U87 transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . were collected analyzed for expression of LXRβ mRNA ( O ) or LXRβ by capillary electrophoresis ( P ) . Shown are means SD ( n = 5 ) . Statistical significance was determined by two - sided student ' s t . .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.


1x106 HCT116 colon carcinoma were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 - 200 mg / kg ) , ABT263 ( 100 mg / kg ) or both agents ( 3 days per week for 1 . 5 . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0063 , * * * * p < 0 . 0001 . Statistical significance was determined by one - way ANOVA . B , E , GBM43 PDXs were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 ) . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0031 . Statistical significance was determined by one - way ANOVA . C , F , A375 BRAF V600E mutated melanomas were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 ) . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 0109 ( GW3965 vs Combination ) , * p = 0 . 0166 ( ABT263 vs Combination ) , * * p = 0 . 0047 . Statistical significance was determined by one - way ANOVA .

O B-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

1x106 HCT116 colon carcinoma cells were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100-200 mg/kg), ABT263 (100 mg/kg) or both agents (3 days per week for 1.5 weeks. Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0063, ****p<0.0001. Statistical significance was determined by one-way ANOVA. B, E, GBM43 PDXs were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0031. Statistical significance was determined by one-way ANOVA. C, F, A375 BRAF V600E mutated melanomas were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.0109 (GW3965 vs Combination), *p=0.0166 (ABT263 vs Combination), **p=0.0047. Statistical significance was determined by one-way ANOVA.


Related to HCT116 colonic xenograft model , representative histopathological ( hematoxylin eosin stain ) are shown from each treatment group . Scale bar : 50 µm . Sections from same treatment groups as in G were stained with TUNEL SDHB , repectively . Displayed are representative of . Scale bar 50 µM .

O O B-CELL_LINE O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE 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-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O

Related to the HCT116 colonic xenograft model, representative histopathological images (hematoxylin and eosin stain) are shown from each treatment group. Scale bar: 50 µm. Sections from the same treatment groups as in G were stained with TUNEL and SDHB, repectively. Displayed are representative images of the staining. Scale bar 50 µM.


Sections from experiment in 7A were quantified for apoptotic

Sections from the experiment in 7A were quantified for apoptotic bodies


, Sections from experiment in 7A were quantified for , number of TUNEL positive

O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

, Sections from the experiment in 7A were quantified for , number of TUNEL positive cells


Sections from experiment in 7A were quantified for number of mitosis per multiple high - power fields .

O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

Sections from the experiment in 7A were quantified for number of mitosis per multiple high-power fields.


Total carbonyl - bearing detected by shotgun redox proteomic measurement in three biological replicates each of E . coli D . radiodurans with without irradiation . left axis is number of - unique detected as carbonylated . right axis is number of sites in total detected as carbonylated ( red ) or not oxidized ( black ) in peptides bearing at least one carbonyl . Stripes indicate carbonylated carbonylatable sites detected only in irradiated samples .

O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O

Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples.


Volcano plots for relative abundance changes measured by mass spectrometry in E . coli ( left ) D . radiodurans ( right ) after irradiation using same biological replicates as in Fig 2A . Black - circled points are those with significant changes ( paired , 2 - sided t - p - value < 0 . 05 ) of > 2 - fold or < 0 . 5 - fold . Red points are with at least one carbonylated peptide detected . Fold change p - value cutoffs considered for significance are indicated by dashed lines .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.


Prevalence of RKPT residues prevalence of carbonylated form in experimentally measured peptides combining 3 biological replicates of both conditions for each organism . Ratios are given above each pair of bars . proportions are significantly different between each RKPT their respective carbonylation state by two - z - of two proportions ( p - values < 0 . 01 ; see Materials Methods ) , meaning carbonylated proportions are not determined simply by relative prevalence of RKPT . See also Appendix Fig S1 .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1.


Prevalence of canonical before irradiation of E . coli D . radiodurans , combining 3 biological replicates for each condition . Ratios are given above each pair of bars . proportions are significantly different between species by two - z - of two proportions ( p - values < 0 . 01 ) . See also Fig EV1 EV2 .

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Prevalence of all canonical amino acids before irradiation of E. coli and D. radiodurans, combining all 3 biological replicates for each condition. Ratios are given above each pair of bars. All proportions are significantly different between species by two-tailed z-test of two proportions (p-values < 0.01). See also Fig EV1 and EV2.


A , B . RT - qPCR of Zfp207 Oct4 in mouse ESCs during ( A ) retinoic acid ( RA ) - induced ( B ) embryoid ( EB ) - mediated differentiation . Nestin was used as a neuronal differentiation marker to monitor RA - mediated differentiation . mRNA are relative to expression at day 0 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-ORGANISM B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O

A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.


J , K . ( J ) AP of shScr Zfp207 - depleted ( sh1 sh2 ) mouse ESCs . Scale bars , 20 µM . ( K ) of fully differentiated ( FD ) , partially differentiated ( PD ) undifferentiated ( UN ) ESC colonies in shScr , sh1 sh2 .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O 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-CELL_TYPE O O O O O O O

J, K. (J) AP staining of shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Scale bars, 20 µM. (K) Percentage of fully differentiated (FD), partially differentiated (PD) and undifferentiated (UN) ESC colonies in shScr, sh1 and sh2.


M , N . ( M ) Western blot of ZFP207 , OCT4 , NANOG , SOX2 in shScr , sh1 sh2 ESCs ( N ) RT - qPCR of Oct4 , Nanog , Sox2 in shScr , sh1 sh2 ESCs ; data is relative to shScr .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O

M, N. (M) Western blot of ZFP207, OCT4, NANOG, and SOX2 in shScr, sh1 and sh2 ESCs and (N) RT-qPCR analysis of Oct4, Nanog, and Sox2 in shScr, sh1 and sh2 ESCs; data is relative to shScr.


( O ) Western blot of OCT4 during a 9 - h cycloheximide ( CHX ) time course treatment in shScr , sh1 sh2 ESCs .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O

(O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs.


( Q ) Western blot of OCT4 during 4 8 h treatment with proteasome inhibitor MG132 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O

(Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132.


D . RT - qPCR of Zfp207 , pluripotency genes ( Oct4 , Nanog Sox2 ) , mesodermal markers ( Msx1 Brachyury , T ) , endodermal markers ( Foxa2 , Sox17 ) , neural - associated genes ( Pax6 , Sox11 Nestin ) in shScr , sh1 sh2 ESCs along time - course of EB - mediated differentiation . mRNA are relative to expression of shScr at day 0 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-TISSUE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O

D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.


B . Immunostaining of NESTIN ( green ) TUJ1 ( red ) of neural progenitors generated from shScr on day 8 10 of neuroectodermal differentiation . Nuclei were counterstained with DAPI . Scale bar , 20 μM .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-TISSUE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O

B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.


( E ) of signal in shScr , sh1 sh2 at day 5 of neuroectodermal differentiation .

O O O O O O O O O O O O O O B-TISSUE O O

(E) quantification of the signal in shScr, sh1 and sh2 at day 5 of neuroectodermal differentiation.


G . RT - qPCR of Zfp207 neural - associated markers in shScr , sh1 sh2 along course of neural differentiation . mRNA are relative to shScr at day 0 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

G. RT-qPCR of Zfp207 and neural-associated markers in shScr, sh1 and sh2 along the course of neural differentiation. mRNA levels are relative to shScr at day 0.


( A ) Volcano plots of common differentially expressed genes in shScr Zfp207 - depleted ( sh1 sh2 ) mouse ESCs . Up - regulated ( Up ) down - regulated ( Down ) genes are indicated in red blue , respectively ( p < 0 . 05 ; > 1 . 5 - fold ) . Zfp207 is depicted in black . Grey dots indicate non - significant ( NS ) < 1 . 5 - fold differential expressed genes .

O O O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O 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-GENEPROD 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) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes.


A . of column z - scores of log2 transformed values of genes involved in alternative splicing ( AS ) in shScr , sh1 sh2 mouse ESCs .

O 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-ORGANISM B-CELL_TYPE O

A. Heatmap of column z-scores of log2 transformed values of genes involved in alternative splicing (AS) in shScr, sh1 and sh2 mouse ESCs.


F . RT - PCR of Zfp207 - regulated AS events . For Mta1 , AS1 ( alternative splicing 1 ) AS2 ( alternative splicing 2 ) . # 1 # 2 indicate distinct biological replicates . * Denotes an isoform that was not taken in consideration for . structure of each isoform is indicated ( not to scale ) . Alternative exons are blue . spliced in ( PSI ) was quantified for each condition .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-PCR of Zfp207-regulated AS events. For Mta1, AS1 (alternative splicing 1) and AS2 (alternative splicing 2). #1 and #2 indicate distinct biological replicates. *Denotes an isoform that was not taken in consideration for the quantification. The structure of each isoform is indicated (not to scale). Alternative exons are blue. The percent spliced in (PSI) was quantified for each condition.


( E ) FISH of poly ( A ) + distribution in shScr , sh1 sh2 stained with 5 ´ Cys3 - oligo dT ( 50 ) . Nuclei were counterstained with DAPI . Scale bar , 20 µM .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O

(E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM.


of ( F ) , ( G ) , ( H ) of / poly ( A ) + FISH signals . ( I ) Relative total in sh1 sh2 compared to shScr . Data are presented as mean ± of n ≥ 3 independent biological experiments . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( shScr vs . sh1 or sh2 ) ; unpaired Student ' s t - .

O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of (F) nuclear, (G) cytoplasmic, and (H) of nuclear/cytoplasmic poly(A)+ FISH signals. (I) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr. Data are presented as mean ± SEM of n ≥ 3 independent biological experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2); unpaired Student's t-test.


( a ) Flow cytometry of LPS - stimulated B stained with LysoTracker Red .

(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.


( b ) Autophagic flux of GFP - LC3 splenic B stimulated with LPS then left untreated ( UT ) or treated for 1 h with bafimolycin A1 ( BafA1 ) , assessed by live - microscopy ( left ) automated of spots ( right ) . Scale bar ( left ) , 5 μm .

O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.


( c ) Immunoblot of processing of endogenous unconjugated LC3 - I to lipid - conjugated LC3 - II in LPS - stimulated B with ( + NH4Cl ) or without treatment with NH4Cl ( left ) . Actin serves as a loading control throughout . Right , of LC3 - II band in absence of NH4Cl , presented relative to that of actin .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O

(c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin.


( d ) Quantitative RT - PCR of transcripts encoding autophagy - related molecules in ASCs 3 d after stimulation with LPS , presented relative to that in unstimulated B .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O

(d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells.


( e ) Immunoblot of p62 in LPS - stimulated B ( bottom ) . Above , of band ( as in c ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O

(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).


( f ) Live - microscopy of CD138 + PCs purified from spleen of GFP - LC3 mice 5 d after injection of NP - Ficoll ( PCs ) of splenic B ( B ) bone marrow CD138 + PCs ( BM PCs ) purified from unstimulated GFP - LC3 mice ( right ) automated of spots ( left ) . Scale bar ( right ) , 5 μm . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) . Data are representative of three or more independent experiments ( a , c - e ) or one experiment or more with > 40 ( b , f ; average s . e . m . ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c-e) or one experiment or more with >40 cells (b,f; average and s.e.m.).


( a ) Proliferation of B purified from Atg5f / fCD19 - Cre Atg5f / f control mice activated for 3 d with LPS , assessed by flow cytometry of dilution of dye CFSE . ( b ) of B purified as in a activated for various times ( horizontal axis ) with LPS , assessed by of forward scatter ( FSC ) by flow cytometry . ( c ) CD138 expression of B purified activated as in a . APC , allophycocyanin ; PE , phycoerythrin .

O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin.


( d ) Immunoblot of ER in lysates of Atg5f / f Atg5f / fCD19 - Cre B 3 d after stimulation with LPS ( left ) , of band ( right ; as in Fig . 1c ) . IgM - H , IgM heavy chain . NS , not significant ; * P 0 . 05 * * P 0 . 01 ( Student ' s t - ) .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).


( a ) Electron microscopy of Atg5f / f Atg5f / fCD19 - Cre B 3 d after stimulation with LPS ( left middle ) , of ER by stereology ( right ) . Scale bars ( left middle ) , 1 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm.


( b ) G6Pase electron - microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS ( left ) , of G6Pase + ( ER ) , relative to total ( right ) . Scale bars ( left ) , 1 μm .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm.


( c ) Quantitative RT - PCR of transcripts encoding spliced XBP - 1 ( sXBP - 1 ) , total XBP - 1 ( tXBP - 1 ) BiP in differentiating PCs at 0 d 3 d after stimulation with LPS , presented relative to that of unstimulated Atg5f / f B at day 3 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O

(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.


( d ) Quantitative RT - PCR of CHOP mRNA in Atg5f / f Atg5f / fCD19 - Cre B stimulated for 3 d with LPS ( left middle ) , in Atg5f / f B stimulated for 3 d with LPS treated for final 24 h with tunicamycin ( Atg5f / f 24 h tun ; 0 . 5 μg / ml ; positive control ) , presented relative to that in Atg5f / f B stimulated for 3 d with LPS .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O

(d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS.


( e ) Immunoblot ( left ) of ER in Atg5f / f B left untreated ( UT ) or treated with lysosomal protease inhibitors ( 10 μM leupeptin E - 64d ) for final 8 h of third day of LPS - induced differentiation ( Leu + E - 64d ( 8 h ) ) . Right , of band ( presented as in Fig . 1c ) . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) .

O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Enzyme - linked immunosorbent ( ELISA ) of IgM in supernatants of Atg5f / fCD19 - Cre Atg5f / f B stimulated for 3 d with LPS plated for 4 h ( as trypan blue - negative at a density of 1 × 106 per ml in dedicated media ) , presented relative to results obtained for Atg5f / f .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O O O

(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.


( b , c ) Immunoglobulin assembly ( b ) total intracellular ( IC ) secreted ( Sec ) IgM heavy chain ( IgM - H , c ) in Atg5f / f Atg5f / fCD19 - CreB stimulated for 3 d with LPS , then - labeled chased ( time , above lanes ) , followed by immunoprecipitation of μ - chain from lysates SDS - PAGE ( left ) in nonreducing conditions ( b ) or reducing conditions ( c ) . Right , of intracellular IgM polymers by densitometry ( b ) of secretion efficiency , estimated as amount of radioactive secreted μ - chain at end of chase relative to intracellular μ - chain at end of ( c ) . AU , arbitrary units .

O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.


O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Quantitative RT - PCR of mRNA encoding Blimp - 1 secreted μ - chain in Atg5f / f Atg5f / fCD19 - Cre B stimulated for 3 d with LPS , presented relative to that of Atg5f / f .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O

(d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells.


( e ) Quantitative RT - PCR of transcripts encoding spliced XBP - 1 , BiP , Blimp - 1 secreted μ - chain in Atg5f / f B stimulated for 3 d with LPS given no further treatment ( − tun ) or treated for final 24 h with tunicamycin ( tun 24 h ; 0 . 5 μg / ml ) , presented relative to results obtained with LPS - stimulated given no further treatment . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Intracellular ATP in fresh lysates of Atg5f / f Atg5f / fCD19 - Cre splenic B activated for 3 d with LPS ( 3 ) or left untreated ( 0 ) .

O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-TISSUE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).


( b ) of Atg5f / f Atg5f / fCD19 - Cre splenic B activated for various times ( horizontal axis ) with LPS , quantified by flow cytometry by propidium iodide ( PI ) .

O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-TISSUE B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O

(b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining.


( c ) Quantitative PCR of genomic in differentiating B from Atg5f / f Atg5f / fCD19 - Cre mice , assessing nonrecombinant ( Undeleted ; left ) recombinant ( Deleted ; right ) loxP - flanked Atg5 alleles ; results are presented relative to those of unstimulated Atg5f / fCD19 - Cre B .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O

(c) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f/f and Atg5f/fCD19-Cre mice, assessing nonrecombinant (Undeleted; left) and recombinant (Deleted; right) loxP-flanked Atg5 alleles; results are presented relative to those of unstimulated Atg5f/fCD19-Cre B cells.


( d ) Quantitative RT - PCR of Atg5 mRNA in differentiating Atg5f / f Atg5f / fCD19 - Cre B at 0 3 d after stimulation with LPS , presented relative to that in untreated Atg5f / fCD19 - Cre B . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O 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) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) ELISA of anti - pneumovax IgM titers in diluted serum 1 day before 4 , 10 14 d after intraperitoneal immunization of Atg5f / f Atg5f / fCD19 - Cre mice with 1 μg pneumovax .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O

(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax.


( b ) ELISA of anti - pneumovax IgG3 titers in serum 14 d after injection as in a . Results are presented as absorbance ( A ) . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Frequency of CD138hiB220lo ( numbers above outlined areas , bottom ) among IRF4hi population ( numbers above outlined areas , top ) , assessed by flow cytometry ( left ) . 488 , Alexa Fluor 488 . Right , of bone marrow PCs ( each symbol represents an mouse ; small horizontal lines indicate mean ( s . e . m . ) .

O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O O O O O O O O

(a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left). 488, Alexa Fluor 488. Right, quantification of bone marrow PCs (each symbol represents an individual mouse; small horizontal lines indicate mean (and s.e.m.).


( b ) Quantitative RT - PCR of mRNA encoding IRF4 Blimp - 1 in CD138hiB220lo bone marrow PCs B220hiCD138lo spleen B lymphocytes ( control ) sorted from Atg5f / f Atg5f / fCD19 - Cre mice , to confirm PC phenotype ; results are presented relative to that of histone H3 mRNA ( loading control ) . ( c ) Quantitative PCR of genomic assessing undeleted deleted loxP - flanked Atg5 alleles in sorted B PCs ( as in b ; presented as in Fig . 5c ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-GENEPROD B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O

(b) Quantitative RT-PCR analysis of mRNA encoding IRF4 and Blimp-1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes (control) sorted from Atg5f/f and Atg5f/fCD19-Cre mice, to confirm PC phenotype; results are presented relative to that of histone H3 mRNA (loading control). (c) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP-flanked Atg5 alleles in sorted B cells and PCs (as in b; presented as in Fig. 5c).


( d ) Immunofluorescence of endogenous LC3 ( left , top row ) immunoglobulin content ( left , bottom row ) in bone marrow PCs sorted from Atg5f / f Atg5f / fCD19 - Cre mice , quantitative RT - PCR of Atg5 mRNA in bone marrow PCs spleen B populations ( right ) sorted from Atg5f / f Atg5f / f CD19 - Cre mice . Nuclei ( left ) are stained blue with Hoechst . Scale bar ( left ) , 5 μm . * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ( Student ' s t - ) .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-TISSUE B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-ORGANISM O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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) Immunofluorescence of endogenous LC3 (left, top row) and immunoglobulin content (left, bottom row) in bone marrow PCs sorted from Atg5f/f and Atg5f/fCD19-Cre mice, and quantitative RT-PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations (right) sorted from Atg5f/f and Atg5f/f CD19-Cre mice. Nuclei (left) are stained blue with Hoechst. Scale bar (left), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


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 recruitment of aerobic metabolism that is representative of endurance exercise . ( A , D ) Kaplan - Meier curves representing values corresponding to of mice still running at a given time point . ( B , E ) Median values of time that mice ran are represented in min to max graphical representation ; 15 . 5 min for wild - type ( WT ) 14 . 33 min for SOD1G86R in ( B ) ; 59 . 33 min for WT 65 . 43 min for SOD1G86R in ( E ) . ( C , F ) Mean run ± by WT SOD1G86R mice in ( C ) by WT SOD1G86R mice in ( F ) .

O O O O O O O O O O O O B-EXP_ASSAY O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O O O O O

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

B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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 of . Spontaneous electrical activity was considered as positive EMG only for peak - to - peak amplitudes > 50 μV . Left : of mice presenting with positive EMG in each group are represented . Right : Representative examples of negative ( EMG − ) positive ( EMG + ) electromyography profiles ( n = 10 for WT SOD1G86R , respectively , at 9 - 13 n = 8 7 for WT SOD1G86R , respectively , at 15 ) .

B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O

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

O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O B-GENEPROD B-ORGANISM O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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

O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 . mean fold change ± compared to - matched WT are represented with * P = 0 . 019 at 65 days of ( n = 5 / genotype ) * P = 0 . 041 at 105 days of ( n = 4 / genotype ) , two - way ANOVA followed by Fisher ' s LSD post hoc . Data shown are representative of two independent experiments having similar results .

B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 phosphorylated glycogen synthase . Right : represents mean of optical densities of glycogen synthase / P - glycogen synthase ± . At 65 days of n = 6 / genotype , * P = 0 . 0162 , at 105 days of n = 5 / genotype , * P = 0 . 0163 , two - way ANOVA followed by Fisher ' s LSD post hoc .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-EXP_ASSAY O O B-GENEPROD B-TISSUE I-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of genes involved in lipid handling evaluated by qPCR at indicated ages in tibialis anterior of WT SOD1G86R mice . Graphs represent mean fold change ± from - 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 Cpt - 1β * * P = 0 . 01 at 65 days * P = 0 . 02 at 105 days ( n = 6 7 for WT SOD1G86R , respectively , at 65 days ; n = 10 8 for WT SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc ) .

O O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O B-GENEPROD B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of ( A ) Pparß / δ , ( B ) Foxo1 , ( C ) citrate synthase , ( D ) Pgc - 1α were evaluated by qPCR at indicated ages in tibialis anterior of WT SOD1G86R mice . Graphs represent mean fold change ± from - 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 8 for WT SOD1G86R , respectively , at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc ) .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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-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 respective actin that was used for normalization . PGC - 1α in tibialis anterior muscle tissue are represented as mean fold change ± from - 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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

B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O B-GENEPROD B-ORGANISM O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O B-SMALL_MOLECULE 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 from T0 for each experimental group ± . # # P = 0 . 0045 * * * P = 0 . 0003 ( n = 9 / genotype in CT groups , n = 9 8 for WT SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc ) .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE 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).


( B ) Immunoblot of whole samples of HA - tagged full - ( FL ) IFT54 , its deletion mutants as indicated , each expressed in ift54 null mutant ; wild type ( WT ) ift54 mutant were used as control . Anti - HA was use to visualize transgenic , antibodies to FLA8 / KIF3B were used as a loading control .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(B) Immunoblot analysis of whole cell samples of the HA-tagged full-length (FL) IFT54, and its deletion mutants as indicated, each expressed in the ift54 null mutant; wild type (WT) and ift54 mutant cells were used as control. Anti-HA was use to visualize the transgenic proteins, antibodies to FLA8/KIF3B were used as a loading control.


( C ) Histogram showing ciliary distribution in populations of ift54 mutant transformed with deletion mutant constructs as indicated . Wild type ( WT ) were used as a control .

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(C) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated. Wild type (WT) cells were used as a control.


( D ) Representative DIC of wild type ( WT ) , ift54 ift54 transformed with deletion constructs as indicated . Arrows indicate cilia bulges . Scale bar , 5 μm .

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(D) Representative DIC images of wild type (WT), ift54 and ift54 cells transformed with the deletion constructs as indicated. Arrows indicate cilia bulges. Scale bar, 5 μm.


( A ) Immunostaining of IFT54 mutated in various deletion mutants as indicated ; wild - type ( WT ) were used as control . Specimens were stained with anti - HA anti - α - tubulin antibodies followed by imaging using an epifluorescence microscope . Arrows indicate accumulation at ciliary tip ( left ) or proximal end of cilia ( right ) . Scale bars , 5 μm .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(A) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated; wild-type (WT) cells were used as control. Specimens were stained with anti-HA and anti-α-tubulin antibodies followed by imaging using an epifluorescence microscope. Arrows indicate protein accumulation at ciliary tip (left) or proximal end of cilia (right). Scale bars, 5 μm.


( B , C ) Immunostaining of IFT43 IFT38 in deletion mutants as indicated . were fixed stained with antibodies against IFT43 ( an IFT - A subunit ) ( B ) or against IFT38 ( an IFT - B subunit ) ( C ) followed by imaging using both DIC epifluorescence microscopy . IFT43 IFT38 accumulated at ciliary tip in IFT54Δ261 - 275 mutant but at proximal end of cilia in IFT54Δ342 - 356 mutant . Arrows indicate ciliary bulges . WT , wild - type . Scale bars , 5 μm .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O

(B, C) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated. Cells were fixed and stained with antibodies against IFT43 (an IFT-A subunit) (B) or against IFT38 (an IFT-B subunit) (C) followed by imaging using both DIC and epifluorescence microscopy. IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261-275 mutant but at proximal end of cilia in IFT54Δ342-356 mutant. Arrows indicate ciliary bulges. WT, wild-type cells. Scale bars, 5 μm.


( A ) Immunostaining of with antibody against IFT dynein subunit D1bLIC . D1bLIC is absent in cilia bulges at ciliary tip in IFT54Δ261 - 275 mutant while enriched at cilia bulges at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type . Arrows indicate ciliary bulges . Scale bar , 5 μm . ( B ) Immunostaining of with antibody against kinesin - II subunit FLA10 . FLA10 is enriched in cilia bulges at ciliary tip in IFT54Δ261 - 275 mutant or at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type . insets show enhanced / or enlarged . Arrows indicate ciliary bulges . Scale bar , 5 μm .

O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

(A) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC. D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261-275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. Arrows indicate ciliary bulges. Scale bar, 5 μm. (B) Immunostaining of cells with antibody against kinesin-II subunit FLA10. FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261-275 mutant or at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. The insets show enhanced and/or enlarged images. Arrows indicate ciliary bulges. Scale bar, 5 μm.


( C , D ) Immunoblot of whole ( C ) isolated cilia ( D ) of wild type three IFT54 deletion mutants . membranes were probed with antibodies against IFT motor as indicated . Wild - type of various IFT components were detected in whole samples of deletion strains ( C ) . IFT121 , IFT38 FLA8 accumulated in cilia of both IFT54Δ261 - 275 IFT54Δ342 - 356 mutants . D1bLIC was relatively reduced in IFT54Δ261 - 275 mutant cilia but was substantially increased in IFT54Δ342 - 356 mutant cilia ( D ) . Ratios of amounts from one representative experiment are in parentheses .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD 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-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

(C, D) Immunoblot analysis of whole cells (C) and isolated cilia (D) of wild type and the three IFT54 deletion mutants. The membranes were probed with antibodies against IFT complex and motor proteins as indicated. Wild-type levels of the various IFT components were detected in the whole cell samples of the deletion strains (C). IFT121, IFT38 and FLA8 accumulated in cilia of both IFT54Δ261-275 and IFT54Δ342-356 mutants. D1bLIC was relatively reduced in IFT54Δ261-275 mutant cilia but was substantially increased in IFT54Δ342-356 mutant cilia (D). Ratios of protein amounts from one representative experiment are in parentheses.


( A ) Kymograms showing trajectories of IFT trains inside cilia as visualized with IFT43 - YFP . An ift43 rescue expressing IFT43 - YFP ( as a control ) IFT54Δ342 - 356 mutant expressing IFT43 - YFP were analyzed by live imaging via TIRF microscopy .

O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43-YFP. An ift43 rescue strain expressing IFT43-YFP (as a control) and the IFT54Δ342-356 mutant expressing IFT43-YFP were analyzed by live imaging via TIRF microscopy.


( B - C ) Velocities ( B ) frequencies ( C ) of anterograde retrograde IFT of IFT43 - YFP in control IFT54Δ342 - 356 mutant . n represents number of cilia assayed from three independent experiments . Values show mean ± S . D . Unpaired two - Student ' s t , * * * , p < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of IFT43-YFP in control and the IFT54Δ342-356 mutant. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, ***, p<0.0001.


( D ) Deletion of residues 342 - 356 of IFT54 increases its interaction with kinesin - II . extracts from wild type ( WT ) , ift54 expressing HA - tagged full - ( FL ) IFT54 or IFT54Δ342 - 356 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with indicated antibodies . normalized ratios of FLA8 FLA10 in immunoprecipitates ( IFT54 versus mutant ) are 1 : 3 . 90 1 : 3 . 13 , respectively .

O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O

(D) Deletion of residues 342-356 of IFT54 increases its interaction with kinesin-II. Cell extracts from wild type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ342-356 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 versus the mutant) are 1:3.90 and 1:3.13, respectively.


( E ) ATP treatment induces dissociation of IFT54 but not IFT54Δ342 - 356 mutant from kinesin - II . Immunoprecipitation experiments were performed as shown in ( D ) in presence or absence of ATP . normalized ratios of FLA8 FLA10 in immunoprecipitates ( IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 37 : 1 . 70 : 1 . 38 1 : 0 . 35 : 1 . 47 : 1 . 45 , respectively .

O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) ATP treatment induces dissociation of IFT54 but not IFT54Δ342-356 mutant from kinesin-II. Immunoprecipitation experiments were performed as shown in (D) in the presence or absence of ATP. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.37:1.70:1.38 and 1:0.35:1.47:1.45, respectively.


( F ) of interaction of IFT54 IFT54Δ342 - 356 mutant with kinesin - II by GST - pulldown . expressed GST , GST - tagged IFT54 or IFT54Δ342 - 356 mutant was mixed respectively with recombinant kinesin - II purified from insect in presence or absence of ATP followed by GST pulldown immunoblotting . normalized ratios of KAP , FLA10 FLA8 in pull - down samples ( GST - IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 51 : 1 . 31 : 1 . 44 , 1 : 0 . 55 : 1 . 29 : 1 . 19 1 : 0 . 50 : 1 . 31 : 1 . 29 , respectively .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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) Analysis of the interaction of IFT54 and IFT54Δ342-356 mutant with kinesin-II by GST-pulldown assay. Bacterial expressed GST, GST-tagged IFT54 or IFT54Δ342-356 mutant was mixed respectively with recombinant kinesin-II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting. The normalized ratios of KAP, FLA10 and FLA8 in the pull-down samples (GST-IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.51:1.31:1.44, 1:0.55:1.29:1.19 and 1:0.50:1.31:1.29, respectively.


( G ) Interaction of IFT54 with kinesin - II is conserved in mammalian . extracts from HEK293T expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ535 - 549 ( corresponding deletion mutant of IFT54Δ342 - 356 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP KIF3A antibodies , respectively . Please note , deletion mutant had an increased interaction with kinesin - II subunit KIF3A relative to control . normalized of KIF3A in immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 1 . 76 .

O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O

(G) Interaction of IFT54 with kinesin-II is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ535-549 (corresponding deletion mutant of IFT54Δ342-356) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and KIF3A antibodies, respectively. Please note, the deletion mutant had an increased interaction with kinesin-II subunit KIF3A relative to the control. The normalized ratio of KIF3A in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:1.76.


( A ) Kymograms showing trajectories of IFT trains inside cilia as visualized with D1bLIC - YFP . Wild - type ( WT ) IFT54Δ261 - 275 mutant expressing D1bLIC - YFP were analyzed by live imaging via TIRF microscopy .

O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC-YFP. Wild-type (WT) and IFT54Δ261-275 mutant cells expressing D1bLIC-YFP were analyzed by live imaging via TIRF microscopy.


( B , C ) Velocities ( B ) frequencies ( C ) of anterograde retrograde IFT of D1bLIC - YFP . n represents number of cilia assayed from four independent experiments . Values show mean ± S . D . Unpaired two - Student ' s t , n . s . : no significance ; * * * , p < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of D1bLIC-YFP. n represents the number of cilia assayed from four independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( D ) Kymograms of IFT43 - YFP of WT mutant cilia .

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(D) Kymograms of IFT43-YFP of WT and mutant cilia.


( E , F ) Velocities ( E ) frequencies ( F ) of anterograde retrograde IFT of IFT43 - YFP . n represents number of cilia assayed from three independent experiments . Values show mean ± S . D . Unpaired two - Student ' s t , n . s . : no significance ; * * * , p < 0 . 0001 .

(E, F) Velocities (E) and frequencies (F) of anterograde and retrograde IFT of IFT43-YFP. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( G ) Yeast two hybrid for interaction between IFT54 IFT dynein subunits . Yeast that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine , tryptophan , histidine , adenine ( - 4 ) or lacking leucine tryptophan ( - 2 ) . DHC1bT , DHC1b tail domain ; Empty AD or BD vectors were used as control .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

(G) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits. Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or lacking leucine and tryptophan (-2). DHC1bT, DHC1b tail domain; Empty AD or BD vectors were used as control.


( H ) Interaction of IFT54 IFT54Δ261 - 275 with D1bLIC by GST - pulldown . expressed GST , GST - IFT54 or GST - IFT54Δ261 - 275 were mixed respectively with His - D1bLIC followed by GST pulldown immunoblotting with anti - GST anti - His antibodies . normalized of His - D1bLIC in pull - down samples ( GST - IFT54 versus mutant ) is 1 : 0 . 32 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(H) Interaction of IFT54 and IFT54Δ261-275 with D1bLIC by GST-pulldown assay. Bacterial expressed GST, GST-IFT54 or GST-IFT54Δ261-275 were mixed respectively with His-D1bLIC followed by GST pulldown and immunoblotting with anti-GST and anti-His antibodies. The normalized ratio of His-D1bLIC in the pull-down samples (GST-IFT54 versus mutant) is 1:0.32.


( I ) Co - immunoprecipitation of IFT54 IFT54Δ261 - 275 with D1bLIC . extracts from wild - type ( WT ) , ift54 expressing HA - tagged full - ( FL ) IFT54 or IFT54Δ261 - 275 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with indicated antibodies . Please note , IFT54Δ261 - 275 mutant exhibited weaker interaction with D1bLIC relative to control . normalized of D1bLIC in immunoprecipitates ( IFT54 - HA versus mutant ) is 1 : 0 . 37 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(I) Co-immunoprecipitation of IFT54 and IFT54Δ261-275 with D1bLIC. Cell extracts from wild-type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ261-275 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. Please note, IFT54Δ261-275 mutant exhibited weaker interaction with D1bLIC relative to the control. The normalized ratio of D1bLIC in the immunoprecipitates (IFT54-HA versus mutant) is 1:0.37.


( J ) Interaction of IFT54 with D1bLIC is conserved in mammalian . extracts from HEK293T expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ460 - 474 ( corresponding deletion mutant of IFT54Δ261 - 275 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP DYNC2LI1 , human homologue of D1bLIC , antibodies , respectively . Please note , deletion mutant weakened interaction with IFT dynein subunit DYNC2LI1 relative to control . normalized of DYNC2LI1 in immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 0 . 49 .

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(J) Interaction of IFT54 with D1bLIC is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ460-474 (corresponding deletion mutant of IFT54Δ261-275) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and DYNC2LI1, human homologue of D1bLIC, antibodies, respectively. Please note, the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control. The normalized ratio of DYNC2LI1 in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:0.49.


( A ) EM of KMnO4 - fixed pex3 atg1 WT grown for 16 h on MM - M / G . inset shows a cluster of vesicles ( enlarged from boxed region ) .

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(A) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region).


( B ) iEM of pex3 atg1 using α - Pex14 antibodies .

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(B) iEM analysis of pex3 atg1 cells using α-Pex14 antibodies.


( C D ) FM of pex3 atg1 producing Pex14 - mCherry ER marker BiPN30 - eGFP - HDEL ( C ) , or Pex14 - mGFP complemented with Mitotracker orange ( D ) .

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(C and D) FM images of pex3 atg1 cells producing Pex14-mCherry and the ER marker BiPN30-eGFP-HDEL (C), or Pex14-mGFP complemented with Mitotracker orange staining (D).


( E ) Electron tomography of a serial - sectioned pex3 atg1 ( a ) containing a perinuclear cluster ( b , arrows ) . ( d - f ) 10 - nm - thin digital slices through tomogram reconstruction ( viewing direction indicated in c ) revealed vesicles ( black arrows ) reticular structures ( red arrows ) . surface - rendered reconstruction in g shows reticulovesicular structures in 3D viewed at right angles from d . CW , wall ; M , mitochondrion ; N , nucleus ; P , peroxisome ; V , vacuole . Bars : ( A ) 500 nm ; ( A , inset ; B ) 100 nm ; ( C D ) 2 . 5 µm ; ( E ) 250 nm .

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(E) Electron tomography analysis of a serial-sectioned pex3 atg1 cell (a) containing a perinuclear membrane cluster (b, arrows). (d-f) 10-nm-thin digital slices through the tomogram reconstruction (viewing direction indicated in c) revealed vesicles (black arrows) and reticular structures (red arrows). The surface-rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d. CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bars: (A) 500 nm; (A, inset; and B) 100 nm; (C and D) 2.5 µm; (E) 250 nm.


( A ) iEM of pex3 using α - Pex14 antibodies , identifying structures ( arrows ) in vicinity of mitochondria . CW , wall ; M , mitochondrion . ( B ) of Pex14 - mGFP spots in pex3 pex3 atg1 .

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(A) iEM analysis of pex3 cells using α-Pex14 antibodies, identifying structures (arrows) in the vicinity of mitochondria. CW, cell wall; M, mitochondrion. (B) Quantification of Pex14-mGFP spots in pex3 and pex3 atg1 cells.


( C ) FM of pex3 atg1 or pex3 , producing Pex14 - mGFP complemented with FM4 - 64 vacuolar . inset ( enlarged from boxed region ) shows optimized intensities for pex3 , highlighting Pex14 - mGFP spot vacuolar mGFP .

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(C) FM images of pex3 atg1 or pex3 cells, producing Pex14-mGFP complemented with FM4-64 vacuolar staining. The inset (enlarged from the boxed region) shows optimized intensities for pex3 cells, highlighting the Pex14-mGFP spot and vacuolar mGFP.


( D ) WB of grown for 16 h on MM - M / G using α - Pex11 or α - Pex14 antibodies . Pyruvate carboxylase 1 ( Pyc1 ) was used as a loading control . Error bars indicate . Bars : ( A ) 100 nm ; ( C ) 1 µm .

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(D) WB analysis of cells grown for 16 h on MM-M/G using α-Pex11 or α-Pex14 antibodies. Pyruvate carboxylase 1 (Pyc1) was used as a loading control. Error bars indicate SEM. Bars: (A) 100 nm; (C) 1 µm.


( A ) FM of pex3 atg1 grown for 16 h on MM - M / G . produced Pex14 - mCherry C - terminal mGFP fusions of indicated .

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(A) FM images of pex3 atg1 cells grown for 16 h on MM-M/G. Cells produced Pex14-mCherry and C-terminal mGFP fusions of the indicated proteins.


( B ) WB of WT ( 1 ) pex3 atg1 ( 2 ) , grown for 6 or 16 h on MM - M / G .

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(B) WB analysis of WT (1) and pex3 atg1 cells (2), grown for 6 or 16 h on MM-M/G.


( C ) FM showing mGFP - in pex3 atg1 producing Pex14 - mCherry ( control ) or Pex14 - mCherry together with indicated mGFP fusion . were grown for 6 h on MM - M / G .

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(C) FM images showing mGFP-fluorescence in pex3 atg1 cells producing Pex14-mCherry (control) or Pex14-mCherry together with the indicated mGFP fusion protein. Cells were grown for 6 h on MM-M/G.


( D E ) iEM of pex3 atg1 using α - Pex5 ( D ) or α - alcohol oxidase antibodies ( E ) .

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(D and E) iEM of pex3 atg1 cells using α-Pex5 (D) or α-alcohol oxidase antibodies (E).


( F ) fractionation of two indicated strains ( seperated by horizontal broken ) . Postnuclear supernatants ( PNS ) were subjected to differential centrifugation resulting in a 30 , 000 g organelle pellet ( P ) supernatant fraction ( S ) .

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(F) Cell fractionation of the two indicated strains (seperated by the horizontal broken line). Postnuclear supernatants (PNS) were subjected to differential centrifugation resulting in a 30,000 g organelle pellet (P) and supernatant fraction (S).


( G ) Flotation of organelle pellet showing distribution of indicated in top ( 1 ) to bottom ( 10 ) fractions .

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(G) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top (1) to bottom (10) fractions.


( H ) Colocalization of Pex8 - GFP Pex14 - mCherry in pex10 .

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(H) Colocalization of Pex8-GFP and Pex14-mCherry in pex10 cells.


( A ) FM of pex3 with Pex14 - mCherry upon Pex3 - eGFP reintroduction after shifting from MM - Glu with ammonium sulfate to MM - M / G with methylamine .

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(A) FM images of pex3 cells with Pex14-mCherry upon Pex3-eGFP reintroduction after shifting cells from MM-Glu with ammonium sulfate to MM-M/G with methylamine.