Representative Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 and S521 ) in response to a 15 min treatment with increasing doses of thapsigargin ( . 01 , . 1 , 1 or 10 µM ) or . 5 mM AICAR . DMSO was used as a vehicle control . 14 - 3 - 3 was used as a loading control . n = 3 independent experiments .

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Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin ( + ) or vehicle ( - ) for 15 min .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . The mClover2 : tagRFP - T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. The mClover2:tagRFP-T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . STIM1 OASF expressed in live L6 myoblasts before ( - ) or after ( + ) treatment with A - 769662 .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . L6 myoblasts with S257 mutated to A or E .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. L6 myoblasts with S257 mutated to A or E.


L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts ,

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L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts,


L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Ca2 + levels over time ( fold of 0 mM Ca2 + + EGTA basal ) and ( C ) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2 + and treated with 2 µM thapsigargin during the indicated times .

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L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.


Negative geotaxis climbing assays in Drosophila ( flies ) . Time required for 50 % of flies to climb to the target line before or after 2 . 5 hr of continuous climbing exercise ( A ) in the yw strain with muscle - specific STIM knockdown using two independent RNAi sequences ( RNAi26 - 1 or RNAi8 - 4 ) compared to GFP - expressing controls , or ( B ) in the w1118 strain with muscle - specific STIM knockdown using RNAiGD16187 compared to non - targeted RNAiGD12145 controls .

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Negative geotaxis climbing assays in Drosophila (flies). Time required for 50% of flies to climb to the target line before or after 2.5 hr of continuous climbing exercise (A) in the yw strain with muscle-specific STIM knockdown using two independent RNAi sequences (RNAi26-1 or RNAi8-4) compared to GFP-expressing controls, or (B) in the w1118 strain with muscle-specific STIM knockdown using RNAiGD16187 compared to non-targeted RNAiGD12145 controls.


Time to fatigue , when flies were no longer able to climb , during a 3 - min period after negative geotaxis stimulation in STIM whole - body knockout flies overexpressing wild type ( WT ) or mutant ( S257A or S257E ) human STIM1 - mRuby3 .

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Time to fatigue, when flies were no longer able to climb, during a 3-min period after negative geotaxis stimulation in STIM whole-body knockout flies overexpressing wild type (WT) or mutant (S257A or S257E) human STIM1-mRuby3.


TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast . Growth of yap1Δahp1Δ cells carrying empty vector ( ev ) , wild type peroxiredoxin gene ( AHP1 ) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


TBH cytotoxicity assay in vivo . Basic growth and the synthetic effect in wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH .

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TBH cytotoxicity assay in vivo. Basic growth and the synthetic effect in wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH.


H2S production using Biggy agar plates - Wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains were grown on Biggy medium , and H2S production was

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H2S production using Biggy agar plates- Wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains were grown on Biggy medium, and H2S production was recorded


In vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control .

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In vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control.


Northern - blot analysis for thiolation levels of tRNA < sub > UUC < / sub > < sup > Glu < / sup > in the genetic background of the indicated yeast strains . Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride . ( bottom ) Quantified thiolation levels from 3 independent biological replicates .

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Northern-blot analysis for thiolation levels of tRNAUUCGlu in the genetic background of the indicated yeast strains. Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu. APM: ([N-Acryloyl-amino] phenyl) mercuric chloride. (bottom) Quantified thiolation levels from 3 independent biological replicates.


( A ) Volcano plot shows genes where RNA reads are statistically significant under - or over - expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis . Decreased genes are represented in red , the up - regulated ones are in green while blue dots highlight most outlying genes ( defined by absolute ( log2 ( fold change ) + log10 ( padj ) ) > 10 ) . Genes that were not statistically significant are represented in black . Grey and black gene names indicate top 30 % of the most dysregulated genes with a highlight on ones involved in metabolism .

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(A) Volcano plot shows genes where RNA reads are statistically significant under- or over-expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis. Decreased genes are represented in red, the up-regulated ones are in green while blue dots highlight most outlying genes (defined by absolute (log2(fold change) + log10(padj))>10).Genes that were not statistically significant are represented in black. Grey and black gene names indicate top 30% of the most dysregulated genes with a highlight on ones involved in metabolism.


( D ) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients . The data depicted is between the affected and recovered biopsies of the same patient ( F6 / 1M ) .

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(D) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients. The data depicted is between the affected and recovered biopsies of the same patient (F6/1M).


( E ) Comparative proteome profiling of proteins that are significantly under - or over - expressed in RIRCD affected versus control individuals ( p - Anova < 0 . 05 - statistically significant - horizontal line ) . Proteins which are decreased are represented in red while the up - regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black . Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis ( the cut - off values were determined based on the 2 × standard deviation and the normal distribution from all identified protein ' s log2 ratio in which the bell curve is symmetric around the mean , therefore , an average log2 ratio of a protein which < − 2 . 2 or > 0 . 98 was considered as regulated ) .

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(E) Comparative proteome profiling of proteins that are significantly under- or over-expressed in RIRCD affected versus control individuals (p-Anova <0.05-statistically significant - horizontal line). Proteins which are decreased are represented in red while the up-regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black. Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis (the cut-off values were determined based on the 2× standard deviation and the normal distribution from all identified protein's log2 ratio in which the bell curve is symmetric around the mean, therefore, an average log2 ratio of a protein which <−2.2 or > 0.98 was considered as regulated).


( A ) Western blotting analysis depicts mitochondrial proteins ( NDUFA9 - Complex I , SDHA - Complex II , MTCO1 - Complex IV and MTCO2 - Complex IV ) levels in control derived muscle ( n = 3 ) , patient derived muscle during the symptomatic phase ( n = 3 ) and patient derived muscle after recovery ( n = 1 ) . VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20 . ( B ) Densitometry analysis of the immunoblotting from ( A ) showing an increase in SDHA ( Complex II ) , while NDUFA9 ( Complex I ) probably as a compensatory mechanism and MT - CO2 ( Complex IV ) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes , thus being destabilized by the defective mitochondrial mistranslation .

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(A) Western blotting analysis depicts mitochondrial proteins (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) levels in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20. (B) Densitometry analysis of the immunoblotting from (A) showing an increase in SDHA (Complex II), while NDUFA9 (Complex I) probably as a compensatory mechanism and MT-CO2 (Complex IV) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes, thus being destabilized by the defective mitochondrial mistranslation.


( C ) Mitochondrial DNA copy numbers were determined in affected ( n = 3 ) and recovered individuals ( n = 2 ) and compared to control individuals ( n = 4 , 3 months , 3 years , 4 years and 7 years ) using qPCR showing a significant increase in recovered muscle .

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(C) Mitochondrial DNA copy numbers were determined in affected (n=3) and recovered individuals (n=2) and compared to control individuals (n=4, 3 months, 3 years, 4 years and 7 years) using qPCR showing a significant increase in recovered muscle.


( D ) Western blotting analysis of proteins involved in the amino - acid sensing and integrated stress response : GCN2 , SLC7A11 and ATF4 in control derived muscle ( n = 3 ) , patient derived muscle during the symptomatic phase ( n = 3 ) and patient derived muscle after recovery ( n = 1 ) . All samples were normalized to VCL which was used as a loading control . ( E ) Densitometry analysis of proteins from ( D ) showing a significant increase of major transducers of ISR such as GCN2 and ATF4 . Consistent with ISR activation is also the increase in SLC7A1 .

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(D) Western blotting analysis of proteins involved in the amino-acid sensing and integrated stress response: GCN2, SLC7A11 and ATF4 in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). All samples were normalized to VCL which was used as a loading control. (E) Densitometry analysis of proteins from (D) showing a significant increase of major transducers of ISR such as GCN2 and ATF4. Consistent with ISR activation is also the increase in SLC7A1.


( F ) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls ( RIRCD n = 5 ; controls n = 6 ) and in recovered muscle compared to affected muscle ( n = 1 , affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates ) and schematic representation of the metabolic pathways impacted in patient muscle ( bottom , right panel ) .

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(F) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls (RIRCD n=5; controls n=6) and in recovered muscle compared to affected muscle (n=1, affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates) and schematic representation of the metabolic pathways impacted in patient muscle (bottom, right panel).


( A ) Patient n = 4 ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) and control fibroblasts ( n = 2 ) were grown for 12 days in media containing 0 . 02 mM cysteine and 5 % dialyzed FBS . Proteins belonging to mitochondrial complexes ( NDUFA9 - Complex I , SDHA - Complex II , MTCO1 - Complex IV and MTCO2 - Complex IV ) were analysed via Western Blotting . VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20 . ( B ) Densitometry analysis of proteins from ( A ) showing no significant changes in OXPHOS proteins upon glutamine / glutamic acid depletion . Control bar represents an average of the two control fibroblast lines employed ( F008 and F154 ) .

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(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20. (B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154).


( C ) Patient ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5 % dialyzed FBS . Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting . VCL was used as a loading control while the densitometry analysis ( D ) was done based on the mitochondrial proteinTOM20 . Control bar represents an average of the two control fibroblast lines employed ( F008 and F154 ) . Although we could observe significant changes upon cysteine depletion in all conditions , the phenotype was exacerbated in the digenic fibroblasts compared to the controls .

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

(C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.


( A ) Volcano plot analysis of transcriptomic changes where statistically significant genese are delimited by a horizontal line . Decreased genes are represented in red while the up - regulated ones are in green , while genes that were not statistically significant are represented in black .

(A) Volcano plot analysis of transcriptomic changes where statistically significant genese are delimited by a horizontal line. Decreased genes are represented in red while the up-regulated ones are in green, while genes that were not statistically significant are represented in black.


( B ) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues , in accordance to the mitochondrial translational defect .

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(B) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues, in accordance to the mitochondrial translational defect.


( B ) TLC analysis of ganglioside species in human serum . ( C ) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum . Data expressed as mean ± SD , n = 6 .

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(B) TLC analysis of ganglioside species in human serum. (C) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum. Data expressed as mean ± SD, n = 6.


( B ) TLC analysis of ganglioside species in human serum .

(B) TLC analysis of ganglioside species in human serum.


( E ) Quantification by LC - MS / MS analysis of serum GM3 species with differing acyl chain structures . Total abundance of 10 species was defined as 1 . Data expressed as mean ± SD , n = 6 .

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(E) Quantification by LC-MS/MS analysis of serum GM3 species with differing acyl chain structures. Total abundance of 10 species was defined as 1. Data expressed as mean ± SD, n = 6.


( A ) Heat map analysis of serum GM3 species in various pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Colors indicate fold change average of each species relative to control ( defined as 1 ) , as shown in key at right . Order of pathological phases corresponds to increments of HOMA - IR and serum CRP .

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(A) Heat map analysis of serum GM3 species in various pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP.


( B - D ) Properties of various GM3 species as a function of pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Data shown are relative abundances of total LCFA species ( 16 : 0 , 18 : 0 , 20 : 0 ) ( B ) , total VLCFA species ( 22 : 0 , 23 : 0 , 24 : 0 , h24 : 0 ) ( C ) , and total unsaturated VLCFA species ( 22 : 1 , 24 : 1 , h24 : 1 ) ( D ) relative to total of ten major GM3 species ( defined as 1 ) in each subject .

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(B-D) Properties of various GM3 species as a function of pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject.


( E - H ) Properties of various GM3 species as a function of BMI : LCFA - GM3 ( E ) , VLCFA - GM3 ( F ) , unsaturated VLCFA - GM3 ( G ) , and α - hydroxyVLCFA - GM3 ( h24 : 0 ) ( H ) . Colors indicate disease severity : light blue , no abnormal scores ( n = 25 ) ; orange , early - phase obesity ( n = 74 ) ; purple , severe obesity ( n = 23 ) .

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(E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23).


( I , J ) Spearman ' s correlations for GM3 h24 : 0 vs . ALT ( I ) and vs . HOMA - IR ( J ) .

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(I, J) Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J).


( K ) Plots of α - hydroxylation rate ( h24 : 0 / 24 : 0 ) vs . serum CRP . Colors indicate range of CRP value ( mg / dL ) : light blue , 0 . 01 - 0 . 02 ( n = 21 ) ; orange , 0 . 03 - 0 . 09 ( n = 56 ) ; gray , 0 . 10 - 0 . 29 ( n = 29 ) ; red , 0 . 3 - 1 . 0 ( diagnostically abnormal ; n = 15 ) .

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(K) Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dL): light blue, 0.01-0.02 (n=21); orange, 0.03-0.09 (n=56); gray, 0.10-0.29 (n=29); red, 0.3-1.0 (diagnostically abnormal; n=15).


( B , C ) GM3 - mediated enhancement and inhibition of proinflammatory cytokine production from LPS - stimulated monocytes ( LPS : 0 . 06 , 0 . 13 , 0 . 25 ng / mL ) . TNF - α ( B ) and IL - 6 ( C ) production in culture supernatant was measured by ELISA .

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(B, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA.


( D , E ) Co - stimulation of monocytes by LPS plus GM3 species or complex ganglioside species ( 1 . 5 , 3 . 0 , 4 . 5 µM ) . TNF - α ( D ) and IL - 6 ( E ) production were shown in heat maps .

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(D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps.


( F ) Co - stimulation of monocytes by LPS plus GM3 species or precursor GSL species . TNF - α , IL - 6 , and IL - 12 / 23 production were shown in heat maps .

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(F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps.


( G ) Inhibitory effect of LCFA and unsaturated VLCFA - GM3 on LVCFA - GM3 species . IL - 6 production in culture supernatant was measured by ELISA .

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(G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.


( A ) Co - stimulation of human monocytes by GM3 species plus various TLR ligands : LPS ( 0 . 13 ng / mL ) , TLR4 / MD - 2 , Pam3CSK4 ( 0 . 5 µg / mL ) , TLR1 / 2 , Flagellin ( 50 ng / mL ) , TLR5 , R848 ( 0 . 5 µg / mL ) , TLR7 / 8 , MALP - 2 ( 1 . 0 ng / mL ) , TLR2 / 6 . IL - 6 production in culture supernatant was quantified by ELISA ( shown in a heat map ) .

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(A) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map).


( B ) Co - stimulation of monocytes by GM3 species ( 16 : 0 , 24 : 0 , 24 : 1 ) plus synthetic TLR4 ligands LA506 ( 15 ng / mL ) , LA505 ( 150 ng / mL ) , or LA504 ( 150 ng / mL ) .

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(B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL).


( C , D ) Production of IL - 6 ( C ) and TNF - α ( D ) in culture supernatant following co - stimulation of monocytes by GM3 species plus human HMGB1 .

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(C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1.


( E ) Overexpression of hTLR4 , hTLR4 / hMD - 2 , hTLR4 ( P714H ) / MD - 2 in HEK293T cells , and co - stimulation by GM3 22 : 0 with LPS ( 5 ng / mL ) . TLR4 activation was monitored by NF - κB luciferase reporter assay ( termed " Relative Luc Activity " on Y - axis ) .

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(E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis).


( F , Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL )

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(F, Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL)


G ) Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL ) and further addition of soluble human CD14 ( 1 µg / mL )

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G) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) and further addition of soluble human CD14 (1 µg/mL)


( H ) Stimulation of Mal - overexpressing HEK293T cells by GM3 species .

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(H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species.


( I ) Co - stimulation of hTLR4 / hMD - 2 by LPS ( 5 ng / mL ) plus various mixtures of GM3 species .

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(I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species.


( A ) Co - stimulation of RAW macrophages by GM3 species plus various TLR ligands : LPS ( 0 . 5 , 1 . 0 ng / mL ) , bovine thymus HMGB1 ( 0 . 25 µg / mL ) , Pam3CSK4 ( 50 ng / mL ) , Poly I : C ( 10 µg / mL ) , R848 ( 4 ng / mL ) , and CpG type - B ( 20 nM ) . TNF - α production in culture supernatant was quantified by ELISA .

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(A) Co-stimulation of RAW macrophages by GM3 species plus various TLR ligands: LPS (0.5, 1.0 ng/mL), bovine thymus HMGB1 (0.25 µg/mL), Pam3CSK4 (50 ng/mL), Poly I:C (10 µg/mL), R848 (4 ng/mL), and CpG type-B (20 nM). TNF-α production in culture supernatant was quantified by ELISA.


( B ) Co - stimulation of RAW macrophages by low - and high - dose LPS ( 0 , 5 , 50 ng / mL ) plus GM3 22 : 0 ( 5 µM ) . Time course of TNF - α production in culture supernatant was quantified by ELISA .

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(B) Co-stimulation of RAW macrophages by low- and high-dose LPS (0, 5, 50 ng/mL) plus GM3 22:0 (5 µM). Time course of TNF-α production in culture supernatant was quantified by ELISA.


( C ) Co - stimulation of BMDMs from C3H / HeN or C3H / HeJ mice by GM3 species plus LPS ( 0 . 5 ng / mL ) , and TNF - α production in culture supernatant .

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(C) Co-stimulation of BMDMs from C3H/HeN or C3H/HeJ mice by GM3 species plus LPS (0.5 ng/mL), and TNF-α production in culture supernatant.


( d ) Co - stimulation of mTLR4 / mMD - 2 - expressing HEK293T cells by GM3 species plus LPS ( 2 . 5 ng / mL ) , and further addition of soluble mouse CD14 - Fc fusion protein ( 1 µg / mL ) .

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(d) Co-stimulation of mTLR4/mMD-2-expressing HEK293T cells by GM3 species plus LPS (2.5 ng/mL), and further addition of soluble mouse CD14-Fc fusion protein (1 µg/mL).


( E Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and complex ganglioside species ( E ; 2 . 5 , 5 . 0 , 10 µM ) , ( shown in heat maps ) .

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(E Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and complex ganglioside species (E; 2.5, 5.0, 10 µM), (shown in heat maps).


F ) Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and precursor GSL species ( F ; 2 . 5 , 5 . 0 , 10 µM ) ( shown in heat maps ) .

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F) Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and precursor GSL species (F; 2.5, 5.0, 10 µM) (shown in heat maps).


( A ) GM3 molecular species of 6 - wk - old control C57 / BL6 mice and ob / ob mice were analyzed respectively by LC - MS / MS ( n = 3 ) .

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(A) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3).


( B ) GM3 molecular species of normal - diet ( ND ) and high - fat - diet ( HFD ) C57 / BL6 mice were analyzed by LC - MS / MS ( n = 4 ) .

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(B) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4).


( C ) TLC analysis of acidic GSL fraction ( equivalent to 0 . 1 mg protein ) from epididymal fat pads of C3H / HeN ( A ) and C3H / HeJ mice ( B ) on ND or HFD for 8 wk .

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(C) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk.


( D ) GM3 molecular species of C3H / HeN mice ( ND , HFD ) and C3H / HeJ mice ( ND , HFD ) were analyzed by LC - MS / MS ( n = 4 ) .

(D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).


( C , D ) Alanine scanning for basic residues involved in signal transduction via VLCFA - GM3 ( n = 5 ) ( C ) , and combinations of effective mutations ( n = 6 ) ( D ) . Signal transduction was monitored by NF - κB reporter assay .

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(C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay.


( E - G ) Comparative inhibitory effects of GM3 16 : 0 on mTLR4 / mMD - 2 ( E ) , mTLR4 / hMD - 2 ( domain - swapped complex ) ( F ) , and hTLR4 / hMD - 2 ( G ) ( n = 3 ) .

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(E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3).


( H ) Cross - linked SDS - PAGE analysis of recombinant mTLR4 ( extracellular domain ) / mMD - 2 complexed with GM3 18 : 0 , GM3 species enhancing mTLR4 activation .

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(H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.


Microscope images of the tissue and microinjection pipette before ( top ) and after ( bottom ) annotation of the tissue surface and microinjection micropipette tip by the experimenter . Scalebars : top 100µm , bottom 10µm . Ap indicates the apical surface and BL indicates the basal lamina .

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Microscope images of the tissue and microinjection pipette before (top) and after (bottom) annotation of the tissue surface and microinjection micropipette tip by the experimenter. Scalebars: top 100µm, bottom 10µm. Ap indicates the apical surface and BL indicates the basal lamina.


( Left ) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection . Dashed lines indicate locations in the slice where microinjections were attempted at different pressures . Microinjections were attempted at pressure of 75 mbar ( left area ) , 100 mbar ( center area ) , and 125 mbar ( right area ) . ( Right ) Percentage of successful injections achieved by the Autoinjector at 75 mbar ( 35 % , n = 314 total , 110 successful ) , 100 mbar ( 35 % , n = 185 total , 65 successful ) , and 125 mbar ( 8 % , n = 287 total , 23 successful ) .

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(Left) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different pressures. Microinjections were attempted at pressure of 75 mbar (left area), 100 mbar (center area), and 125 mbar (right area). (Right) Percentage of successful injections achieved by the Autoinjector at 75 mbar (35%, n = 314 total, 110 successful), 100 mbar (35%, n = 185 total, 65 successful), and 125 mbar (8%, n = 287 total, 23 successful).


( Left ) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection . Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar . Microinjections were attempted at depths of 10 μm ( left area ) , 15 μm ( center area ) , and 25 μm ( right area ) into the apical surface . ( Right ) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm ( 68 % , n = 163 total , 111 successful ) , 15 μm ( 22 % , n = 170 total , 37 successful ) , and 25 μm ( 11 % , n = 169 total , 19 successful ) into the apical surface .

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(Left) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar. Microinjections were attempted at depths of 10 μm (left area), 15 μm (center area), and 25 μm (right area) into the apical surface. (Right) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm (68%, n = 163 total, 111 successful), 15 μm (22%, n = 170 total, 37 successful), and 25 μm (11%, n = 169 total, 19 successful) into the apical surface.


Cells targeted at a constant spacing of 30 μm , scale bar is 10 μm .

Cells targeted at a constant spacing of 30 μm, scale bar is 10 μm.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI . Fluorescence images of tissue after microinjection with dextran - A555 ( magenta ) . The tissue was stained for Tbr2 ( green ) and TuJI ( white ) . Microinjected cell at 0 h ; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2 . Two - daughter cell progeny 24 h after microinjection ; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2 ; the bottom cell is a newborn BP positive for Tbr2 . Both cells are negative for the neuronal marker TuJI ( white ) . Four - daughter cell progeny 48 h after microinjection ; note that daughter 1 and 2 reside in the VZ , while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ . Distribution of microinjected cells and their progeny in the VZ , SVZ and CP ( cortical plate ) . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface ( 0 µm ) and expressed as % of total . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white). Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white). Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI . Expression of Tbr2 in microinjected cells and their progeny at 0 , 24 an 48 h . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . Expression of TuJI in microinjected cells and their progeny at 0 , 24 an 48 h . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dx - A488 along with mRNA for RFP Organotypic slices of mouse E14 . 5 dorsal telencephalon injected with Dextran - A488 ( green ) and RFP poly - A + - mRNA . After 24h in culture the progeny expresses RFP ( magenta ; blue : DAPI , n = 17 cells injected , 8 RFP positive ) . Percentage of RFP - positive and RFP - negative cells ( n = 17 cells injected ) .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dx-A488 along with mRNA for RFP Organotypic slices of mouse E14.5 dorsal telencephalon injected with Dextran-A488 (green) and RFP poly-A+-mRNA. After 24h in culture the progeny expresses RFP (magenta; blue: DAPI, n = 17 cells injected, 8 RFP positive). Percentage of RFP-positive and RFP-negative cells (n = 17 cells injected).


Automated microinjection was performed on organotypic slices of mouse E14 . 5 - E15 . 5 dorsal telencephalon using a solution containing Dextran - A555 ( not shown ) and Alexa488 ( green ) . Slices were fixed after microinjection ( n = 71 cells total ) and were stained for Tbr2 ( white , H - J ) . ( A - C ) Representative examples of a non - coupled cell ( A , cartoon on the right ) , a 2 - cell cluster ( B , cartoon on the right ) and a 5 - cell cluster ( C , cartoon on the right ) . The asterisks indicate the Dx - A555 - positive , microinjected cell . Percentage of microinjected cells found in a coupled cluster ( coupled , green ) . Cluster size , expressed as % of total . Distribution of coupled cells expressed as the distance from the ventricular surface ( 0 µm = ventricle surface . Distribution of coupled cells divided into 11 bins and expressed as % of total . the images on the left are maximum intensity projections ( MIP ) of 18 , 17 , 23 , 12 and 30 - focal planes ,

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

Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell. Percentage of microinjected cells found in a coupled cluster (coupled, green). Cluster size, expressed as % of total. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface. Distribution of coupled cells divided into 11 bins and expressed as % of total. the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectively


A representative picture of a 2 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 2 . Microinjected cells are stained for Tbr2 . A representative picture of ( H ) a 2 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 2 ) and ( I ) a 7 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 7 ) , the latter containing one Tbr2 positive cell . Microinjected cells are stained for Tbr2 . The asterisks indicate the Dx - A555 - positive , microinjected cell . comparison of Tbr2 - positive cells ( expressed as % of total ) among all the cells in the VZ ( VZ ; n = 1003 from 4 different confocal images from 3 independent experiments ; error bar represents standard deviation ) and in coupled clusters in the VZ ( coupled clusters , VZ ; n = 104 from two independent experiments ) . Coupled cells were scored based one presence / absence of apical and / or basal polarity cues , and results are expressed as % of total . the images on the right are single optical sections corresponding to the nucleus ( white dotted line ) ; scale bars are 20 µm for MIP and 10 µm for single focal planes . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments). Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total. the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 dorsal telencephalon using Dx - A555 Phase contrast image of automated microinjection into mouse neurons . Scale bar is 100 µm .

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

Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 dorsal telencephalon using Dx-A555 Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green , Slices were fixed after microinjection and stained for Reelin ( white , C ) Left : overview image of a mouse microinjected area ( arrow indicates a pyramidal neuron , arrowhead indicates a Cajal - Retzius neuron ; image is MIP of 20 focal planes ) . Microinjected neurons show different morphology and positivity for the Cajal - Retzius marker Reelin ( white ) . Right , top row : the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin . Right , bottom row : the Cajal - Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin . Images on the top and bottom row left ( LY MIP ) are a MIP of 2 and 3 focal planes , respectively . The three images on the top and bottom right ( LY , Reelin and DAPI ) are single optical sections . Scale bar is 20 µm for MIP and 10 µm for single focal planes . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection and stained for Reelin (white, C) Left: overview image of a mouse microinjected area (arrow indicates a pyramidal neuron, arrowhead indicates a Cajal-Retzius neuron; image is MIP of 20 focal planes). Microinjected neurons show different morphology and positivity for the Cajal-Retzius marker Reelin (white). Right, top row: the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin. Right, bottom row: the Cajal-Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin. Images on the top and bottom row left (LY MIP) are a MIP of 2 and 3 focal planes, respectively. The three images on the top and bottom right (LY, Reelin and DAPI) are single optical sections. Scale bar is 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green Slices were fixed after microinjection and stained for SatB2 ( white , Overview image of a mouse microinjected area . The arrow indicates a pyramidal neuron ; the image is MIP of 30 focal planes , scale bar : 50 µm . Row on the right : the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper - layers marker SatB2 ( white ) . Images are single focal planes . Scale bar is 10 µm . Proportion of neurons positive or negative for Reelin ( n = 12 cells total ) . Proportion of neurons positive or negative for SatB2 ( n = 39 cells in total ) . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green Slices were fixed after microinjection and stained for SatB2 (white, Overview image of a mouse microinjected area. The arrow indicates a pyramidal neuron; the image is MIP of 30 focal planes, scale bar: 50 µm. Row on the right: the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper-layers marker SatB2 (white). Images are single focal planes. Scale bar is 10 µm. Proportion of neurons positive or negative for Reelin (n = 12 cells total). Proportion of neurons positive or negative for SatB2 (n = 39 cells in total). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of human ( G , H ) neurons . Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green , Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon . Top , MIP of 20 focal planes ( scale bar : 50µm ) . Bottom : high magnification ( scale bar : 10 µm ) . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection of human (G,H) neurons. Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon. Top, MIP of 20 focal planes (scale bar: 50µm). Bottom: high magnification (scale bar: 10 µm). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


FACS gating strategy representative of 5 independent experiments adopted to sort CD11b + CD45int microglia distinctly from CD11b + CD45high resident macrophages and CD11b - CD45high lymphocytes .

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

FACS gating strategy representative of 5 independent experiments adopted to sort CD11b+CD45int microglia distinctly from CD11b+CD45high resident macrophages and CD11b-CD45high lymphocytes.


Analysis of relative transcript levels of CD11b + CD45int FACS - sorted microglia compared with whole brain tissue by qPCR . Gene expression levels of microglia ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 ) , astrocyte ( Gfap , Gjb6 , Ntsr2 , Aldh1l1 ) , oligodendrocyte ( Mobp , Mog , Cldn1 ) , and neuron ( Tubb3 , Vglut1 , NeuN ) markers . Bars represent mean ( n = 4 ; pool of 1 female and 1 male per biological replicate ) of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ) . N . D . not detected .

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

Analysis of relative transcript levels of CD11b+CD45int FACS-sorted microglia compared with whole brain tissue by qPCR. Gene expression levels of microglia (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12), astrocyte (Gfap, Gjb6, Ntsr2, Aldh1l1), oligodendrocyte (Mobp, Mog, Cldn1), and neuron (Tubb3, Vglut1, NeuN) markers. Bars represent mean (n=4; pool of 1 female and 1 male per biological replicate) of relative expression (Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test). N.D. not detected.


Representative quantification of CD206 expression in CD11b + CD45int microglia and CD11b + CD45high resident macrophages . Values denote the percentage of the mean ± SEM of 5 independent experiments .

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

Representative quantification of CD206 expression in CD11b+CD45int microglia and CD11b+CD45high resident macrophages. Values denote the percentage of the mean ± SEM of 5 independent experiments.


Representative images of 2 independent experiments showing microglia , resident macrophages and lymphocytes acquired with ImageStream imaging cytometer ( Amnis ) based on CD45 , CD11b and CD206 expression levels ( scale bar represents 7 µm ) .

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

Representative images of 2 independent experiments showing microglia, resident macrophages and lymphocytes acquired with ImageStream imaging cytometer (Amnis) based on CD45, CD11b and CD206 expression levels (scale bar represents 7 µm).


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of microglia homeostatic ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 , Mef2c ) , phagocytic ( Tyrobp , Trem2 ) , and inflammatory ( Il1β , Tnf , Ccl2 , Mrc1 , Arg1 ) markers were analyzed by qPCR . Bars represent mean of relative expression ( % of saline ; Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ; n = 4 ) .

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

Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of microglia homeostatic (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12, Mef2c), phagocytic (Tyrobp, Trem2), and inflammatory (Il1β, Tnf, Ccl2, Mrc1, Arg1) markers were analyzed by qPCR. Bars represent mean of relative expression (% of saline; Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test; n=4).


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS - injected mouse brains .

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

Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains.


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b + CD45int cells from saline or LPS - injected mice .

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 B-CONTROLLED_VAR O O O O

Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b+CD45int cells from saline or LPS-injected mice.


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia ( n = 4 ) and isolated bone marrow monocytes ( n = 2 ) by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ) .

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

Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia (n=4) and isolated bone marrow monocytes (n=2) by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test).


Primary adult microglia were cultivated in the presence of TGF - β ( 50 μg / ml ) and M - CSF ( 10 ng / ml ) , while neonatal cells were stimulated for 24 hours with TGF - β ( 50 μg / ml ) followed by 6 hours stimulation with LPS ( 1 ng / mL ) or left untreated . Expression levels of microglia homeostatic ( Olfml3 , Tmem119 , Gpr34 ) and inflammatory ( Il1β , Tnf , Ccl2 ) genes were analysed by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ; n = 3 ) .

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

Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).


Heatmap showing clustering analysis of 1 , 247 single cells , featuring 100 most variable genes ( FDR < 0 . 05 ) . Single - cell RNA - seq results are obtained from 2 mice per group ( 1 female and 1 male each ) .

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

Heatmap showing clustering analysis of 1,247 single cells, featuring 100 most variable genes (FDR<0.05). Single-cell RNA-seq results are obtained from 2 mice per group (1 female and 1 male each).


Expression of specific homeostatic ( Tmem119 , P2ry12 , Siglech ) and inflammatory ( Ccl2 , Gpr84 ) genes overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) .

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

Expression of specific homeostatic (Tmem119, P2ry12, Siglech) and inflammatory (Ccl2, Gpr84) genes overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).


Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b + CD45int microglia of saline or LPS - injected mouse brains . For the unconjugated TMEM119 antibody , negative stands for primary antibody without secondary antibody . For P2RY12 antibody , negative represents isotype PE control .

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Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b+CD45int microglia of saline or LPS-injected mouse brains. For the unconjugated TMEM119 antibody, negative stands for primary antibody without secondary antibody. For P2RY12 antibody, negative represents isotype PE control.


Heatmap showing examples of specific genes mainly up - regulated in " main LPS " ( Manf ) or " subset LPS " ( Ash1l ) and down - regulated in " main LPS " ( Mef2c ) or " subset LPS " ( Lamp1 ) overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) .

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

Heatmap showing examples of specific genes mainly up-regulated in "main LPS" (Manf) or "subset LPS" (Ash1l) and down-regulated in "main LPS" (Mef2c) or "subset LPS" (Lamp1) overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).


Pseudotime dynamics of inflammatory ( Ccl12 , Ccl2 , Gpr84 ) and homeostatic ( Mef2c , P2ry12 , Siglech ) genes in dependence on inferred cell states .

Pseudotime dynamics of inflammatory (Ccl12, Ccl2, Gpr84) and homeostatic (Mef2c, P2ry12, Siglech) genes in dependence on inferred cell states.


( H ) The bur1 - ΔC mutant exhibits a synthetic growth defect with the vac17Δ mutant . Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [ URA3 ] BUR1 . Plasmids tested were pRS415 [ LEU2 ] ( mock ) , pRS415 BUR1 , or pRS415 bur1 - ΔC . Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC + 5 - FOA or SC - Leu - Ura plates . Plates were incubated at 24 ̊ C for 3 days .

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

(H) The bur1-ΔC mutant exhibits a synthetic growth defect with the vac17Δ mutant. Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [URA3] BUR1. Plasmids tested were pRS415 [LEU2] (mock), pRS415 BUR1, or pRS415 bur1-ΔC. Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura plates. Plates were incubated at 24 ̊C for 3 days.


( A , B ) The bur1 - ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of the cell cycle . Yeast were grown in YPD medium and collected in log phase growth . DNA content was measured using propidium iodide ( PI ) staining and assessed by flow cytometry . p - value determined with a one - way ANOVA and Tukey post hoc test . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 cells counted in each strain / experiment . ( C , D ) A bur1 - 267 mutant also exhibits a delay in progression through G1 phase of the cell cycle . DNA content was measured using PI staining and assessed by flow cytometry . p - value determined with a one - way ANOVA and Tukey post hoc test . * * * * ( p - value < 1 x 10 - 4 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 cells counted in each strain / experiment .

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

(A, B) The bur1-ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of the cell cycle. Yeast were grown in YPD medium and collected in log phase growth. DNA content was measured using propidium iodide (PI) staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. (C, D) A bur1-267 mutant also exhibits a delay in progression through G1 phase of the cell cycle. DNA content was measured using PI staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. **** (p-value < 1 x 10-4). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.


( E , F ) The bur1 - ΔC vac17Δ double mutant shows accumulation of Whi5 in the nucleus . WT , vac17Δ , bur1 - ΔC , and bur1 - ΔC vac17Δ cells , which express Whi5 - 3xGFP and Nup188 - mCherry from endogenous loci , were incubated at 24 ̊ C . Cells were scored for the presence of Whi5 - 3xGFP in within the nucleus ( Nup188 - mCherry ) . Error bars ; SD calculated from at least four independent experiments with at least 100 cells counted in each strain / experiment . p - value determined with a one - way ANOVA and Tukey post hoc test . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 cells counted in each strain / experiment . Scale bar , 2 μm .

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(E, F) The bur1-ΔC vac17Δ double mutant shows accumulation of Whi5 in the nucleus. WT, vac17Δ, bur1-ΔC, and bur1-ΔC vac17Δ cells, which express Whi5-3xGFP and Nup188-mCherry from endogenous loci, were incubated at 24 ̊C. Cells were scored for the presence of Whi5-3xGFP in within the nucleus (Nup188-mCherry). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. Scale bar, 2 μm.


( B ) The bur1 - ∆ C vac17 ∆ double mutant cells have the ability to generate a vacuole de novo , which suggests that the cell - cycle delay is due to a defect in Bur1 - dependent signaling . WT , vac17Δ , bur1 - ∆ C , and bur1 - ∆ C vac17 ∆ cells which express Vph1 - GFP from its endogenous locus , were pulse labeled with the vacuole specific dye FM4 - 64 . WT and bur1 - ∆ C cells have both FM4 - 64 and Vph1 - GFP signals in both mother and daughter cells . vac17Δ and bur1 - ∆ C vac17 ∆ cells have both Vph1 - GFP and FM4 - 64 on the vacuole in mother cells , however the daughter cells solely have a Vph1 - GFP labeled vacuole , indicating that the vacuole was generated de novo , rather than inherited from the mother vacuole . Arrowheads ; new vacuoles in daughter cells . Scale bar , 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The bur1-∆C vac17∆ double mutant cells have the ability to generate a vacuole de novo, which suggests that the cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with the vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both Vph1-GFP and FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.


( A ) In vivo phosphorylation of Sch9 is reduced in bur1 - ∆ C vac17 ∆ cells . WT , bur1 - ∆ C , vac17 ∆ , or bur1 - ∆ C vac17 ∆ cells expressing SCH9 - HA from the URA3 locus were grown in YPD . Cell lysates were analyzed by immunoblot with antibodies directed against HA ( top ) and Pgk1 ( loading control ) . ( B ) In vivo phosphorylation of Sch9 is reduced in bur1 - 267 cells . WT , vac17 ∆ , bur1 - 267 , or bur1 - 267 vac17 ∆ cells expressing SCH9 - HA from the URA3 locus were grown in YPD . Cell lysates were analyzed by immunoblot with antibodies directed against HA ( top ) and Pgk1 ( loading control ) .

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(A) In vivo phosphorylation of Sch9 is reduced in bur1-∆C vac17∆ cells. WT, bur1-∆C, vac17∆, or bur1-∆C vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control). (B) In vivo phosphorylation of Sch9 is reduced in bur1-267 cells. WT, vac17∆, bur1-267, or bur1-267 vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control).


( C ) bur1 - ∆ C shows high rapamycin sensitivity . WT , tor1 ∆ , bur1 - ∆ C , tor1 ∆ bur1 - ∆ C or vac17 ∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0 , 2 , 4 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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(C) bur1-∆C shows high rapamycin sensitivity. WT, tor1∆, bur1-∆C, tor1∆ bur1-∆C or vac17∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0, 2, 4 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) In vitro , Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself . pRS425 BUR1 - HA and pVT102 - Ura BUR2 , the essential cyclin for BUR1 , were co - expressed in a bur1Δ mutant , immunoprecipitated using anti - HA antibody and protein A beads , and then incubated with [ γ - 32P ] ATP and GST , GST - N - Sch9 ( 1 - 390 ) or GST - C - Sch9 ( 391 - 824 ) . Proteins were eluted with SDS sample buffer , separated by SDS - PAGE , and transferred to nitrocellulose membranes . Membranes exposed to x - ray film ( top ) or stained with Ponceau S ( bottom ) . Red arrowhead ; phosphorylated GST - C - Sch9 . Gray arrowhead ; auto - phosphorylated Bur1 .

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(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.


( D ) Sch9 - 2D3E , but not Sch9 - 3A - 2D3E , partially suppressed the rapamycin sensitivity of tor1 ∆ cells . tor1 ∆ which expresses pRS426 ( mock ) , pRS425 TOR1 , pVT102 - Ura ( mock ) , pVT102 - Ura Sch9 , pVT102 - Ura Sch9 - 2D3E , or pVT102 - Ura Sch9 - 3A - 2D3E , were cultured in liquid SC - Ura medium and serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 - 3 days .

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(D) Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells. tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days.


( E ) The Sch9 - 2D3E mutant did not suppress the rapamycin sensitivity of bur1 - ∆ C cells . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pVT102 - Ura ( mock ) , pVT102 - Ura SCH9 , pVT102 - Ura sch9 - 2D3E , pVT102 - Ura sch9 - 3A - 2D3E , pVT102 - Ura sch9 - 3D / E , or pVT102 - Ura sch9 - 3D / E - 2D3E were cultured in liquid SC - Ura medium and serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 3 days .

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(E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.


( A ) Bur1 fused SV40 - NLS does not rescue the rapamycin sensitivity of bur1 - ∆ C compared to Bur1 without the NLS . bur1 - ∆ C which expresses pRS416 ( mock ) , pRS416 BUR1 , pRS416 BUR1 - mNG , pRS416 BUR1 - mNG - NLS , pRS416 bur1 - ∆ C , pRS416 bur1 - ∆ C - mNG - NLS or pRS416 bur1 - ∆ N , were cultured in liquid SC - Ura medium and serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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(A) Bur1 fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416 bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) Adding nuclear - export signal ( NES ) of PKI to Bur1 and / or deletion of the importin α - dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pRS426 bur1 - ∆ N , pRS426 BUR1 - mNG , pRS426 BUR1 - mNG - NES , or pRS426 bur1 - ∆ N - mNG - NES , were cultured in liquid SC - Ura medium and serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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

(B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the importin α-dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426 BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


A , C ) After 24 , 48 , and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner . Untreated cells were measured as controls ( n = 12 , mean ± S . D . ) .

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A,C) After 24, 48, and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner. Untreated cells were measured as controls (n = 12, mean ± S.D.).


B , D ) Dynamic proliferation curves for Cal - 78 and SW - 1353 cells in the presence of 0 ( red ) , 2 . 5 nM ( green ) , 5 nM ( blue ) , and 10 nM ( purple ) bortezomib . Data shown are representatives from three independent experiments ( n = 3 , measured in biological quadruplicates ) .

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B,D) Dynamic proliferation curves for Cal-78 and SW-1353 cells in the presence of 0 (red), 2.5 nM (green), 5 nM (blue), and 10 nM (purple) bortezomib. Data shown are representatives from three independent experiments (n = 3, measured in biological quadruplicates).


A , C ) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3 / 7 activity than untreated cells . Untreated control cells served as reference value ( ratio = 1 ) .

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A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1).


B , D ) Cleavage of caspase - 3 was detected after 24 h of bortezomib treatment by flow cytometry . The y - axis denotes cell counts and the x - axis represents fluorescence intensity of the APC antibody . The black filled histogram represents untreated control cells , the striated histogram represents 5 nM , and the checkered histogram shows 10 nM bortezomib treated cells .

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B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.


A , B ) Relative gene expression analysis of the pro - and anti - apoptotic markers Bax , Bak , Bcl - 2 , and Bcl - xl in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h ( n = 10 ) .

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A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10).


C ) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27 , HSP60 , and HSP70 as well as HTRA , Livin , p27 , and p53 in both cell lines .

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C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines.


D ) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment .

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D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.


A , B ) Relative gene expression analysis of IGF1R , Fas , and the death receptors TRAILR - 1 and TRAILR - 2 after bortezomib treatment with the respective IC50 concentrations for 48 h . Untreated control cells served as reference value ( ratio = 1 ) .

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A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1).


C ) The human apoptosis antibody protein array supported the important role of the IGF binding proteins ( IGFBP - 1 to IGFBP - 6 ) , the TNF receptor family , and the TRAIL receptors in both cell lines .

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C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines.


D ) Western blot analysis showed the downregulation of Fas and TNF - R1 .

D) Western blot analysis showed the downregulation of Fas and TNF-R1.


A ) Relative gene expression and B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

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A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

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B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


C ) Western blot analysis for the expression of LC3BI - II . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II .

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C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II.


D ) Quantification of the relative LC3B protein expression .

D) Quantification of the relative LC3B protein expression.


A ) Effect of bortezomib treatment on LC3B immunostaining of Cal - 78 and SW - 1353 cells . Double immunolabeling with DAPI ( blue ) and anti - LC3B ( green ) antibodies was performed as described in the methods section . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II ( bar : 20 μm ) .

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A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm).