( C ) ChIP performed with U2 - OS cells irradiated ( 20 Gy ) 1 . 5h or 7 days prior to assay , or left untreated ( UT ) . Normalized relative enrichment of NFKBIA is shown ( relative to input and two non - recruiting reference regions ) . Left panel : p65 antibody . n = 5 biological replicates with 3 technical repeats per biological replicate . Right : Antibody against p65 pSer468 . n = 2 biological replicates with three technical repeats per biological replicate . Statistical significance was determined by ANOVA with Tukey multiple comparisons test . SD shown . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 .

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(C) ChIP performed with U2-OS cells irradiated (20 Gy) 1.5h or 7 days prior to assay, or left untreated (UT). Normalized relative enrichment of NFKBIA is shown (relative to input and two non-recruiting reference regions). Left panel: p65 antibody. n = 5 biological replicates with 3 technical repeats per biological replicate. Right: Antibody against p65 pSer468. n = 2 biological replicates with three technical repeats per biological replicate. Statistical significance was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001


( D ) Kinase proteome array was performed with nuclear fractions of U2 - OS cells that were either left untreated ( UT ) or irradiated ( 20 Gy ) at time points indicated prior to harvest . Kinase binding to membrane was quantitated ( n = 2 ) and only significant results ( p < 0 . 05 ) are shown . Significance was determined by ANOVA with Tukey multiple comparisons test .

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(D) Kinase proteome array was performed with nuclear fractions of U2-OS cells that were either left untreated (UT) or irradiated (20 Gy) at time points indicated prior to harvest. Kinase binding to membrane was quantitated (n = 2) and only significant results (p < 0.05) are shown. Significance was determined by ANOVA with Tukey multiple comparisons test.


( E ) U2 - OS cells left untreated ( ut ) or irradiated ( 20 Gy ) 7 days before harvesting , as indicated . Two days prior to harvest cells were transfected with siRNA against GSK3β or scrambled control ( Scr ) . Whole cell lysates were analyzed by SDS - PAGE and western blotting . A representative example from n = 3 experiments is shown .

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(E) U2-OS cells left untreated (ut) or irradiated (20 Gy) 7 days before harvesting, as indicated. Two days prior to harvest cells were transfected with siRNA against GSK3β or scrambled control (Scr). Whole cell lysates were analyzed by SDS-PAGE and western blotting. A representative example from n = 3 experiments is shown.


( F ) U2 - OS cells were left untreated or exposed overnight to 10 mM LiCl , with or without prior irradiation , as indicated , and analyzed as in ( E ) . Representative data from n = 3 experiments are shown .

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(F) U2-OS cells were left untreated or exposed overnight to 10 mM LiCl, with or without prior irradiation, as indicated, and analyzed as in (E). Representative data from n = 3 experiments are shown.


( G ) RT - qPCR of U2 - OS cells irradiated ( 20 Gy ) either 1 . 5h or 7 days prior to harvest . CHIR denotes treatment with GSK3 inhibitor CHIR - 99021 ( 10nM overnight ) . DMSO overnight treatment was used as control . Quantitation was performed and statistical significance obtained from n = 3 replicates , using ANOVA with Tukey multiple comparisons test . SD shown . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 .

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(G) RT-qPCR of U2-OS cells irradiated (20 Gy) either 1.5h or 7 days prior to harvest. CHIR denotes treatment with GSK3 inhibitor CHIR-99021 (10nM overnight). DMSO overnight treatment was used as control. Quantitation was performed and statistical significance obtained from n = 3 replicates, using ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.


( B ) SDS - PAGE western blot of whole cell lysates of U2 - OS cells irradiated ( 20 Gy ) at time points indicated prior to harvest . ut , untreated . LDHA . Representative gel shown from n = 4 biological replicates .

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(B) SDS-PAGE western blot of whole cell lysates of U2-OS cells irradiated (20 Gy) at time points indicated prior to harvest. ut, untreated. LDHA. Representative gel shown from n = 4 biological replicates.


( C ) U2 - OS cells transfected with TRAF6 siRNA or scrambled control siRNA and TRAF6 and IKKβ levels analyzed by western blotting ( top panels ) . NF - κB activity was determined by EMSA in untreated cells ( ut ) and after IR at indicated time points ( lower panel ) . ns , unspecific band . Representative gels are shown from n = 2 biological replicates .

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(C) U2-OS cells transfected with TRAF6 siRNA or scrambled control siRNA and TRAF6 and IKKβ levels analyzed by western blotting (top panels). NF-κB activity was determined by EMSA in untreated cells (ut) and after IR at indicated time points (lower panel). ns, unspecific band. Representative gels are shown from n = 2 biological replicates.


( D ) U2 - OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα , NIK , IL - 1α and PARP1 . Treatment with proteasomal inhibitor MG132 ( 10 μM ) started 4 hours prior to harvest . NIK serves as positive control for efficient proteasome inhibition . Quantitation from n = 4 biological replicates and SD is shown for IκBα ( right panel ) . Brown - Forsynthe ANOVA test : p = 0 . 0005 .

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(D) U2-OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα, NIK, IL-1α and PARP1. Treatment with proteasomal inhibitor MG132 (10 μM) started 4 hours prior to harvest. NIK serves as positive control for efficient proteasome inhibition. Quantitation from n = 4 biological replicates and SD is shown for IκBα (right panel). Brown-Forsynthe ANOVA test: p = 0.0005.


( E ) U2 - OS CRISPR IKBKB knockout and control cell lines were irradiated ( 20 Gy ) and harvested at indicated time points . NF - κB activity was analyzed by EMSA ( lower panel ) . ns indicates an non - specific band . Upper panel , western blot analysis of actin and IKKβ . Representative gels shown from n = 3 replicates .

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(E) U2-OS CRISPR IKBKB knockout and control cell lines were irradiated (20 Gy) and harvested at indicated time points. NF-κB activity was analyzed by EMSA (lower panel). ns indicates an non-specific band. Upper panel, western blot analysis of actin and IKKβ. Representative gels shown from n = 3 replicates.


( F ) Irradiated U2 - O2 control and IKBKB knockout cells ( as above ) were analyzed by RT - qPCR at the time points indicated for expression of NF - κB target genes identified by RNA - Seq ( Dataset EV1 ) . Significantly regulated targets , cutoff set at p = 0 . 05 ( as determined by ANOVA with Tukey multiple comparison ) , are shown .

(F) Irradiated U2-O2 control and IKBKB knockout cells (as above) were analyzed by RT-qPCR at the time points indicated for expression of NF-κB target genes identified by RNA-Seq (Dataset EV1). Significantly regulated targets, cutoff set at p = 0.05 (as determined by ANOVA with Tukey multiple comparison), are shown.


A - F ( A - C ) Exercise performance on a treadmill apparatus using an intense exercise ( incremental speed paradigm ) that relies solely on anaerobic metabolism . ( D - F ) Exercise performance on a treadmill apparatus using a moderate exercise ( constant sub - maximal speed ) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise . ( A , D ) Kaplan - Meier curves representing values corresponding to the percentage of mice still running at a given time point . ( B , E ) Median values of the time that mice ran are represented in the min to max graphical representation ; 15 . 5 min for wild - type ( WT ) and 14 . 33 min for SOD1G86R in ( B ) ; 59 . 33 min for WT and 65 . 43 min for SOD1G86R in ( E ) . ( C , F ) Mean distance run ± SEM by WT and SOD1G86R mice in ( C ) and by WT and SOD1G86R mice in ( F ) .

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A-F (A-C) Exercise performance on a treadmill apparatus using an intense exercise (incremental speed paradigm) that relies solely on anaerobic metabolism. (D-F) Exercise performance on a treadmill apparatus using a moderate exercise (constant sub-maximal speed) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise. (A, D) Kaplan-Meier curves representing values corresponding to the percentage of mice still running at a given time point. (B, E) Median values of the time that mice ran are represented in the min to max graphical representation; 15.5 min for wild-type (WT) and 14.33 min for SOD1G86R in (B); 59.33 min for WT and 65.43 min for SOD1G86R in (E). (C, F) Mean distance run ± SEM by WT and SOD1G86R mice in (C) and by WT and SOD1G86R mice in (F).


Grip test was performed to measure mousemuscle strength at different time points during disease progression . Graph represents the mean of percentage from initial force measured at 11 weeks ± SEM . At 13 weeks , * P = 0 . 03 ; at 14 weeks , P = 0 . 06 ; at 15 weeks , * P = 0 . 04 ( n = 8 for WT and SOD1G86R , respectively , at 11 - 15 weeks , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Grip test was performed to measure mousemuscle strength at different time points during disease progression. Graph represents the mean of percentage from initial force measured at 11 weeks ± SEM. At 13 weeks, *P = 0.03; at 14 weeks, P = 0.06; at 15 weeks, *P = 0.04 (n = 8 for WT and SOD1G86R, respectively, at 11-15 weeks, two-way ANOVA followed by Fisher's LSD post hoc test).


Electromyography ( EMG ) recordings were performed weekly during disease development , starting from 9 weeks of age . Spontaneous electrical activity was considered as positive EMG only for peak - to - peak amplitudes > 50 μV . Left : The percentage of mice presenting with positive EMG in each age group are represented . Right : Representative examples of negative ( EMG − ) and positive ( EMG + ) electromyography profiles ( n = 10 for WT and SOD1G86R , respectively , at 9 - 13 weeks and n = 8 and 7 for WT and SOD1G86R , respectively , at 15 weeks ) .

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Electromyography (EMG) recordings were performed weekly during disease development, starting from 9 weeks of age. Spontaneous electrical activity was considered as positive EMG only for peak-to-peak amplitudes > 50 μV. Left: The percentage of mice presenting with positive EMG in each age group are represented. Right: Representative examples of negative (EMG−) and positive (EMG+) electromyography profiles (n = 10 for WT and SOD1G86R, respectively, at 9-13 weeks and n = 8 and 7 for WT and SOD1G86R, respectively, at 15 weeks).


Relative mRNA levels of denervation markers AChR ( subunits α and γ ) and MuSK were evaluated by qPCR at the indicated ages ( 65 and 105 days ) in tibialis anterior ( upper panels ) and soleus ( lower panels ) of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT ( n > 5 ) . * * * P - values versus WT : < 0 . 0001 for AChRα , AChRγ , and MuSK in tibialis anterior ; and * * P - values versus WT : 0 . 001 for AChRα , and * * * P - values versus WT : 0 . 0007 for AChRγ , 0 . 0002 for MuSK in soleus ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 and 6 for WT and SOD1G86R , respectively , at 105 days ; two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Relative mRNA levels of denervation markers AChR (subunits α and γ) and MuSK were evaluated by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT (n > 5). ***P-values versus WT: < 0.0001 for AChRα, AChRγ, and MuSK in tibialis anterior; and **P-values versus WT: 0.001 for AChRα, and ***P-values versus WT: 0.0007 for AChRγ, 0.0002 for MuSK in soleus (n=7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days; two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of muscle atrophy markers MuRF1 and Atg - 1 were measured by qPCR at the indicated ages ( 65 and 105 days ) in tibialis anterior ( upper panels ) and soleus ( lower panels ) . Graphs represent mean fold change ± SEM from age - matched WT . * * * P - values versus WT : < 0 . 0001 for MuRF1 and Atg - 1 in tibialis anterior ; and * * P - values versus WT : 0 . 007 for MuRF1 and P = 0 . 006 for Atg - 1 in soleus ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days , n = 5 and 6 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Relative mRNA levels of muscle atrophy markers MuRF1 and Atg-1 were measured by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels). Graphs represent mean fold change ± SEM from age-matched WT. ***P-values versus WT: < 0.0001 for MuRF1 and Atg-1 in tibialis anterior; and **P-values versus WT: 0.007 for MuRF1 and P = 0.006 for Atg-1 in soleus (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days, n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Enzymatic activity of phosphofructokinase in whole tibialis anterior muscle cytosolic homogenates is expressed as mean fold change ± SEM from age - matched WT at 65 days of age * P = 0 . 016 ( WT n = 6 , SOD1G86Rn = 7 ) and 105 days of age * * * P < 0 . 0001 ( n = 5 / genotype ) , two - way ANOVA followed by Fisher ' s LSD post hoc test . Data shown are representative of two independent experiments having similar results .

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Enzymatic activity of phosphofructokinase in whole tibialis anterior muscle cytosolic homogenates is expressed as mean fold change ± SEM from age-matched WT at 65 days of age *P = 0.016 (WT n = 6, SOD1G86Rn = 7) and 105 days of age ***P < 0.0001 (n = 5/genotype), two-way ANOVA followed by Fisher's LSD post hoc test. Data shown are representative of two independent experiments having similar results.


Pyruvate was measured in whole tibialis anterior muscle tissue homogenates . The mean fold change ± SEM compared to age - matched WT are represented with * P = 0 . 019 at 65 days of age ( n = 5 / genotype ) and * P = 0 . 041 at 105 days of age ( n = 4 / genotype ) , two - way ANOVA followed by Fisher ' s LSD post hoc test . Data shown are representative of two independent experiments having similar results .

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Pyruvate was measured in whole tibialis anterior muscle tissue homogenates. The mean fold change ± SEM compared to age-matched WT are represented with *P = 0.019 at 65 days of age (n = 5/genotype) and *P = 0.041 at 105 days of age (n = 4/genotype), two-way ANOVA followed by Fisher's LSD post hoc test. Data shown are representative of two independent experiments having similar results.


Left : Representative Western blot showing glycogen synthase and phosphorylated glycogen synthase levels . Right : Quantification represents the mean ratio of optical densities of glycogen synthase / P - glycogen synthase ± SEM . At 65 days of age n = 6 / genotype , * P = 0 . 0162 , and at 105 days of age n = 5 / genotype , * P = 0 . 0163 , two - way ANOVA followed by Fisher ' s LSD post hoc test .

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Left: Representative Western blot showing glycogen synthase and phosphorylated glycogen synthase levels. Right: Quantification represents the mean ratio of optical densities of glycogen synthase/P-glycogen synthase ± SEM. At 65 days of age n = 6/genotype, *P = 0.0162, and at 105 days of age n = 5/genotype, *P = 0.0163, two-way ANOVA followed by Fisher's LSD post hoc test.


Left : Representative microphotographs of PAS staining from WT and SOD1G86R tibialis anterior cross - sections at 65 and 105 days of age showing glycogen - negative ( light pink ) and glycogen - positive ( dark pink ) fibers . Scale bar : 200 μm . Right : Quantification of glycogen - negative ( − ) and glycogen - positive ( + ) fibers at 65 and 105 days of age . For ( − ) fibers : * * P - values versus WT : 0 . 0026 at 65 days and 0 . 0074 at 105 days , for ( + ) fibers : # # P - values versus WT : 0 . 0025 at 65 days and 0 . 0073 at 105 days ( n = 5 / genotype at 65 days and n = 4 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Left: Representative microphotographs of PAS staining from WT and SOD1G86R tibialis anterior cross-sections at 65 and 105 days of age showing glycogen-negative (light pink) and glycogen-positive (dark pink) fibers. Scale bar: 200 μm. Right: Quantification of glycogen-negative (−) and glycogen-positive (+) fibers at 65 and 105 days of age. For (−) fibers: **P-values versus WT: 0.0026 at 65 days and 0.0074 at 105 days, for (+) fibers: ##P-values versus WT: 0.0025 at 65 days and 0.0073 at 105 days (n = 5/genotype at 65 days and n = 4/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of Pdk4 and Pdk2 evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± from age - matched WT . Pdk4 : * * P - values versus WT : 0 . 014 at 65 days and 0 . 018 at 105 days ; Pdk2 : * * * P - value versus WT < 0 . 0001 at 105 days . n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test .

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Relative mRNA levels of Pdk4 and Pdk2 evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± from age-matched WT. Pdk4: **P-values versus WT: 0.014 at 65 days and 0.018 at 105 days; Pdk2: ***P-value versus WT < 0.0001 at 105 days. n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test.


Relative mRNA levels of genes involved in lipid handling evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . P - values versus WT : Lpl * * P = 0 . 0009 , Cd36 * P = 0 . 051 , Acsf2 * * * P < 0 . 0001 at 65 days , * * P = 0 . 0059 at 105 days and Cpt - 1β * * P = 0 . 01 at 65 days and * P = 0 . 02 at 105 days ( n = 6 and 7 for WT and SOD1G86R , respectively , at 65 days ; n = 10 and 8 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Relative mRNA levels of genes involved in lipid handling evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Lpl **P = 0.0009, Cd36 *P = 0.051, Acsf2 ***P < 0.0001 at 65 days, **P = 0.0059 at 105 days and Cpt-1β **P = 0.01 at 65 days and *P = 0.02 at 105 days (n = 6 and 7 for WT and SOD1G86R, respectively, at 65 days; n = 10 and 8 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative levels of ( left ) ATP and ( right ) NADH and NAD + measured in total tibialis anterior homogenates of WT and SOD1G86R mice . Left : Graphs represent mean fold change ± SEM in ATP from age - matched WT . * * P = 0 . 002 ( n = 9 and 12 for WT and SOD1G86R , respectively , at 65 days ; n = 10 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) . Right : The amounts of NADH and NAD + relative to protein content in whole tibialis anterior homogenates are represented as mean ± SEM . * P = 0 . 027 and * * P = 0 . 001 ( n = 5 , Student ' s t - test ) .

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Relative levels of (left) ATP and (right) NADH and NAD+ measured in total tibialis anterior homogenates of WT and SOD1G86R mice. Left: Graphs represent mean fold change ± SEM in ATP from age-matched WT. **P = 0.002 (n = 9 and 12 for WT and SOD1G86R, respectively, at 65 days; n = 10/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test). Right: The amounts of NADH and NAD+ relative to protein content in whole tibialis anterior homogenates are represented as mean ± SEM. *P = 0.027 and **P = 0.001 (n = 5, Student's t-test).


A - D Relative mRNA levels of ( A ) Pparß / δ , ( B ) Foxo1 , ( C ) citrate synthase , and ( D ) Pgc - 1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . P - values versus WT : Pparβ / δ * * P = 0 . 0016 at 65 days ; Foxo1 * * * P = 0 . 0001 at 105 days , citrate synthase * * * P < 0 . 0001 at 105 days , Pgc - 1α * P = 0 . 0104 at 105 days ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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A-D Relative mRNA levels of (A) Pparß/δ, (B) Foxo1, (C) citrate synthase, and (D) Pgc-1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Pparβ/δ **P = 0.0016 at 65 days; Foxo1 ***P = 0.0001 at 105 days, citrate synthase ***P < 0.0001 at 105 days, Pgc-1α *P = 0.0104 at 105 days (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


E Representative Western blot of PGC - 1α and respective actin that was used for normalization . PGC - 1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age - matched WT . * * P - values versus WT : 0 . 02 ( n = 6 / genotype at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

E Representative Western blot of PGC-1α and respective actin that was used for normalization. PGC-1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age-matched WT. **P-values versus WT: 0.02 (n = 6/genotype at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


F Relative mRNA levels of ERRα , Mfn2 , and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . P - values versus WT : ERRα * * * P < 0 . 0001 , Mfn2 * * P = 0 . 002 and Nrf1 * * P = 0 . 009 ( n = 6 - 7 and 7 - 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 and 6 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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F Relative mRNA levels of ERRα, Mfn2, and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: ERRα ***P < 0.0001, Mfn2 **P = 0.002 and Nrf1 **P = 0.009 (n = 6-7 and 7-8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Mitochondrial DNA ( mtDNA ) was quantified using qPCR . Relative mtDNA levels are expressed as the ratio between mitochondrial - encoded gene Cox1 and the nuclear - encoded gene cyclophilin A . Graphs represent mean fold change ± SEM from age - matched WT for tibialis anterior ( left panel ) and soleus ( right panel ) of WT and SOD1G86R mice . * * * P < 0 . 0001 ( n = 5 and 4 for WT and SOD1G86R , respectively , at 65 days ; n = 6 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Mitochondrial DNA (mtDNA) was quantified using qPCR. Relative mtDNA levels are expressed as the ratio between mitochondrial-encoded gene Cox1 and the nuclear-encoded gene cyclophilin A. Graphs represent mean fold change ± SEM from age-matched WT for tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. ***P < 0.0001 (n = 5 and 4 for WT and SOD1G86R, respectively, at 65 days; n = 6/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of Gpx1 were measured by qPCR at the indicated ages in tibialis anterior ( left panel ) and soleus ( right panel ) of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . * * * P < 0 . 0001 ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 9 and 8 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Relative mRNA levels of Gpx1 were measured by qPCR at the indicated ages in tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. ***P < 0.0001 (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 9 and 8 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


A - I Relative mRNA levels of ( A ) Pdk4 , ( B ) Pparβ / δ , ( C ) Foxo1 , ( D ) Pfk1 , ( E ) Acsf2 , ( F ) citrate synthase , ( G ) PGC - 1α , ( H ) Mfn2 , and ( I ) Gpx1 were evaluated by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . P - values versus WT : Pdk4 * * * P = 0 . 002 and # # P = 0 . 0039 , Pparβ / δ # P = 0 . 0178 , Foxo1 * * P = 0 . 0033 and # # P = 0 . 0038 , Pfk1 * * * P = 0 . 0002 and # # P = 0 . 0080 , Acsf2 * P = 0 . 0437 , citrate synthase # # # P = 0 . 0004 , Pgc - 1α * * P = 0 . 0084 and # # # P = 0 . 0009 , Mfn2 * P = 0 . 0272 , $ P = 0 . 0145 and # # # P = 0 . 0002 and Gpx1 * * * P < 0 . 0001 and # # # P < 0 . 0001 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

A-I Relative mRNA levels of (A) Pdk4, (B) Pparβ/δ, (C) Foxo1, (D) Pfk1, (E) Acsf2, (F) citrate synthase, (G) PGC-1α, (H) Mfn2, and (I) Gpx1 were evaluated by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. P-values versus WT: Pdk4 ***P = 0.002 and ##P = 0.0039, Pparβ/δ #P = 0.0178, Foxo1 **P = 0.0033 and ##P = 0.0038, Pfk1 ***P = 0.0002 and ##P = 0.0080, Acsf2 *P = 0.0437, citrate synthase ###P = 0.0004, Pgc-1α **P = 0.0084 and ###P = 0.0009, Mfn2 *P = 0.0272, $P = 0.0145 and ###P = 0.0002 and Gpx1 ***P < 0.0001 and ###P < 0.0001 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Grip strength is represented as mean of percent from T0 for each experimental group ± SEM . # # P = 0 . 0045 and * * * P = 0 . 0003 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Grip strength is represented as mean of percent from T0 for each experimental group ± SEM. ##P = 0.0045 and ***P = 0.0003 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of muscular atrophy markers Murf1 and Atg - 1 were measured by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . * * P = 0 . 0079 and * * * P = 0 . 0018 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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Relative mRNA levels of muscular atrophy markers Murf1 and Atg-1 were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.0079 and ***P = 0.0018 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of denervation markers AChRα , AChRγ , and MuSK were measured by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . * * P = 0 . 015 for AChRα , # P = 0 . 028 and * * P = 0 . 074 for AChRγ , # P = 0 . 042 and * * P = 0 . 0029 for MuSK ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Relative mRNA levels of denervation markers AChRα, AChRγ, and MuSK were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.015 for AChRα, #P = 0.028 and **P = 0.074 for AChRγ, #P = 0.042 and **P = 0.0029 for MuSK (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


( A ) Internalization of apoptotic or necrotic GFP - expressing RAW264 . 7 cells . ( B ) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres ( B ) in primary M2 ( IL - 4 ) and M0 macrophages . Cytochalasin D ( Cyto ) ( 6 µM ) was used as an inhibitor of phagocytosis , 1 h before phagocytosis . Data are shown as means of relative fluorescence units ( RFU ) ± standard error of the mean ( SEM ) .

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(A) Internalization of apoptotic or necrotic GFP-expressing RAW264.7 cells. (B) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 µM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM).


( C - E ) Real - time fluorescence assays for intraphagosomal proteolysis ( C ) , acidification ( D ) and lipolysis ( E ) show substantially increased proteolysis , acidification and lipolysis in the phagosomes of M2 ( IL - 4 ) macrophages . The kinetics of proteolysis , acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence . Beads were added to macrophages at 0 min . Shaded area in ( C ) & ( D ) represents SEM . ( E ) is a representative of three independent experiments . Leupeptin ( 100nM ) and Bafilomycin ( 100nM ) treatment serve as negative controls in ( C ) and ( D ) , respectively .

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(C-E) Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2(IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. Shaded area in (C) & (D) represents SEM. (E) is a representative of three independent experiments. Leupeptin (100nM) and Bafilomycin (100nM) treatment serve as negative controls in (C) and (D), respectively.


( B ) Volcano plot of the phagosome proteome data . 1 , 766 proteins were quantified of which 121 proteins were significantly up - regulated and 62 proteins were down - regulated in M2 ( IL - 4 ) macrophages . Selected proteins are indicated .

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(B) Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2(IL-4) macrophages. Selected proteins are indicated.


( C ) Heatmap of proteomics data shows high reproducibility between biological replicates . Selected proteins are highlighted .

(C) Heatmap of proteomics data shows high reproducibility between biological replicates. Selected proteins are highlighted.


( A ) Immunoblot ( IB ) analysis showing the recruitment of TAK1 , MKK7 , JNK and TAB1 / TAB2 to the phagosome in M2 ( IL - 4 ) macrophages , while MKK4 is not recruited . Rab7a , a phagosomal marker , was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates . The white line in the JNK blot shows other bands were cut out .

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(A) Immunoblot (IB) analysis showing the recruitment of TAK1, MKK7, JNK and TAB1/TAB2 to the phagosome in M2(IL-4) macrophages, while MKK4 is not recruited. Rab7a, a phagosomal marker, was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates. The white line in the JNK blot shows other bands were cut out.


( B ) Quantitation of three independent IB experiments by imageJ of non - saturated blots for TAK1 , MKK7 , TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2 ( IL - 4 ) macrophages . Error bars represent SEM . * p < 0 . 01 , * * p < 0 . 001 ( Student ' s T - test )

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(B) Quantitation of three independent IB experiments by imageJ of non-saturated blots for TAK1, MKK7, TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2(IL-4) macrophages. Error bars represent SEM. *p<0.01, **p<0.001 (Student's T-test)


( C ) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2 ( IL - 4 ) macrophages compared to M0 macrophages .

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(C) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2(IL-4) macrophages compared to M0 macrophages.


Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1 , TAB2 , TAK1 and MKK7 to the phagosome of M2 ( IL - 4 ) macrophages , indicating a K63 - polyubiquitylation dependent translocation for these proteins .

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Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2(IL-4) macrophages, indicating a K63-polyubiquitylation dependent translocation for these proteins.


( B ) Selected ubiquitylated phagosomal proteins identified by TUBE - MS approach .

(B) Selected ubiquitylated phagosomal proteins identified by TUBE-MS approach.


( D ) Tab2 - TUBE pull - downs from phagosome extracts of M2 ( IL - 4 ) macrophages treated with the K63 specific deubiquitylase ( DUB ) AMSH - LP or the unspecific DUB USP2 .

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(D) Tab2-TUBE pull-downs from phagosome extracts of M2(IL-4) macrophages treated with the K63 specific deubiquitylase (DUB) AMSH-LP or the unspecific DUB USP2.


MSR1 immunoprecipitation from M2 ( IL - 4 ) macrophage phagosomes shows that TAB1 , TAB2 , TAK1 and MKK7 bind to polyubiquitylated MSR1 . TAK1 shows a specific pattern of post - translational modifications indicative of its activation .

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MSR1 immunoprecipitation from M2(IL-4) macrophage phagosomes shows that TAB1, TAB2, TAK1 and MKK7 bind to polyubiquitylated MSR1. TAK1 shows a specific pattern of post-translational modifications indicative of its activation.


( A ) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL .

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(A) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL.


( B ) Msr1 + / + ( WT ) and Msr1 - / - ( KO ) M0 macrophages and M2 ( IL - 4 ) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL ( 50 μg / ml , 30 min ) were analysed for the phosphorylated and the total forms of JNK1 / 2 and MSR1 . ITGAM ( CD11b ) serves as a loading control . Both fucoidan and oxLDL activate JNK in a MSR1 - dependent manner .

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

(B) Msr1+/+ (WT) and Msr1-/- (KO) M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1-dependent manner.


( C ) IL4 - activated MSR1 knock - out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg / ml Fucoidan for 1 hr . Mutation of the ubiquitylation site K27 abolishes MSR1 signalling .

(C) IL4-activated MSR1 knock-out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml Fucoidan for 1 hr. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling.


( D ) qPCR data of Tnfa , Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2 ( IL - 4 ) BMDMs shows an MSR1 - dependent increase of pro - inflammatory cytokines in response to MSR1 ligation by fucoidan .

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(D) qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2(IL-4) BMDMs shows an MSR1-dependent increase of pro-inflammatory cytokines in response to MSR1 ligation by fucoidan.


( E ) Inhibition of JNK by JNK - IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation , showing that it is JNK - dependent .

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(E) Inhibition of JNK by JNK-IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK-dependent.


( F ) Flow cytometry analysis of cell surface markers in WT and MSR1 - / - M0 macrophages and M2 ( IL - 4 ) macrophages untreated or stimulated with the MSR1 ligands fucoidan ( 50 μg / ml , 24 h ) . Data shows MSR1 - dependent increase of the early activation markers CD54 , CD69 and CD86 and a decrease of the M2 marker CD301b / Mgl2 . CD11b serves as a control .

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(F) Flow cytometry analysis of cell surface markers in WT and MSR1-/- M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data shows MSR1-dependent increase of the early activation markers CD54, CD69 and CD86 and a decrease of the M2 marker CD301b/Mgl2. CD11b serves as a control.


( A ) Immunohistochemistry analysis of MSR1 in patient ovarian , uterus and sarcoma cancers showing tumour - associated macrophages .

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(A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages.


( B ) Pull - downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour .

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(B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour.


E . The endouc and ela3l ( negative control ) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one - cell stage . Upper panels : observation of GFP signal expressed in huORFZ embryos at 72 hpf . Lower panels : magnification of pictures corresponding to the upper panels .

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E. The endouc and ela3l (negative control) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one-cell stage. Upper panels: observation of GFP signal expressed in huORFZ embryos at 72 hpf. Lower panels: magnification of pictures corresponding to the upper panels.


C - E . Western blot analysis of proteins , as indicated , in ( C ) zebrafish embryos , ( D ) HEK 293T cells and ( E ) HeLa cells . GAPDH and α - tubulin served as internal controls . Protein levels relative to each internal control are presented below each lane .

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C-E. Western blot analysis of proteins, as indicated, in (C) zebrafish embryos, (D) HEK 293T cells and (E) HeLa cells. GAPDH and α-tubulin served as internal controls. Protein levels relative to each internal control are presented below each lane.


D . Zebrafish embryos at one - cell stage were microinjected with plasmids as indicated , followed by extraction of embryonic proteins at the 96 - hpf stage . Protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants were detected using Western blot analysis . GAPDH served as an internal control . E . HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . The α - tubulin served as an internal control . Protein levels relative to each internal control are presented in each lane .

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

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated, followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.


D . Zebrafish embryos at one - cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96 - hpf stage . Protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants were detected using Western blot analysis . GAPDH served as an internal control . E . HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . Here , α - tubulin served as an internal control . Protein levels relative to each internal control were presented in each lane .

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot . Here, α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.


B . HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO , thapsigargin ( TH ) , ISRIB , or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . The α - tubulin served as an internal control . Protein levels relative to each internal control were presented at each lane .

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

B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane.


D , E . MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , GADD34 , and Endouc - Flag using Western blot . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane .

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

D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.


A . MEF wild - type ( WT ) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin ( TH ) for 4 hr followed by analysis of the protein level of p - PKR , total PKR , and Endouc - Flag . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane . The results obtained from the two trials are shown on the left and right .

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

A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.


B . MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p - eIF2α , total eIF2α , CHOP , and Endouc - Flag . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane . The results obtained from the two trials are shown on the left and right .

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

B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.


D , E . The cleavage activity of Endouc was studied using materials presented at each lane . EndoucK242A : Endouc mutant ; Extract : incubated in cell extracts ; and Buf : reaction buffer . RNA probe without adding reaction buffer served as a negative control . The predicted cleavage sites ( U ) were mutated to generate mutant huORFchop transcript ( unlabelled huORF - mt ) and labelled by biotin ( biotin - huORF - mt ) . Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe .

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

D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe.


A . HEK 293T cells were transfected with pCS2 vector ( black ) , pCS2 - Endouc ( red ) , and pCS2 - EndoucK242A ( green ) for 24 hr and subjected to either DMSO ( control ) or thapsigargin ( TH ; stress ) treatment for 1 hr . The lysates of transfected cells were applied to polysome profile analysis ( PPA ) . The sucrose gradient from top to bottom was shown from left to right , respectively . The sedimentation of 40S , 60S , 80S , and polysomes was indicated . Lysate from control , Endouc - expressing , and EndoucK242A - expressing cells were applied to PPA . The resulting fractions were TCA - precipitated , analysed by SDS - PAGE , and subjected to Western blot using antibodies against Flag and S6 .

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

A. HEK 293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 hr and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 hr. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysate from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analysed by SDS-PAGE, and subjected to Western blot using antibodies against Flag and S6.


B . The luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1 . These data suggested that the change of Fluc / Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity . The data were averaged from three independent experiments and presented as mean ± S . D . ( n = 3 ) . Student ' s t test was used to determine significant differences between each group ( * * : P < 0 . 005 ) .

B. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005).


D . The luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1 . These data suggested that the change of Fluc / Rluc ratio shown in Figures C was entirely dependent on the change of Fluc activity . The data were averaged from three independent experiments and presented as mean ± S . D . ( n = 3 ) . Student ' s t test was used to determine significant differences between each group ( * * * : P < 0 . 001 ) .

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

D. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures C was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (***: P <0.001).


A - F . Schematic representation of capped ( m7G - capped ) and A - capped ( non - functional capped ) monocistronic mRNAs with poly ( A ) - tail - encoded firefly luciferase ( Fluc ) and fused upstream with ( A ) c - myc IRES , ( B ) no insertion , ( C ) non - specific 105 - nt luc RNA ( Non105 ) , ( D ) huORFchop - 1 - 105 - nt , ( E ) huORFchop - 69 - 105 - nt , and ( F ) huORFchop - 81 - 105 - nt as shown above each bar graph . The RNA transfection assay of each transcript equipped with m7G - capped or A - capped monocistronic mRNA in HEK 293T cells was compared . To normalize the luc activities of capped and A - capped Fluc transcripts , the m7G - capped Renilla luc ( Rluc ) mRNA was used as an internal control . The luc activity of cells transfected with Cap - monocistronic mRNA in each graph was set as 100 % .

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

A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%.


M - O : Schematic describing the measurement of a cell ' s approach angle to the nearest point on the AER ( M ) . Merged , immunofluorescent images of WT ( top ) , slit3sq49 ( centre ) and s1pr2te273 ( below ) mesenchymal cells in sqet37Et background stained for EGFP ( green ) , Gamma tubulin ( red ) and DAPI ( blue ) . White lines run from the centre of nucleus to the nearest point on the AER , through the MTOC ( N ) . Graph depicting the approach angles to AER of leading ( Tier 1 ) , following ( Tier 2 ) and trailing ( Tier 3 ) cells of WT , slit3sq49 and s1pr2te273 embryos ( O ) . A minimum of 30 cells were measured for each tier of each genotype . * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; ANOVA with Fisher ' s post - test .

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

M-O: Schematic describing the measurement of a cell's approach angle to the nearest point on the AER (M). Merged, immunofluorescent images of WT (top), slit3sq49 (centre) and s1pr2te273 (below) mesenchymal cells in sqet37Et background stained for EGFP (green), Gamma tubulin (red) and DAPI (blue). White lines run from the centre of nucleus to the nearest point on the AER, through the MTOC (N). Graph depicting the approach angles to AER of leading (Tier 1), following (Tier 2) and trailing (Tier 3) cells of WT, slit3sq49 and s1pr2te273 embryos (O). A minimum of 30 cells were measured for each tier of each genotype. *p<0.05; **p<0.005; ***p<0.001; ANOVA with Fisher's post-test.


C - E : Lateral Nomarski images of 48hpf larval fins which are WT ( C ) or slit3 + / sq49 heterozygotes ( D ) and are injected with 300µM fn1a morpholino . Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes ( quantified in E ) .

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

C-E: Lateral Nomarski images of 48hpf larval fins which are WT (C) or slit3+/sq49 heterozygotes (D) and are injected with 300µM fn1a morpholino. Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes (quantified in E).


F - G : Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone ( F ) or with 300µM fn1a morpholino ( G ) . H : Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos ( 125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone ) are injected into WT , fn1a morphants ( 300µM MO ) , fn1a + / - heterozygotes and fn1a - / - mutants . Loss of a single or both copies of fn1a exacerbates reduced gna13 levels , as does knockdown of fn1a .

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

F-G: Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone (F) or with 300µM fn1a morpholino (G). H: Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos (125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone) are injected into WT, fn1a morphants (300µM MO), fn1a+/- heterozygotes and fn1a-/- mutants. Loss of a single or both copies of fn1a exacerbates reduced gna13 levels, as does knockdown of fn1a.


Representative images and quantification of somal intensity per unit area of GFP - RBM3 i - neurons at 37 ° C or after 72h cooling at 32 ° C . Nuclei and cells are outlined by white and yellow dashed lines , respectively . N = 207 ( 37 ° C ) and 220 ( 32 ° C ) cells . Scale bar : 5 μm .

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

Representative images and quantification of somal intensity per unit area of GFP-RBM3 i-neurons at 37°C or after 72h cooling at 32°C. Nuclei and cells are outlined by white and yellow dashed lines, respectively. N= 207 (37°C) and 220 (32°C) cells. Scale bar: 5 μm.


Western blots and quantification of RBM3 and GFP normalised to GAPDH in GFP - RBM3 i - neurons at 37 ° C or 32 ° C ( 72h ) .

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

Western blots and quantification of RBM3 and GFP normalised to GAPDH in GFP-RBM3 i-neurons at 37°C or 32°C (72h).


( A - B ) Median GFP intensity of BFP - positive GFP - RBM3 i - neurons measured by flow cytometry upon the sgRNA / Cas9 - mediated KO of top 30 positive ( A ) or negative ( B ) regulator candidates . Statistical analysis is performed between the specific and non - targeting sgRNA groups for positive regulators ( A ) or between the specific sgRNA and reporter groups for negative regulators ( B ) within the 37 ° C or 32 ° C ( 72h ) population .

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

(A-B) Median GFP intensity of BFP-positive GFP-RBM3 i-neurons measured by flow cytometry upon the sgRNA/Cas9-mediated KO of top 30 positive (A) or negative (B) regulator candidates. Statistical analysis is performed between the specific and non-targeting sgRNA groups for positive regulators (A) or between the specific sgRNA and reporter groups for negative regulators (B) within the 37°C or 32°C (72h) population.


( C ) qRT - PCR of RBM3 mRNA level normalised to 18s rRNA in i - neurons at 37 ° C or 32 ° C ( 72h ) .

(C) qRT-PCR of RBM3 mRNA level normalised to 18s rRNA in i-neurons at 37°C or 32°C (72h).


RT - PCR of RBM3 mRNA ( Exon 2 - 4 ) in i - neurons at 37 ° C or 32 ° C ( 72h ) in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are calculated based on the intensity of PE - included ( red arrows ) and PE - skipped ( green arrow ) isoforms .

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

RT-PCR of RBM3 mRNA (Exon 2-4) in i-neurons at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are calculated based on the intensity of PE-included (red arrows) and PE-skipped (green arrow) isoforms.


qRT - PCR using a combination of primers targeting Exon 3a , Exon 3 or Exon 4 - 5 quantifies the PSI values of RBM3 PE ( Exon 3a , including both 3a - L and 3a - S ) relative to Exon 3 or Exon 4 - 5 at 37 ° C or 32 ° C ( 72h ) in the presence or absence of SMG1 inhibitor .

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

qRT-PCR using a combination of primers targeting Exon 3a, Exon 3 or Exon 4-5 quantifies the PSI values of RBM3 PE (Exon 3a, including both 3a-L and 3a-S) relative to Exon 3 or Exon 4-5 at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor.


RT - PCR of RBM3 mRNA ( Exon 2 - 4 ) in HeLa cells at 37 ° C and 32 ° C ( 48h ) in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are depicted in the graph on the right .

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

RT-PCR of RBM3 mRNA (Exon 2-4) in HeLa cells at 37°C and 32°C (48h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.


Schematics and RT - PCR of RBM3 minigene ( Exon 1 - 4 ) , flanked by unique sequences ( thick black bars ) to distinguish it from endogenous transcripts during PCR amplification , expressed in HeLa cells at 37 ° C or 32 ° C ( 48h ) , in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are depicted in the graph on the right .

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

Schematics and RT-PCR of RBM3 minigene (Exon 1-4), flanked by unique sequences (thick black bars) to distinguish it from endogenous transcripts during PCR amplification, expressed in HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.


( A - B ) qRT - PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA in WT i - neurons at 37 ° C ( A ) or 32 ° C ( 72h ) ( B ) , when NMD is blocked by SMG1 inhibitor . Statistical analysis is performed between the specific and non - targeting sgRNA groups .

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

(A-B) qRT-PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA in WT i-neurons at 37°C (A) or 32°C (72h) (B), when NMD is blocked by SMG1 inhibitor. Statistical analysis is performed between the specific and non-targeting sgRNA groups.


PSI values of RBM3 Exon 3a - L in control and HNRNPH1 - knocked down K562 and HepG2 cells . RNA - Seq data from ENCODE Project , 2 isogenic replicates are included .

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

PSI values of RBM3 Exon 3a-L in control and HNRNPH1-knocked down K562 and HepG2 cells. RNA-Seq data from ENCODE Project, 2 isogenic replicates are included.


( D - E ) RT - PCR of endogenous RBM3 ( D ) and expressed RBM3 minigene ( E ) in control ( scramble siRNA ) or HNRNPH1 - knocked down HeLa cells at 37 ° C or 32 ° C ( 48h ) , in the presence or absence of SMG1 inhibitor . The ratio of PSI values between HNRNPH1 KD and control is shown for only the SMG1 i - treated conditions .

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

(D-E) RT-PCR of endogenous RBM3 (D) and expressed RBM3 minigene (E) in control (scramble siRNA) or HNRNPH1-knocked down HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. The ratio of PSI values between HNRNPH1 KD and control is shown for only the SMG1 i-treated conditions.


( F - G ) RT - PCR of endogenous RBM3 ( F ) and expressed RBM3 minigene ( G ) in SMG1 inhibitor - treated control ( untransfected ) or FLAG - HNRNPH1 - overexpressing ( OX ) HEK293T cells at 37 ° C . PSI values of RBM3 PE are shown in the graphs on the right respectively .

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

(F-G) RT-PCR of endogenous RBM3 (F) and expressed RBM3 minigene (G) in SMG1 inhibitor-treated control (untransfected) or FLAG-HNRNPH1-overexpressing (OX) HEK293T cells at 37°C. PSI values of RBM3 PE are shown in the graphs on the right respectively.


WT i - neurons are transduced with lentiviral constructs expressing BFP ( Control ) or HNRNPH1 - T2A - BFP ( HNRNPH1 overexpression , OX ) at 37 ° C and 32 ° C , followed by RT - PCR of endogenous RBM3 with 24h SMG1 inhibitor treatment . PSI values of RBM3 PE are shown in the graph on the right .

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

WT i-neurons are transduced with lentiviral constructs expressing BFP (Control) or HNRNPH1-T2A-BFP (HNRNPH1 overexpression, OX) at 37°C and 32°C, followed by RT-PCR of endogenous RBM3 with 24h SMG1 inhibitor treatment. PSI values of RBM3 PE are shown in the graph on the right.


Western blot and quantification of HNRNPH1 normalised to GAPDH in WT i - neurons at 37 ° C and 32 ° C ( 72h ) . RBM3 blots of the same samples are shown for comparison .

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

Western blot and quantification of HNRNPH1 normalised to GAPDH in WT i-neurons at 37°C and 32°C (72h). RBM3 blots of the same samples are shown for comparison.


RT - PCR of WT and delGGGG RBM3 minigenes in HeLa cells at 37 ° C or 32 ° C ( 48h ) treated with SMG1 inhibitor . PSI values of RBM3 PE are shown in the graphs on the right .

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

RT-PCR of WT and delGGGG RBM3 minigenes in HeLa cells at 37°C or 32°C (48h) treated with SMG1 inhibitor. PSI values of RBM3 PE are shown in the graphs on the right.


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across ( A ) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA

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Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genes


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the U6 snRNA

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Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the srp7 loci .

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Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the srp7 loci.


Genome - wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp .

Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp.


ChIP - qPCR analysis of Flag - tagged Sen1 at the indicated loci in a population of cycling cells ( mean ± standard deviation from 4 biological replicates ) .

ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates).


ChIP - qPCR analysis of the Flag - tagged RNAP3 subunit Rpc37 and Flag - tagged Sen1 across the SPCTRNAARG . 10 tRNA locus , whose TATA box was either mutated ( TATA - ) or not ( TATA + ) . The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs . The enrichment values were normalized to SPCTRNATHR . 10 ( mean ± standard deviation from 4 biological replicates ; p - value obtained using the Wilcoxon - Mann Whitney statistical test ) .

ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


( left ) ChIP - qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells ( mean ± standard deviation from 4 biological replicates ) . ( right ) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1 . Tubulin was used as a loading control .

(left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control.


( left ) Total RNA from the indicated strains were separated on a 2 , 8 % agarose gel . ( right ) Quantification of overall tRNA levels ( mean ± standard deviation from 3 biological replicates ) .

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(left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).


Strand - specific RT - qPCR was used to quantify the indicated RNAP3 transcripts . Transcript levels were normalized to act1 ( mean ± standard deviation from 3 biological replicates ) .

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Strand-specific RT-qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± standard deviation from 3 biological replicates).


Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA . R - ChIP using a catalytically - inactive RNase H1 ( Rnh1 - D129N ) was used to quantify R - loop formation at RNAP3 - transcribed genes ( mean ± standard deviation from 4 biological replicates ) .

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Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA. R-ChIP using a catalytically-inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± standard deviation from 4 biological replicates).


( left ) ChIP - qPCR of the 13myc - tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci ( mean ± standard deviation of 4 biological replicates ) . ( right ) Western blot analysis of Myc - tagged Rpc25 and Flag - tagged RnhA protein levels in the indicated strains . Tubulin was used as a loading control .

(left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± standard deviation of 4 biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control.


Snapshots of ChIP - seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative ( A ) tRNA gene , ( B ) 5S rRNA gene and ( C ) srp7 . Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1 .

Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.


Average ChIP - seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1 .

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Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.


Scanning of Rpc37 - 3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP - qPCR ( mean ± standard deviation from 6 biological replicates ) .

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Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± standard deviation from 6 biological replicates).


Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct ( schematized on top ) were grown either in the presence of the optimum concentration of adenine ( left ) or in the presence of a limiting concentration of adenine ( right ) . Two independent clones of the same genotype ( # 1 and # 2 ) were used . See text for details . 3F refers to the 3 Flag epitope tag at the C - terminus of Rpc37 .

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Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.


Strand - specific RT - qPCR was used to quantify the levels of read - through transcripts ( see Methods ) . The mean ± standard deviation from 4 biological replicates is represented here . P - values were obtained using the Wilcoxon - Mann Whitney statistical test .

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Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.


Northern blot analysis of the tRNA SPATRNAPRO . 02 using an intron - specific probe ( TCTAAACTCAGCATACAAGTGGGG ) . U5 snRNA was used as a loading control .

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Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.


ChIP - qPCR analysis of Rpc37 around SPCTRNAARG . 10 gene in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ) .

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ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).


Strand specific RT - qPCR was used to quantify read - through transcripts at SPCTRNAARG . 10 ( arg10 ) and SPCTRNASER . 09 ( ser09 ) in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ; p - value obtained using the Wilcoxon - Mann Whitney statistical test ) .

Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


B . Western blot of oocyte lysate ( left ) and biotinylation - purified surface ( right ) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1 / PC2 complex compared to either protein expressed separately . Anti - PC1 C - terminus antibody [ 29 ] recognized both full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 . A higher - glycosylated 130 kDa PC2 ( star ) band was only seen when PC1 is coexpressed .

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B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.


C . Co - IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes . IP was done with an anti - FLAG antibody . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 are indicated . Both 120 and 130 kDa bands of PC2 were seen in the IPed product .

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C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.


D . Representative current - voltage relationship ( I - V ) curves ( left ) and a scatter plot and bar graph ( right ) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording . The current of the GOF PC2 _ F604P is included as a control . Currents at + 60 mV are shown in the bar graph . Each point represents the recorded current from one oocyte . Oocyte numbers for scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD ( * * * P < 0 . 001 , Student ' s t - test ) .

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D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).


B . Representative I - V curves ( left ) and a scatter plot and bar graph ( right ) showing that the PC2 _ AA is a GOF mutant and gave rise to a larger current than PC2 _ F604P . Scatter plot and bar graph shows the average current sizes at + 60 mV . The cations included in the bath solution , 100 mM Na + and 2 mM Ca2 + in this case , are indicated by the thick - lined boxes here and in all the following figures . Oocyte numbers for bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


C . Representative currents of PC2 _ F604P and PC2 _ AA mutants in the divalent ion - free bath solution , which contains 100 mM Na + .

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C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.


D . Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2 + .

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D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.


E . Calcium - activated chloride channel ( CaCC ) blocker MONNA ( 10 μM ) or niflumic acid ( NFA ) ( 1 mM ) partially blocked the current recorded from the PC2 _ AA - injected oocytes in the 70 mM Ca2 + bath solution .

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E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.


A , B . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 100 mM Na + ( A ) , and 100 mM Na + and 2 mM Ca2 + ( B ) . Scatter plot and bar graphs show currents at + 80 mV and - 80 ( or - 70 ) mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) .

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A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).


C . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 100 mM Na + and 2 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

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C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


D . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 70 mM Ca2 + . Scatter plot and bar graphs show currents at + 80 mV and - 70 mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


E . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 70 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

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E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


F . Representative currents of PC2 _ AA ( left ) and PC1 / PC2 _ AA ( right ) in solutions containing the indicated Ca2 + concentrations . All solutions contain 20 μM of MONNA to block CaCC current . Corresponding concentrations of NMDG + were added to compensate the osmolarity . See Fig EV2 for currents of the full voltage scale and the details of solutions used .

F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.


G . Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1 / PC2 _ AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone . For oocytes injected with PC2 alone , five times more concentrated PC2 RNA was injected to increase its surface expression . The purple dashed line indicates the background 45Ca uptake set by the measurement with the water - injected oocytes . Data were averaged from three independent experiments . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

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G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


H . Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments .

H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + ( B ) , Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

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Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( C ) Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

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

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 70 mM Ca2 + ( D ) . Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.