα depletion but not targets of suppressed miRNAs; black). (G) For such subsets of miRNA targets (i.e. from F), the relative number of offset and non-offset mRNAs (as determined by anota2seq analysis) was also compared to the background (same as in F) using Fisher's exact test; and the resulting adjusted p-values (FDRs; across all assessed miRNAs) were visualized as a histogram. (H-I) A similar assessment as in (F-G) but for suppressed mRNAs and induced miRNAs. (J) A similar assessment as in (H) but for randomly selected groups of 10 miRNAs (gray lines) compared to background (black) and targets of all induced miRNA (red; i.e. from H).


Figure 5For each codon , the average frequency ( per thousand ) is compared between transcripts induced but offset vs non - offset ( i . e . abundance mode of regulation ) upon ERα depletion in BM67 cells . Codons for the same amino acid are connected by a gray line . Heatmap of standardized residuals from a chi - squared contingency table test . Shown in red and blue are cells with counts significantly higher and lower , respectively , than expected counts under the null hypothesis ( i . e . independence between mode for regulation of gene expression and codon composition ) . A correspondence analysis for average codon frequency among transcripts annotated to each GO term . Each gray dot corresponds to one GO term . The gene expression modes observed upon ERα depletion ( Fig 1E ) were then projected into the same dimensions based on the average codon frequencies of included transcripts . A correspondence analysis for codon frequency in each regulated mRNA ( from Fig 1E ) . Each dot represents one mRNA and is colored according to its mode of regulation . Codons identified as over - represented among translationally offset mRNAs are also indicated . Unsupervised clustering of gene level codon usage normalized by amino acid counts . All regulated mRNAs ( from Fig 1E ) are shown in rows and all codons in columns . Codons identified as over - represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue , respectively .

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

Figure 5For each codon, the average frequency (per thousand) is compared between transcripts induced but offset vs non-offset (i.e. abundance mode of regulation) upon ERα depletion in BM67 cells. Codons for the same amino acid are connected by a gray line. Heatmap of standardized residuals from a chi-squared contingency table test. Shown in red and blue are cells with counts significantly higher and lower, respectively, than expected counts under the null hypothesis (i.e. independence between mode for regulation of gene expression and codon composition). A correspondence analysis for average codon frequency among transcripts annotated to each GO term. Each gray dot corresponds to one GO term. The gene expression modes observed upon ERα depletion (Fig 1E) were then projected into the same dimensions based on the average codon frequencies of included transcripts. A correspondence analysis for codon frequency in each regulated mRNA (from Fig 1E). Each dot represents one mRNA and is colored according to its mode of regulation. Codons identified as over-represented among translationally offset mRNAs are also indicated. Unsupervised clustering of gene level codon usage normalized by amino acid counts. All regulated mRNAs (from Fig 1E) are shown in rows and all codons in columns. Codons identified as over-represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue, respectively.


Figure 6Immunoblotting of BM67 cell extract for ELP3 after stable shRNA - mediated ERα knockdown in BM67 cells . Graphs represent mean ± standard deviation of n = 3 . qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 5 . qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 4 . Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector , Elp3 - null BM67 cells and Elp3 - null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site . Graphs represent mean ± standard deviation of n = 3 - 4 .

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

Figure 6Immunoblotting of BM67 cell extract for ELP3 after stable shRNA-mediated ERα knockdown in BM67 cells. Graphs represent mean ± standard deviation of n=3.qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5.qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=4.Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector, Elp3-null BM67 cells and Elp3-null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site. Graphs represent mean ± standard deviation of n=3-4.