Figure 2Flow cytometry analysis of Rex1 - GFPd2 cells following addition of 10mM 2 - DG during the 48h EpiLC induction . Representative GFP intensity distributions are depicted . Average proportions of Rex1 - GFPd2 positive ( GFP + ) cells are quantified from two independent biological replicates . Expression analysis by qRT - PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2 - DG treatment . Relative expression levels , normalized to control culture conditions , are shown . Graphs represent averages from triplicate ( duplicate for Klf4 and Tfcp2l1 ) independent biological experiments . Colony - forming ability following 10mM 2 - DG supplementation during EpiLC stimulation . Representative images of alkaline phosphatase ( AP ) - stained colonies are displayed . Scale bar , 250 um . The average colony formation , normalized to control culture conditions , quantified from two independent biological replicates , is shown .

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Figure 2Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates.Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments.Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown.


Figure 3Pseudotime expression profiles for the αKG - regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency . Tricarboxylic acid ( TCA ) cycle enzymes and metabolites produced within the TCA cycle are illustrated . Representative flow cytometry profiles of Rex1 - GFPd2 cells following 4mM dm - αKG supplementation during the EpiLC induction are depicted . Graphs show average fractions of Rex1 - GFPd2 positive ( GFP + ) cells from six independent biological assays . qRT - PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm - αKG . Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells . Colony forming ability succeeding 4mM dm - αKG treatment during the 48h EpiLC induction . Characteristic images of AP - stained colonies are shown . Scale bar , 250 um . Colony formation is normalized to control - treated cells and quantified from quadruplicate experiments . Representative super - resolution images of TOM - 20 immune - labelled mitochondria in ESCs following 48h culture in 2i / Lif / KSR media , and EpiLC - inducing conditions in the presence of 4mM dm - αKG and DMSO , respectively , are displayed . Scale bar , 3um . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . ( F ) Characteristic bright field images of Rex1 - GFPd2 cells after 10 days of culture in 2i / Lif / KSR and N2B27 / Lif / KSR , in the presence of dm - αKG and DMSO , respectively . Scale bar , 10um . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . Flow cytometer - based quantification of Rex1 - GFPd2 positive ( GFP + ) cells . Representative GFP intensity distributions are displayed . The average fractions of GFP + cells are measured from duplicate experiments . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . qRT - PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2 . 5 day - intervals during the 10 - day culture in N2B27 / Lif / KSR with dm - αKG or DMSO . Expression data are normalized to time - matched ESCs in 2i / Lif / KSR - culture conditions and are averaged over two independent biological experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Pseudotime expression profiles for the αKG-regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency. Tricarboxylic acid (TCA) cycle enzymes and metabolites produced within the TCA cycle are illustrated.Representative flow cytometry profiles of Rex1-GFPd2 cells following 4mM dm-αKG supplementation during the EpiLC induction are depicted. Graphs show average fractions of Rex1-GFPd2 positive (GFP+) cells from six independent biological assays.qRT-PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm-αKG. Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells.Colony forming ability succeeding 4mM dm-αKG treatment during the 48h EpiLC induction. Characteristic images of AP-stained colonies are shown. Scale bar, 250 um. Colony formation is normalized to control-treated cells and quantified from quadruplicate experiments.Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. (F) Characteristic bright field images of Rex1-GFPd2 cells after 10 days of culture in 2i/Lif/KSR and N2B27/Lif/KSR, in the presence of dm-αKG and DMSO, respectively. Scale bar, 10um.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. Flow cytometer-based quantification of Rex1-GFPd2 positive (GFP+) cells. Representative GFP intensity distributions are displayed. The average fractions of GFP+ cells are measured from duplicate experiments.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. qRT-PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2.5 day-intervals during the 10-day culture in N2B27/Lif/KSR with dm-αKG or DMSO. Expression data are normalized to time-matched ESCs in 2i/Lif/KSR-culture conditions and are averaged over two independent biological experiments.


Figure 4FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 embryoids specified in the presence of 4mM dm - αKG and PGC cytokines . Representative flow cytometer profiles are depicted . Graphs show the average fractions of GFP + cells from duplicate experiments . P1 - GFP , Prdm1 - GFP . FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 2 embryoids aggregated under addition of LIF ( 10ng ml - 1 ) and BMP4 ( 500ng ml - 1 ) , dm - αKG ( 4mM ) , or DMSO . Representative flow cytometer profiles are displayed . Average fractions of GFP + cells are calculated from duplicate assays . P1 - GFP , Prdm1 - GFP . FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 PGCLC aggregates specified from 4mM dm - αKG - treated ( t = 48h to t = 72h ) EpiLCs . Representative flow cytometer profiles are depicted . The average fractions of GFP + cells , quantified from triplicate experiments , are shown . P1 - GFP , Prdm1 - GFP . Transcript analysis by qRT - PCR of PGC specifiers , demethylating enzymes , mesoderm , endoderm , and ESC regulators in FACS - sorted day - 4 Prdm1 - GFP embryoids induced from 4mM dm - αKG - treated 72h EpiLCs . Expression levels are normalized to Prdm1 - GFP negative cells from control embryoids . Graphs represent averages from triplicate experiments . + , Prdm1 - GFP positive cells ; - , Prdm1 - GFP negative cells . Western blot analysis for H3K9me2 , H3K27me3 , and DNMT3b in 4mM dm - αKG - treated EpiLCs . H3 is used as a loading control . 48h , dm - αKG and DMSO , respectively , supplementation from t = 24h to t = 48h during the EpiLC differentiation . 72h , dm - αKG and DMSO , respectively , supplementation from t = 48h to t = 72h during the EpiLC differentiation . ChIP - qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state ( Esrrb , Arid5b ) and PGC fate ( Tfap2c ) , respectively , in naïve ESCs and at t = 48h following EpiLC induction in the presence of 4mM dm - αKG and DMSO , respectively . Graphs show enrichment of H3K9me2 , H3K27me3 , and IgG control , respectively , relative to DMSO - treated EpiLCs .

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Figure 4FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. P1-GFP, Prdm1-GFP.FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. P1-GFP, Prdm1-GFP.FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. P1-GFP, Prdm1-GFP.Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells.Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation.ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs.


Figure 1 ( A ) SA - β - gal staining of U2 - OS cells , harvested at indicated times post irradiation ( IR ) ; h , hours ; D , days ; UT , untreated . n = 3 . Scale bar : 50 μm . Bar Graph : Significance was determined by Brown Forsythe ANOVA with Dunnett ' s 3T test , using n = 3 biological replicates . SD shown . * * * p < 0 . 001 , n . s . = not significant . ( B ) Nuclear fractions from U2 - OS cells were harvested at indicated time points post IR ( 20 Gy ) as in ( A ) . Top panel : NF - κB DNA binding was analyzed by EMSA . Lower panels : Western blot of indicated NF - κB subunits and PARP1 as loading control . The two phases of nuclear translocation ( C ) Gene expression analysis of two NF - κB phases following damage . RNA - Seq analysis ( n = 5 biological replicates per group ) of U2 - OS cells , either untreated or analyzed 1 . 5 hours or 7 days after irradiation ( 20 Gy ) , as indicated . The heatmap shows significantly regulated genes ( log2 value > 0 . 5 and p value < 0 . 05 ) . Black arrow points to NFKBIA . Significance determined by ANOVA with Bonferroni correction for multiple testing . See also Dataset EV1 . ( D ) Female mice 12 - 16 weeks old ( n = 6 mice per condition ( with three technical repeats per mouse ) , were irradiated ( 5 Gy ) and sacrificed after 1 hour ( first phase ) , 7 days ( second phase ) or left untreated . Skin or renal tissue RNA was analyzed by RT - qPCR , shown as a mean + / - SD . Significance was confirmed by ANOVA with Tukey multiple comparisons test . SD shown : * = p < 0 . 05 , * * = p < 0 . 01 . ( E ) In situ hybridization using an anti - sense IκBα / Nfkbia riboprobe on longitudinal skin sections from male and female mice ( 5 - 8 weeks of age ) treated as in ( D ) for the time indicated . Representative sections shown from n = 6 mice per condition . Bu , bulge region ( dashed lines ) , HS , hair shaft , IFE , interfollicular epidermis . Scale bar : 50 μm . ( F ) Quantitation of ( E ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for Nfkbia were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * * = p < 0 . 01 . ( G ) Skin sections as in ( E ) analyzed by immunofluorescence with IκBα ( red ) or IL - 6 antibody ( green ) and nuclear DAPI staining ( blue ) . Dotted lines delineate hair follicles , Bu , bulge region , HS , hair shaft , IFE , interfollicular epidermis , SG , sebaceous gland . Scale bar : 50 μm . ( H ) Quantitation of ( G ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for IκBα or IL - 6 were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * = p < 0 . 05 , * * * = p < 0 . 001 .

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Figure 1(A) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm. (F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01. (G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.


Figure 2 ( A ) Left panel : IκBα western blot of U2 - OS cells . Lane 1 , untreated ( ut ) ; Lanes 2 - 4 , single IR ( 20 Gy ) at indicated time points prior to harvest ; Lane 5 , IR at 5 days prior to harvest , plus 1 . 5 hours prior to harvest . Right panel : Samples treated as in the left panel , analyzed by RT - qPCR . Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test . SD shown . * * * = p < 0 . 001 . ( B ) NFKBIA knockdown by dox - inducible shRNA in U2 - OS cells . Cells were pre - treated with Dox to induce knockdown . For knockdown efficiency see Fig . S2F . Cells were irradiated ( 20 Gy ) or not , as indicated and described above . NF - κB activity was analyzed by EMSA . ns , non - specific band . A representative gel from n = 3 biological replicates is shown . ( E ) GSEA was performed comparing the IκBα IEC ‐ KO gene list ( n = 4 villin - Cre x floxed IκBα mice and n = 4 littermate controls ( GSE 139251 ) Mikuda et al , 2020 ( ) , with a gene signature based on a list of SASP factors Coppe et al , 2010 ( ) . NES : normalized enrichment score . FDR : false discovery rate . ( F ) RT - qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin - Cre ( n = 6 ) or villin - Cre x floxed IκBα mice ( n = 6 ) both male and female . Expression is shown as fold change between littermates , paired t - test . SD shown . * p < 0 . 05 * * = P < 0 . 01 .

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Figure 2(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.(E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate.(F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.


Figure 3 ( A ) Nuclear fractions from either untreated or irradiated ( 20 Gy ) U2 - OS cells , 1 . 5 hours or 7 days prior to harvest , were analyzed by EMSA with supershift analysis . For supershifting , lysates were incubated with antibodies or water , as indicated . Arrows point to shift location of a given subunit , due to antibody binding . ns , non - specific band . All antibodies were previously tested for supershift compatibility . Representative gel shown from n = 3 replicates . ( B ) Expression of SASP targets of NF - κB obtained from RNA - seq analysis ( Dataset EV1B ) , was quantitated using RT - qPCR from the shRNA ( RELA ) stably expressing U2 - OS cells . For knockdown efficiency see Appendix Fig S4A . Cells were treated with doxycycline ( Dox ) to induce knockdown of p65 . Heatmap represents targets normalized to the untreated Scrambled control . Expression is shown as log2 change with p values < 0 . 05 . Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing . First - phase and second - phase samples were irradiated ( 20 Gy ) 1 . 5h and 7 Days prior to harvest , respectively . Analysis based on n = 3 biological replicates . ( C ) Cell duplication was measured at time points indicated in U2 - OS cells bearing either scrambled control or Dox - inducible shRNA against RELA ( n = 3 biological replicates ) . Treatment with Dox was initiated at two days prior to IR ( 20 Gy ) or after IR . Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test . SD shown . * = P < 0 . 05 , * * * = p < 0 . 001 .

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

Figure 3(A) Nuclear fractions from either untreated or irradiated (20 Gy) U2-OS cells, 1.5 hours or 7 days prior to harvest, were analyzed by EMSA with supershift analysis. For supershifting, lysates were incubated with antibodies or water, as indicated. Arrows point to shift location of a given subunit, due to antibody binding. ns, non-specific band. All antibodies were previously tested for supershift compatibility. Representative gel shown from n = 3 replicates.(B) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.


Figure 4 ( A ) Nuclear ( Nuc . ) and cytoplasmic ( Cyto . ) fractions of U2 - OS cells were analyzed at times indicated after IR exposure by SDS - PAGE western blotting . PARP1 and LDHA are fractionation controls and phospho - specific p65 signals are indicated . Representative gels from n = 3 biological replicates are shown . Right panel : quantitation of nuclear p65 and phosphorylated p65 species ( n = 3 ) . Fold changes compared to untreated samples ( ut ) and time points post IR are indicated on Y and X axes , respectively . ( B ) U2 - OS cells treated with dox to deplete endogenous p65 , were irradiated ( 20 Gy ) and transfected with plasmids encoding p65 , p65 - S276A or p65 - S468A , as indicated . Nuclear ( N ) and cytoplasmic ( C ) lysates were analyzed by SDS - PAGE at day seven post IR . Representative gel from n = 3 biological replicates is shown . PARP1 and αTubulin serve as fractionation and loading controls . ( 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 . ( 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 . ( 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 .

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

Figure 4(A) Nuclear (Nuc.) and cytoplasmic (Cyto.) fractions of U2-OS cells were analyzed at times indicated after IR exposure by SDS-PAGE western blotting. PARP1 and LDHA are fractionation controls and phospho-specific p65 signals are indicated. Representative gels from n = 3 biological replicates are shown. Right panel: quantitation of nuclear p65 and phosphorylated p65 species (n = 3). Fold changes compared to untreated samples (ut) and time points post IR are indicated on Y and X axes, respectively.(B) U2-OS cells treated with dox to deplete endogenous p65, were irradiated (20 Gy) and transfected with plasmids encoding p65, p65-S276A or p65-S468A, as indicated. Nuclear (N) and cytoplasmic (C) lysates were analyzed by SDS-PAGE at day seven post IR. Representative gel from n = 3 biological replicates is shown. PARP1 and αTubulin serve as fractionation and loading controls.(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.(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(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.(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.(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.


Figure 1A - 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 ) .

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

Figure 1A-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).


Figure 2Grip 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 ) . 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 ) .

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

Figure 2Grip 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).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).


Figure 3Enzymatic 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 . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Enzymatic 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.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).


Figure 4Relative 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 ) . 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 ) .

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

Figure 4Relative 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).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).


Figure 5A - 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 ) . 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 ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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).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).


Figure 6Mitochondrial 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 ) .

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 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 6Mitochondrial 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).


Figure 7A - 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 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

Figure 7A-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).


Figure 8Grip 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 8Grip 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).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).


Figure 1 ( 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 .

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 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

Figure 1(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.


Figure 2 ( 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 .

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

Figure 2(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.


Figure 3 ( 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 ) ( 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 .

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

Figure 3(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)(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.


Figure 4 ( 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 . 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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Figure 4(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.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.


Figure 5 ( 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 . ( 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 . ( 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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Figure 5(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.(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.(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.


Figure 6 ( 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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Figure 6(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.


Figure 1E . 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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Figure 1E. 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.


Figure 2C - 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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Figure 2C-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.


Figure 3D . 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 .

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Figure 3D. 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.


Figure 4D . 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 .

Figure 4D. 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.


Figure 5B . 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 .

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Figure 5B. 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.


Figure 6A . 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 .

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Figure 6A. 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.


Figure 7D , 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 .

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Figure 7D, 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.


Figure 8A . 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 .

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Figure 8A. 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.


Figure 9B . 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 ) .

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Figure 9B. 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).


Figure 10A - 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 % .

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Figure 10A-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%.


Figure 4M - 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 .

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Figure 4M-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.


Figure 5C - 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 .

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Figure 5C-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.


Figure 1Representative 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 ) .

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Figure 1Representative 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).