( A ) The vasculature in P5 retinas from ( A ) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence . ( B ) The fractional distance migrated to the edge of the retina was quantified . There was no statistically significant difference ( p = 0 . 305 , Student ' s 2 - tailed t - test , n = 6 mice for each group ) . The average value of 8 measurements ( 4 retina leaflets measured per eye on both eyes ) was used for each mouse . ( C ) The number of vascular branchpoints was counted . There was no statistically significant difference ( p = 0 . 25 , Student ' s 2 - tailed t - test , n = 6 for control and n = 5 for mutant ) .

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(A) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student's 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student's 2-tailed t-test, n=6 for control and n=5 for mutant).


( D ) Control and mutant adult mouse aortas were stained for GM130 ( green ) to mark the Golgi , CD144 ( red ) to mark ECs and DAPI ( blue ) to label DNA . ( E ) The angle between the Golgi , nucleus and tissue axis was measured and binned as ' left ' , ' right ' , ' upstream ' or ' downstream ' . ( F ) Six areas in the aorta were analyzed . ( G ) There was no difference in Golgi distribution at any single region or overall , nor was there a difference in the variance of the individual un - binned angles . Error bars are + 1 S . E . M . Scale bars are 500μm . n = 3 control and mutant mice for each region except region 2 where n = 3 control and n = 2 mutant mice .

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(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.


( B ) Loss of EC cilia by Tek - Cre increases atherosclerosis in the aortas of Apoe - / - mice on a high - fat high - cholesterol diet as visualized by Oil Red O ( ORO ) staining ( left panels ) whereas loss of cilia genes in blood by Mx1 - Cre has no effect ( right panels ) . ( C ) Cropped views of the boxed areas in ( B ) showing increased atherosclerosis in mice with Tek - Cre but not Mx1 - Cre mediated loss of Ift88 in the aortic arch . ( D - G ) Scatter plots of the percent ORO - positive area of the thoracic aorta in ( D - E ) Tek - Cre and ( F - G ) Mx1 - Cre mice , separated by ( D , F ) females and ( E , G ) males . Tek - Cre Apoe - / - Ift88C / - mice had a 58 . 9 % ( p = 0 . 0135 , Student ' s 2 - tailed t - test , n = 13 control and n = 10 mutant mice ) and 67 % ( p = 0 . 0106 , Student ' s 2 - tailed t - test , n = 17 control and n = 14 mutant ) increase in ORO - positive area versus control littermates in females and males , respectively . Mx1 - Cre Apoe - / - Ift88C / - mice had no significant difference in ORO - positive area in females ( p = 0 . 6643 , Student ' s 2 - tailed t - test , n = 7 control and mutant mice ) or males ( p = 0 . 9381 , Student ' s 2 - tailed t - test , n = 9 control and n = 7 mutant mice ) . Black bars represent the mean and error bars are + / - 1 S . E . M . Aortas depicted in ( B - C ) are red in the scatter plots .

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(B) Loss of EC cilia by Tek-Cre increases atherosclerosis in the aortas of Apoe-/- mice on a high-fat high-cholesterol diet as visualized by Oil Red O (ORO) staining (left panels) whereas loss of cilia genes in blood by Mx1-Cre has no effect (right panels).(C) Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek-Cre but not Mx1-Cre mediated loss of Ift88 in the aortic arch.(D-G) Scatter plots of the percent ORO-positive area of the thoracic aorta in (D-E) Tek-Cre and (F-G) Mx1-Cre mice, separated by (D, F) females and (E, G) males. Tek-Cre Apoe-/- Ift88C/- mice had a 58.9% (p=0.0135, Student's 2-tailed t-test, n=13 control and n=10 mutant mice) and 67% (p=0.0106, Student's 2-tailed t-test, n=17 control and n=14 mutant) increase in ORO-positive area versus control littermates in females and males, respectively. Mx1-Cre Apoe-/- Ift88C/- mice had no significant difference in ORO-positive area in females (p=0.6643, Student's 2-tailed t-test, n=7 control and mutant mice) or males (p=0.9381, Student's 2-tailed t-test, n=9 control and n=7 mutant mice). Black bars represent the mean and error bars are +/- 1 S.E.M. Aortas depicted in (B-C) are red in the scatter plots.


( A ) Transcripts for the inflammatory cytokines Il1b , Il6 , Tnf1 and Tgfb1 , the adhesion molecules Vcam1 and Sele , the lymphocyte marker Nos2 and macrophage marker Cd68 , and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe - / - mice lacking EC cilia by RT - qPCR , while transcripts of the Hh target genes Gli1 , Ptc1 , and Ptc2 were not . Error bars are + 1 S . E . M . n = 8 control and n = 10 mutant mice of mixed sex . P - values calculated by Student ' s 2 - tailed t - test .

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(A) Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1 and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe-/- mice lacking EC cilia by RT-qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 S.E.M. n=8 control and n=10 mutant mice of mixed sex. P-values calculated by Student's 2-tailed t-test.


( B ) Pkd2 localizes to EC cilia in vivo . Adult mouse aortas were stained for Pkd2 , acetylated α - Tubulin , and Hoechst to label DNA . Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells . Arrowheads point out cilia . Example cilium highlighted in inset .

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(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.


( C ) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia . Aortas from control and mutant mice were stained for eNOS , p - eNOS - S1176 , acetylated α - Tubulin , and Hoechst to label DNA .

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(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.


( D ) The ratio of the total fluorescence p - eNOS - S1176 to total eNOS in the upper thoracic aorta ( near the first intercostal artery ) was calculated for control and mutant mice . The relative p - eNOS - S1176 to eNOS ratio was reduced in the mutant to 72 . 7 % the level of control siblings ( p = 0 . 009 , paired 2 - tailed ratio t - test , n = 7 sets of mice ) . Error bar is + 1 S . E . M . Four sets consisted of one control mouse and one mutant mouse . Three sets consisted of two control or mutant mice and the average value of the two was used . Scale bars are 10μm .

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(D) The ratio of the total fluorescence p-eNOS-S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p-eNOS-S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (p=0.009, paired 2-tailed ratio t-test, n=7 sets of mice). Error bar is +1 S.E.M. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used. Scale bars are 10μm.


C . Cell cycle fractions inferred from Cycleflow in IFN - β - treated NSCs . n = 3 biological replicates .

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C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. n = 3 biological replicates.


C , D . Ribo - Seq results depicting translation efficiency as the interaction of log2 fold changes ( LFC ) between footprints mapped to the CDS , referred to as " CDS counts " , and total RNA after 2h or 14h IFN - β treatment . FDR < 10 % , LR - Test in DESeq2 . Genes with a p value < 0 . 1 after FDR correction are highlighted . n = 4 biological replicates .

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C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. n = 4 biological replicates.


I . Representative WB and phosphorylation levels quantification ( log2FC ) of the mTOR - related proteins ribosomal protein S6 kinase ( S6K ) , ribosomal protein S6 ( S6 ) and eIF4E - binding protein 1 ( 4E - BP1 ) from NSCs treated with IFN - β and normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - 4E - BP1S65 * ( p = 0 . 021 ) , p - S6S235 / 236 & p - S6S240 / 244 * * * ( p < 0 . 001 ) , p - S6KT389 * * ( p = 0 . 004 ) . n = biological replicates specified . p ≡ p - value .

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I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.


A . Representative WB image and quantification ( log2FC ) of p - TSC2Thr1462 & p - AktThr308 from IFNβ - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - TSC2T1462 * * * ( p < 0 . 001 ) , p - AktT308 * * ( p = 0 . 003 ) . n = 4 biological replicates . Data information : p ≡ p - value .

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A. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates. Data information: p ≡ p-value.


C . Representative WB images and quantifications ( log2FC ) of p - eIF2αS51 in IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - eIF2αS51 * * ( p = 0 . 003724 ) . n = 4 biological replicates . Data information : p ≡ p - value .

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C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates. Data information: p ≡ p-value.


E . Representative WB image and quantifications ( log2FC ) of p - Rb1Ser780 from IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - Rb1S780 * * * ( p = 5 . 37e - 06 ) . n = 3 biological replicates . Data information : p ≡ p - value .

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E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates. Data information: p ≡ p-value.


I , J . Luciferase activity controlled by the upstream 5 ' UTR fragment from Sox2 ( WT and mutant ) , Rps21 ( WT and mutant ) and Actb . Treatments were applied for 12h . Data is normalized to vehicle and is represented as mean ± SD . n = 6 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test ( p - value specified ) . Data information : p ≡ p - value .

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I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified). Data information: p ≡ p-value.


B . vSVZ niche FACS quantifications of total NSCs ( Total NSCs ) & immunofluorescence quantification of aNSCs ( Fraction of aNSCs ) in WT or IFNAGR KO mice at 2 , 7 and 22 months old ( MO ) . Data from individual mice is plotted . Inferred dynamics are depicted as a blue ( WT ) or red ( IFNAGR KO ) line over time . Sample sizes : WT total at 60 days : 8 mice , WT total at 210 days : 3 mice , WT total at 660 days : 3 mice , WT fraction aNSCs at 60 days : 3 mice , WT fraction aNSCs at 660 days : 4 mice , IFNAGRKO total at 60 days : 8 mice , IFNAGRKO total at 180 days : 3 mice , IFNAGRKO total at 660 days : 3 mice , IFNAGRKO fraction aNSCs at 60 days : 3 mice , IFNAGRKO fraction aNSCs at 660 days : 3 mice .

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B. vSVZ niche FACS quantifications of total NSCs (Total NSCs) & immunofluorescence quantification of aNSCs (Fraction of aNSCs) in WT or IFNAGR KO mice at 2, 7 and 22 months old (MO). Data from individual mice is plotted. Inferred dynamics are depicted as a blue (WT) or red (IFNAGR KO) line over time. Sample sizes: WT total at 60 days: 8 mice, WT total at 210 days: 3 mice, WT total at 660 days: 3 mice, WT fraction aNSCs at 60 days: 3 mice, WT fraction aNSCs at 660 days: 4 mice, IFNAGRKO total at 60 days: 8 mice, IFNAGRKO total at 180 days: 3 mice, IFNAGRKO total at 660 days: 3 mice, IFNAGRKO fraction aNSCs at 60 days: 3 mice, IFNAGRKO fraction aNSCs at 660 days: 3 mice.


( a ) RT - PCR analysis of LC3 , IL - 8 , CHOP and GADD34 in U2OS cells subjected to glutamine deprivation ( − Q ) or exposed to TPG ( 1 μM ) . (

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(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (


( b ) Immunoblot analysis of ER stress - and autophagy - related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment . Samples are run in triplicate .

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(b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.


( c ) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine .

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(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.


( d ) IL - 8 enzyme - linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine . Error bars in all figures represent s . d . of three biological replicates .

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(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.


( a ) Autophagosomes were visualized in U2OS cells stably expressing a GFP - LC3 reporter construct . GFP - labelled puncta were examined after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine , with or without co - addition of 100 nM CCI - 779 ( CCI ) and 400 nM BafA1 . Scale bar , 10 μm . ( b ) Graphical summary of experiments performed as described in a . Percentage of cells with > 10 puncta per cell from three independent experiments is depicted . Bars represent mean ± s . d . from three independent experiments ( > 50 cells per experiment ) . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . * P0 . 05 .

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(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.


( c ) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1 . Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine - deficient medium . Autophagic activity was monitored by detection of p62 and LC3 - II proteins .

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(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.


d ) U2OS mCherry - GFP - LC3 cells were cultured in the presence ( + Q ) or absence ( − Q ) of glutamine for 18 h . Red vesicles denote autolysosomes , whereas yellow vesicles represent autophagosomes . Bars indicate numbers of yellow vesicles ( autophagosomes ) or red vesicles ( autolysosomes ) per cell ± s . d . ( e ) Images of U2OS mCherry - GFP - LC3 cells cultured for 18 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

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d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( f ) Phagophore formation in mCherry - ULK1 . Scale bar , 10 μm . ( g ) mCherry - ATG5 - expressing U2OS cells after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

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(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( a ) Colocalization of lysosomes ( Lysotracker Green ) with phagophores ( mCherry - ULK1 and mCherry - glutamine puncta ) in glutamine - deprived U2OS cells . Scale bar , 25 μm .

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(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.


( b ) Colocalization of LAMP1 - positive lysosomes with GFP - LC3 - labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells . Scale bar , 15 μm .

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(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.


( c ) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment . Nuclei were stained with DAPI . Scale bar , 15 μm .

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(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.


( d ) Immunofluorescence images of cells stained with LAMP1 ( lysosomes ) and the Golgi marker , RCAS1 , following glutamine deprivation . Nuclei were stained with DAPI . Scale bar , 15 μm .

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(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.


( e ) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids . Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62 . (

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(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. (


( f ) Immunofluorescence staining for LAMP1 ( green ) and mTOR ( red ) in U2OS cells subjected to glutamine deprivation ( 24 h ) , in the presence of 100 nM WYE - 125132 ( WYE - 132 ) . − AA denotes cells deprived of all amino acids . Scale bar , 15 μm . Error bars in all figures represent s . d . of three biological replicates . Ess . , essential .

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(f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.


( a ) Immunoblot of autophagy - related proteins in U2OS cells expressing a control TALEN ( U2OS - TAL2 - C1 ) or two independent subcloned TALEN cell lines targeting ATG7 ( U2OS - TAL2 - A7 , U2OS - TAL2 - A8 ) . p62 long and short refer to either long or short exposure times during film development .

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(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.


( b ) U2OS cells expressing an ATG7 - TALEN ( TAL2 - ATG7 ) or control TALEN ( TAL2 - CTL ) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine - replete medium . Cells were then stained with SRB and absorbance was read at 540 nM . Graph shows average results from five clonal sub - lines for each condition . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . P0 . 05 . ( c ) Representative images of SRB - stained clones in

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(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in b


d ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of U2OS - TAL2 - CTL - C1 ( control ) and U2OS - TAL2 - ATG7 - A8 ( ATG7 knockout ) following 24 h glutamine deprivation .

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d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.


( e ) IL - 8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE - 125132 . Scale bar , 15 μm .

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(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.


( b ) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE - 125132 .

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(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.


( c ) IL - 8 enzyme - linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 .

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(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.


( d ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) Immunoblot analysis of autophagy , ER stress - and mTOR - related proteins , and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 .

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(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.


( b ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK ( JNK # 1 and JNK # 2 ) and subjected to 24 h glutamine deprivation .

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(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.


( c ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of conditioned media from the experiment described in b .

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(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.


( d ) Immunoblot analysis of JNK protein expression in JNK siRNA - treated cells .

(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.


e ) RT - PCR analysis of IL - 8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 ( JNKi ) .

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e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).


( f ) IL - 8 ELISA of conditioned medium from the experiment described in e .

(f) IL-8 ELISA of conditioned medium from the experiment described in e.


( g ) IL - 8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 ( IRE - A and IRE - B ) and subjected to glutamine deprivation for 24 h .

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(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.


( h ) RT - PCR analysis of IL - 8 mRNA expression from experiment described in g . ( i ) RT - PCR analysis of CHOP mRNA expression from experiment described in g . ( j ) RT - PCR analysis of IRE1 mRNA expression from experiment described in g .

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(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.


( k ) Immunoblot analysis of experiment described in g showing phosphorylation of JNK . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) on U2OS cells starved of glutamine or treated with BPTES ( 10 μM ) for 24 h .

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(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.


( b ) RT - PCR analysis of IL - 8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG .

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(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.


( c ) Metabolite analysis of U2OS cells starved of glutamine , treated with BPTES or glutamine - starved co - treated with DMαKG .

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(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.


( d ) IL - 8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co - treated with DMαKG .

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(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.


( e ) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( E ) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay . Interaction between bls - CRBN and FLAG - GST - protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology .

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(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.


( F ) In vitro binding assay for thalidomide , pomalidomide and lenalidomide . Interaction between bls - CRBN and FLAG - GST - PLZF in the presence of DMSO ( 3 . 125 , 6 . 25 , 12 . 5 , 25 , 50 , 100 or 200 µM ) thalidomide , pomalidomide or lenalidomide was analysed using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) .

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(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).


( E ) In vitro binding and ubiquitination assay of AGIA - PLZF . Empty vector , AGIA - PLZF or FLAG - CRBN expressing HEK293T cells were lysed and the lysates were mixed . The first immunoprecipitation with anti - AGIA or anti - FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide . The purified AGIA - PLZF or CRL4FLAG - CRBN complex , including AGIA - PLZF and FLAG - CRBN , was incubated with recombinant E1 , E2 and HA - ubiquitin in the presence of DMSO or 200 µM lenalidomide . The second immunoprecipitation was performed using anti - AGIA antibody . Ubiquitination of PLZF was analysed by immunoblot .

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(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.


( B ) In vitro binding assay using truncated PLZF . Thalidomide - dependent interaction between bls - CRBN and FLAG - GST - PLZF - full length ( FL ) or truncated FLAG - GST - PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * * * P < 0 .

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(B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001


( D ) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . ( E ) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP - 1 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . Data information : Data in bar graphs in ( D ) and ( E ) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100 . Error bars donated ± standard deviation ( n = 3 ) and P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ) .

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(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).


( A , B ) Immunoblot analysis of AGIA - MmPLZF ( A ) or - MmSALL4 ( B ) in FLAG - MmCRBN - WT , - MmCRBN - I391V or - MmCRBN - V380E / I391V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . Data information : Data in bar graphs were calculated as relative Mm ) PLZF or SALL4 band intensity with Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ) .

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(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( C , D ) Immunoblot analysis of AGIA - GgPLZF ( C ) or - GgSALL4 ( D ) in FLAG - GgCRBN - WT or - GgCRBN - I390V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . ( E , F ) Immunoblot analysis of AGIA - Mm PLZF ( E ) or - MmSALL4 ( F ) in FLAG - MmCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . ( G , H ) Immunoblot analysis of AGIA - GgPLZF ( G ) or - GgSALL4 ( H ) in FLAG - GgCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . Data information : Data in bar graphs were calculated as relative ( Gg or Mm ) PLZF or SALL4 band intensity with ( Gg or Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ) .

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(C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( E ) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl ( n = 10 ) or shPLZF ( n = 9 ) were analysed by Victoria blue staining . h ; humerus , r ; radius , u ; ulna , fe ; femur , fi ; fibula , t ; tibia . Data in bar graphs were calculated as relative bone length with bone length of shControl as 100 . Error bars denote ± standard deviation ( forelimb and hindlimb shControl ( n = 3 ) , forelimb shPLZF ( n = 5 ) or hindlimb shPLZF ( n = 6 ) ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) .

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(E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).


( F ) Photographs show E4 chicken embryos treated with HBC ( control , n = 10 , C1 , C3 , C5 and C6 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 , T1 , T2 , T6 and T9 ) or 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 , H1 , H2 , H8 and H9 ) corresponding to immunoblot analysis in Fig 7G . Red arrows show treated regions . Data information Data in bar graphs in ( F ) were calculated as relative limb bud length with left limb bud length as one . Error bars denote ± standard deviation ( F ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * P < 0 . 01 , and * * * P < 0 . 001 ) .

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

(F) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001).


( G ) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud . Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC ( n = 10 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 ) and 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 ) . Data information : Data in bar graphs in ( G ) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4 , respectively . The band intensity of HBC was 100 . Error bars denote ± standard deviation ( ( E ) , n = 3 or ( G ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , and * * P < 0 . 01 ) .

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

(G) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).


E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS - GgPLZF was treated with HBC or 1 µg / µl ( 3 . 9 mM ) thalidomide , and the chicken forelimb bud phenotypes were observed in bright field ( BF ) or fluorescence of enhanced GFP ( EGFP ) . Fgf10 / Fgf8 expression was analysed by section by in situ hybridisation . ( Q ) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in ( Q ) were calculated as relative limb bud length with limb bud length of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 ) .

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

E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation. (Q) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).


( R ) Quantitative analysis of Fgf10 / Fgf8 expression Data in bar graphs in ( R ) were calculated as relative Fgf10 / Fgf8 mRNA expressions with Fgf10 / Fgf8 mRNA expressions of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * P < 0 . 05 and * * P < 0 . 01 ) .

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(R) Quantitative analysis of Fgf10/Fgf8 expression Data in bar graphs in (R) were calculated as relative Fgf10/Fgf8 mRNA expressions with Fgf10/Fgf8 mRNA expressions of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01).


A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells . MIC10 KO cells show a drastic reduction of MIC13 , MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged . MIC60 KO cells show a drastic reduction of protein levels of all MICOS components .

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A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components.


B Representative electron micrographs of WT , MIC10 KO and MIC60 KO HAP1 cells . Scale bar 500 nm .

B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm.


C Quantification of CJs per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) .

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C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).


D Quantification of cristae per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) .

D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).


E Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 034 for basal ( using one sample t - test ) and * P = 0 . 018 for maximum respiration ( using unpaired Student ' s t - test ) .

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E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).


F Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 01 for basal ( using one sample t - test ) , * * * P = 0 . 0004 for maximum respiration and * P = 0 . 012 for non - mitochondrial respiration ( using unpaired Student ' s t - test ) .

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F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).


A Representative STED super - resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies ( top panel ) and TOMM70 ( middle panel ) . Bottom panel shows merged images . Arrowheads show colocalization of MIC60 and TOMM70 punctae . Scale bar 500 nm .

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A Representative STED super-resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies (top panel) and TOMM70 (middle panel). Bottom panel shows merged images. Arrowheads show colocalization of MIC60 and TOMM70 punctae. Scale bar 500 nm.


B Quantification of median interpunctae distance ( IPD ) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Median IPD from n = 10 - 16 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . ns , not significant , unpaired Student ' s t - test .

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B Quantification of median interpunctae distance (IPD) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Median IPD from n = 10-16 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ns, not significant, unpaired Student's t-test.


FRAP curves ( curve fitted ) of WT Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown .

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FRAP curves (curve fitted) of WT Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.


C FRAP curves ( curve fitted ) of MIC13 KO Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown .

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C FRAP curves (curve fitted) of MIC13 KO Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.


D Histogram showing average percentage of mobile fractions , obtained from curve fits of all mitochondria expressing GFP - tagged versions of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) in WT and MIC13 KO Hela cells ( data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments , 6 - 10 mitochondria from each experiment ) .

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D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment).


A - D Representative single particle tracks of cells expressing MIC60 - ( A ) and MIC10 - SNAP ( B ) in WT Hela cells and MIC60 - ( C ) and MIC10 - SNAP ( D ) in MIC13 KO Hela cells , stained with silicone - rhodamine dye , and imaged at a rate of 33 ms / frame . Single tracks were colour - coded according to temporal appearance . Scale Bar 5 µm . Insets in each image show zoomed - in images of few representative tracks .

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A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.


E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients ( insD ) in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP stained with silicone - rhodamine . Number of tracks analysed ( obtained from 2 independent experiments ) : n = 2541 , 2540 , 3560 and 1441 in WT Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , MIC13 KO Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , respectively . ( * * * * P ≤ 0 . 0001 for all possible comparisons , unpaired Student ' s t - test ) .

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E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).


F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10 - SNAP . Number of corresponding subtracks analysed ( obtained from 2 independent experiments ) : n = 2561 , 2443 , 3402 , 1360 , 561 and 1110 . Data are mean ± SEM . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired Student ' s t - test .

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F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.


A , C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT ( A ) and MIC13 KO ( C ) Hela cells expressing MIC10 - SNAP stained with silicone - rhodamine . Box in ( A and C ) mark selection shown as a zoom in panel ( B and D ) respectively . Scale bar 500 nm .

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A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.


E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10 - SNAP ( 3 independent experiments , 5 - 8 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * P = 0 . 026 ( merging and splitting events in WT cells ) and * * * * P < 0 . 0001 ( merging and splitting events of WT vs . MIC13 KO ) , unpaired Student ' s t - test .

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E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test.


A Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 2 . 5 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 1 . 3 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing MIC13 - SNAP imaged at a time interval of 1 . 3 s / frame . Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13 - SNAP . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E First image from ( D ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM . Magenta boxes depict intensity maxima at IBM .

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E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.


A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm . B Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in WT Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Green ` Y ´ represent Y - type imminent mergence event . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in WT Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green `Y´ represent Y- type imminent mergence event . Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) showing MIC13 KO Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in MIC13 KO Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s) showing MIC13 KO Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in MIC13 KO Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E First image from ( B ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM . The represented IBM area is superimposed from intensities of MIC13 - SNAP from Figure 6F . Magenta box depicts intensity maxima at cristae membrane apart from IBM .

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E First image from (B) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM. The represented IBM area is superimposed from intensities of MIC13-SNAP from Figure 6F. Magenta box depicts intensity maxima at cristae membrane apart from IBM.


G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I - SNAP ( 3 independent experiments , 3 - 7 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * * * * P < 0 . 0001 , unpaired Student ' s t - test .

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G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I-SNAP (3 independent experiments, 3-7 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. ****P < 0.0001, unpaired Student's t-test.


A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( B ) ( red hot LUT ) . Scale bar 500 nm . B Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green asterisks show merging events and redistribution of TMRM intensity from a high - intensity cristae to a low - intensity cristae ( two such instances are shown at 3 and 7 . 4 s ) . Green arrows pointing inward connected by solid line at 1 . 5 and 5 . 9 s indicate imminent merging events . Scale bar 500 nm .

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A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (B) (red hot LUT). Scale bar 500 nm. B Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green asterisks show merging events and redistribution of TMRM intensity from a high-intensity cristae to a low-intensity cristae (two such instances are shown at 3 and 7.4 s). Green arrows pointing inward connected by solid line at 1.5 and 5.9 s indicate imminent merging events. Scale bar 500 nm.


C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( D ) ( red hot LUT ) . Scale bar 500 nm . D Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green and magenta asterisks show cristae merging and splitting cycles ( shown at 5 . 9 s and 7 . 4 s ) . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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C Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (D) (red hot LUT). Scale bar 500 nm. D Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green and magenta asterisks show cristae merging and splitting cycles (shown at 5.9 s and 7.4 s). Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


E Representative time - lapse images of WT Hela cells stained with NAO , and imaged using Airyscan microscope , indicate cristae merging and splitting dynamics . Yellow and magenta asterisks show merging and splitting events of cristae respectively . Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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E Representative time-lapse images of WT Hela cells stained with NAO, and imaged using Airyscan microscope, indicate cristae merging and splitting dynamics. Yellow and magenta asterisks show merging and splitting events of cristae respectively. Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


A Mitochondrion in WT Hela cells expressing ATP5I - PAGFP was photoactivated , in the area shown by a blue box , at 0 s . Inset of white box at 0 . 98 s clearly shows fluorescent crista ( red arrow ) , absent at 0 . 38 s , 0 . 59 s & 0 . 78 s ( red arrowhead showing the area enclosed by white dotted lines ) , confirming merging of non - fluorescent cristae vesicle with the IBM . Threshold images of insets are shown above the whole images . Scale bar 500 nm .

A Mitochondrion in WT Hela cells expressing ATP5I-PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38 s, 0.59 s & 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non-fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm.


B Dual - colour STED super - resolution images of fused mitochondrion in WT Hela cells ( Left Panel ) showing colocalization of cristae marked with ATP5I - GFP ( using anti - GFP antibody ) and ATP5I - SNAP ( stained with silicone - rhodamine ) . ( Right Panel ) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I - GFP ( using anti - GFP antibody ) or ATP5I - SNAP ( stained with silicone - rhodamine ) . Arrowheads show merged cristae while arrows show cristae that maintain their individual identity . Scale bar 500 nm .

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B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm.


( Left panel ) A10 cells were transfected with Flag - FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . ( Bottom right panel ) A line was drawn through a representative cell to indicate relative intensity of RGB signals . ( Top right panel ) An orthogonal projection was generated by a Z - stack ( 240 nm interval ) image set using the ZEN program ( Zeiss ) depicting the region surrounding the nucleus in the XY , XZ , and YZ

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(Left panel) A10 cells were transfected with Flag-FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β-catenin (green), Flag (FMRP, red), and nucleus (blue). (Bottom right panel) A line was drawn through a representative cell to indicate relative intensity of RGB signals. (Top right panel) An orthogonal projection was generated by a Z-stack (240 nm interval) image set using the ZEN program (Zeiss) depicting the region surrounding the nucleus in the XY, XZ, and YZ planes


B . ( i ) HEK 293T cells were transfected with Flag - FMRP as in A , and endogenous β - catenin protein complexes were immunoprecipitated with β - catenin antibody . Co - immunoprecipitated Flag - FMRP protein was detected by Western blot analysis with the Flag antibody . Non - programmed rabbit IgG was used as a control . ( ii ) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β - catenin or FMRP antibody . The corresponding protein was detected in the precipitated immunocomplex by Western blot . Non - programmed rabbit IgG was used as a

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B. (i) HEK 293T cells were transfected with Flag-FMRP as in A, and endogenous β-catenin protein complexes were immunoprecipitated with β-catenin antibody. Co-immunoprecipitated Flag-FMRP protein was detected by Western blot analysis with the Flag antibody. Non-programmed rabbit IgG was used as a control. (ii) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β-catenin or FMRP antibody. The corresponding protein was detected in the precipitated immunocomplex by Western blot. Non-programmed rabbit IgG was used as a control


( i ) Purified 6x - His - FMRP and various regions of β - catenin proteins fused to GST were incubated with glutathione - agarose beads . Purified GST or GST - fusion proteins were visualized by Western blotting using the GST antibody , and sizes of corresponding GST - fusion proteins were confirmed . Pulled - down FMRP proteins by GST fused β - catenin were detected by immunoblotting with the FMRP antibody . ( ii ) Interaction between FMRP and β - catenin was assessed in the presence or absence of RNA . HEK 293T cells were subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody , in the presence or absence of RNase A ( 10 µg / mL ) . The corresponding protein was detected in the precipitated immunocomplex by Western blot , and non - programmed rabbit IgG was used as a control . RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase

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(i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A


( Top panel ) A10 cells were transfected with GBP - Lifeact and either EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red )

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(Top panel) A10 cells were transfected with GBP-Lifeact and either EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red)


( Top panel ) A10 cells were transfected wit EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) E . A10 cells were transfected as in ( D ) with GBP - Lamin

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(Top panel) A10 cells were transfected wit EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red) E. A10 cells were transfected as in (D) with GBP-Lamin B1


A . Co - sedimentation of protein / RNA complexes on a 10 - 50 % sucrose gradient . Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients ; peaks corresponding to the 40S and 60S subunits , 80S monosome , and polysomes were detected by UV absorbance at 254 nm , and indicated proteins in these fractions were detected by Western blot . Corresponding total cell lysate was used as

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A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input


( i ) Precipitated proteins in A10 cell lysates with m7GTP - agarose beads were identified by Western blot analysis . eIF4E and tubulin were shown as positive and negative controls , respectively . Total lysates were used as input . ( ii ) A10 cell lysates were incubated with GTP - agarose beads , and none of the proteins tested were precipitated with the beads . Total lysates were used as input

(i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control


( i ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to m7GTP - agarose pull - down as in ( C )

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(i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C)


( ii ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to GTP - agarose pull - down as in ( i ) . E . HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP - agarose pull - downs as in ( C )

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(ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C)


F . HEK 293T cells lysates were subjected to m7GTP - agarose pull - downs as in ( C ) in the presence or absence of RNase A ( 10 µg / mL ) . RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis .

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F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.


Primary VSMCs ( left panel ) , A10 ( middle panel ) , and HEK 293T cells ( right panel ) were transfected with siRNAs targeting ctnnb1 ( β - catenin ) or scrambled RNA . After 36 hr , the cells were pulsed with 0 . 5 µM puromycin for 15 min and then harvested . The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody . β - catenin blots indicated efficacy of siRNA and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation . A one - way ANOVA and Tukey post - hoc test was performed , with p - values indicated above the error

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Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error bars


A10 cells were transfected with Flag - FMRP then treated with either cycloheximide or its solvent ( DMSO ) for 4 hr prior to fixation . β - catenin ( green ) , Flag ( FMRP , red ) were visualized by immunofluorescence . Nucleus ( blue ) was identified by DNA staining with Hoechst 33342 . Arrows indicate a loss of β - catenin signal from the perinuclear region that was seen in the control following cycloheximide

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A10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatment