( B ) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins .

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(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.


( E ) HepG2 cells were transfected with FLAG - JFK and treated with 50 μg / ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis . Quantitation was done by densitometry and expressed as signals of ING5 / β - actin .

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(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.


( H ) Bacterially - expressed GST - JFK or JFK mutants was incubated with in vitro transcribed / translated ING5 for in vitro ubiquitination assays .

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(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.


( D ) ChIP - seq track at the representative promoter of target genes . The red rectangles indicate the peak regions / binding sites of ING5 on the target genes promoter .

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(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.


( C ) HepG2 cells were treated with control siRNAs or siRNAs against JFK or / and ING5 , or transfected with vector , JFK , or / and ING5 . Cellular extracts were prepared for western blotting analysis with the indicated antibodies .

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(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.


( D ) HepG2 cells were transfected with vector or JFK , AMPKα1 , or / and ING5 , or treated with control siRNAs or siRNAs against JFK , AMPKα1 , or / and ING5 , Cellular extracts were prepared for AMPK activity assays by ELISA . Error bars represent mean ± SD for triplicate experiments ( * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).


( D ) The measurement of AMPK activity and the rate of fatty acid β - oxidation of wild - type or JfkKO mice . Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA . Mitochondria were isolated from MEFs or liver tissues , and the rate of fatty acid β - oxidation was quantified by spectrophotometric detection for ferricyanide - trapped reduced products . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector control , shIng5 , or shAmpkα1 via tail vein . Seven days after injection , cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content ( E ) . Asterisk ( * ) represents significant comparison to wild - type mice , octothorpe ( # ) represents significant comparison to JfkKO mice . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , # p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) .

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Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).


D . Expression distribution of lncORF in both groups by Ribo - seq .

D. Expression distribution of lncORF in both groups by Ribo-seq.


H . Scatter diagram of lncRNA - seq in HCT116 / L - OHP and HCT116 cells .

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H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.


A . Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation , and PCR verification of the 12 screened lncRNA in both RNC - RNA and total RNA from two groups of CRC cells , among which the translation level of lnc - AP ( marked with a green box ) was the most notably dysregulated in both groups of drug - resistant cells ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance .

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A. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance.


B . PCR analysis of lnc - AP in serum samples from oxaliplatin - resistant ( PD , n = 15 ) and - sensitive ( PR , n = 9 ) CRC patients ( mean ± SEM , t test ) .

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B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).


C . In the public database lncCAR , high expression level of lnc - AP was correlated with long overall survival in CRC patients .

C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients.


E . PCR and WB analysis of HEK293T cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . F . PCR and WB analysis of HCT116 / L - OHP and SW480 / L - OHP cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


J . IF detection of pep - AP in HCT116 / L - OHP and SW480 / L - OHP cells transfected with either HA - lnc - AP OE plasmid . Scale bar , 10 µm .

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J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.


B . CCK - 8 assay of inhibition ratio by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA .

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B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


C . IF detection of H2AFX for DNA damage by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP , and nuclei were stained with DAPI ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) .

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C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).


G . Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 .

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G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01


H . PCR analysis of lnc - AP attenuation and restoration in HCT116 and SW480 cells ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


J . CCK - 8 assay of inhibition ratio by L - OHP in HCT116 and SW480 cells with lnc - AP / pep - AP attenuation and restoration ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA .

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J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


D . Survival analysis correlated with TALDO1 using TCGA data . Upper : the Kaplan curve of overall survival probability for patients with high or low level of TALDO1 . Lower : the expression graph of TALDO1 in patients who were dead or not .

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D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not.


F . IF detection of the colocalization of pep - AP - Flag and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells . Scale bar , 10 µm .

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F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.


G . WB confirmation of the interaction between HA - pep - AP and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells .

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G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.


A . WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells , which was quantified by gray analysis on the right ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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A. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


D . Cellular ROS was measured by flow cytometry and shown in both curve and bar charts ( n = 3 , biological replicates , mean ± SEM , t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . MMP detection . Scale bar , 10 µm . F . IF detection of H2AFX for DNA damage by L - OHP . Scale bar , 10 µm .

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E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.


E . Cellular ROS was measured by flow cytometry and shown in both curve ( left ) and bar ( right ) charts ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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E. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


F . MMP detection with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells . Scale bar , 10 µm .

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F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.


C . Flow cytometry of the cell apoptosis ratio under L - OHP with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells , which was quantified in bar charts on the right ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA .

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C. Flow cytometry of the cell apoptosis ratio under L-OHP with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . CCK - 8 assay of the inhibition ratio by L - OHP with inhibitors of apoptosis Z - VAD - FMK or ferroptosis Fer - 1 in HCT116 / L - OHP and SW480 / L - OHP cells ( n = 3 , biological replicates , mean ± SEM ) . Data information : * Control referred to the corresponding plasmid

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E. CCK-8 assay of the inhibition ratio by L-OHP with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector


B . Alterations of tumor volume in different groups with or without pep - AP OE ( n = 6 , biological replicates , mean ± SEM ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


F WB analysis of pep - AP , TALDO1 , PARP , cleaved - PARP and cleaved - caspase - 3 within the tumors in C ( a ) .

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F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a).


G . Relative NADPH / NADP + ratio within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . H . GSH contents within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector .

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G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


I . IF detection of pep - AP , TALDO1 and H2AFX , TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C ( a ) .

I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( a ) Endogenous LC3 was detected by immunoblot in total cell lysates , quantified by densitometric analysis and normalized to actin . Relative levels were expressed in percentage with control cells set to 100 and shown in graph .

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(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph.


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( b ) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels . Graph shows mRNA levels quantified by densitometric analysis and normalized to actin .

(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin.


( c ) 8xGli - luciferase construct was transfected in HeLa cells for 24 h , followed by treatment with 10 μM purmorphamine , or DMSO as a control , for another 24 h . Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control .

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(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.


( d ) Endogenous LC3 - II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h , either in the absence or presence of trehalose ( 100 mM ) . Where indicated , bafA1 ( 400 nM ) was added for the last 4 h . Quantification by densitometric analysis relative to actin is shown in the graph .

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(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.


( e ) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions , either in the presence of bafA1 or DMSO as a control , and LC3 - II and actin levels were detected .

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(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.


( f ) HA - HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h . The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph . P - values were calculated by odds ratio .

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(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.


( g ) U20S cells stably expressing HaloTag - p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine . After 48 h , cells were lysed , run on a SDS - polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager . Fluorescence was normalized to total protein levels , detected on the same gels by Kryton Fluorescence protein stain . A representative gel and its quantitated normalized levels are shown . In all panels , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test unless indicated : * * * P0 . 001 ; * * P0 . 01 ; * P0 . 05 . SDS - PAGE , SDS - polyacrylamide gel electrophoresis .

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(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.


( a ) HeLa cells were transiently transfected with pcDNA ( vector ) , Ptch1 or Ptch2 constructs for 48 h . Where indicated , cells were treated with bafA1 for the last 4 h . LC3 - II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph .

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(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.


( b ) HeLa were transfected with Ptch1 or Ptch2 together with HA - HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence . P - values were calculated by odds ratio .

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(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.


( c ) Atg5 + / + or Atg5 − / − MEFs were transfected with GFP - HttQ74 for 48 h , cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence .

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(c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h ( d ) Quantification and statistical analysis is shown in graphs .

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h ( e ) . Quantification and statistical analysis is shown in

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs


( f ) siRNA - targeting Ptch2 was transfected into HeLa cells stably expressing mRFP - GFP - LC3 and representative confocal microscopy images are shown . Scale bars represent 26 μm . Percentage of GFP - positive vesicles ( autophagosomes ) , RFP - positive vesicles ( total number of vesicles ) or percentage of autolysosomes ( calculated by subtracting numbers of GFP from RFP vesicles ) , quantified using a Cellomics array scan , was calculated relative to control siRNA - transfected cells . Although an increase in autophagosome biogenesis generally increases both the number of GFP + vesicles and the number of GFP - / RFP + vesicles as a result of an enhanced autophagy flux , defects in autophagosome degradation , such as in bafA1 - treated cells , increase GFP + vesicles but not GFP − / RFP + structures , as GFP remains intact . Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100 . In all panels , unless indicated , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test : * * P0 . 01 ; * P0 . 05 ; NS , not significant .

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(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.


( a ) Smo siRNA - treated cells were transfected with HA - HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA - positive cells by immunofluorescence . Graphs show mean values and error bars represent s . d . of a representative experiment performed in triplicate . P - values were calculated by odds ratio .

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(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio.


( b ) siRNA - targeting Smo was transfected into HeLa cells stably expressing mRFP - GFP - LC3 . Representative confocal microscopy images are shown . Bars represent 26 μm . The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA - transfected cells . Statistical tests were performed on data from three independent experiments in triplicate . * P0 . 05 ; * * * P0 . 001 .

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(b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.


( a ) Wild - type Gli + / + or Gli1 − / − , Gli2 − / − and Gli3 − / − MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

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(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( b ) Gli2 − / − MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a . with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( c ) LC3 - II levels were detected by western blotting of total lysates from wild - type and Gli2 − / − mice embryos ( E12 . 5 ) . For quantification , LC3 - II levels from a total of five wild - type and five Gli2 − / − embryos were normalized to actin , wild - type was set to 100 . Graphs show mean values and error bars represent s . d . Statistical analysis were performed by two - tail Student ' s t - test * * P0 . 01 . Pmph , purmorphamine .

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(c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.


( a , b ) Control ( a ) or hh [ ts2 ] ( b ) larvae were incubated at permissive temperature ( 18 ° C ) to the third instar stage , then cultured at non - permissive temperature ( 29 ° C ) in well - fed conditions for 24 h . Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed .

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(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed.


( c ) Lysotracker Red - labelled fat body containing single - cell flipout clones ( GFP positive ) overexpressing patched . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( d ) Confocal image of a GFP - positive patched - expressing clone , with mCherry - Atg8a expressed in all cells . Fat body was dissected from well - fed larvae and imaged after formaldehyde fixation .

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(d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation.


( e ) GFP - marked flipout clones of CiCell - expressing cells . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( f ) Confocal image of fixed mCherry - Atg8a expressing fat body containing a GFP - marked clone of CiCell - expressing cells .

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(f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells.


( g ) Cells homozygous for the null costal2 [ 2 ] allele ( marked by lack of GFP ) were generated by flp / FRT - mediated recombination in mCherry - Atg8a expressing fat body . Larvae were starved 4 h before dissection and fixation .

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(g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCherry-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation.


( a ) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells . Each circle represents an autophagy gene ; those with largest fold - changes are indicated in red . The central line represents no changes in expression ; above the central line , genes whose expression is increased : below , those with reduced levels . Grey lines indicate 2 - fold increase or decrease . ( b ) Table showing the 10 genes with higher fold changes in expression and the values obtained .

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(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained.


( c ) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h . Actin was used as a protein loading control .

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(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.


( d ) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h . Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI - COR infra - red imager .

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(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.


( e , f ) Wild - type and PERK − / − ( e ) or eIF2α S51A / S51A MEFs ( f ) were treated with DMSO or 10 μM purmorphamine for 24 h and , where indicated , cells were treated with bafA1 for the last 4 h . tubulin - II and tubulin or tubulin , as a loading control , were detected and a representative blot is shown . HBSS , Hank ' s balanced salt solution .

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(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.


A ) Differential interference contrast ( DIC ) image of cortical neurons in culture . The arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP fluorescence before and after exposure to lactacystin ( 10µM ) .

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A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).


C ) A neuron coexpressing Ub - R - GFP and CFP before ( left ) and 10 hours after exposure to lactacystin ( right ) . Top row - GFP fluorescence ; bottom row - CFP fluorescence . Bars : 50µm ( A , B ) , 20 µm ( C ) . D ) Changes in GFP and CFP fluorescence following exposure to lactacystin at t = 1 hour ( vertical dashed line ) . Changes normalized to initial fluorescence in same cells . 9 neurons from 5 experiments ; averages and SEM . E ) Same as in D , except that here neurons were exposed only to carrier solution . 11 neurons from 5 experiments ; averages and SEM ( barely observable ) . F ) Comparison of GFP fluorescence accumulation rates . Linear fits shown as black lines ; fit parameters shown next to fits . Same data as in D , and E .

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C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.


A ) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins ( the postsynaptic scaffold molecule Dlg4 / PSD - 95 , the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A ) following 10 hour incubations in carrier solution ( control , upper panels ) or Lactacystin ( 10 μM ; bottom panels ) . Scale bar ,

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A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm


B ) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution . Numbers within bars represent the number of fields of view analyzed for each condition ( ~ 170 synapses / field of view , on average ) . Error bars , SEM ; P values - two tailed unpaired t - test . Note that a statistically significant accumulation was observed only for RIM , whereas immunolabeling of all other proteins was either unchanged or reduced .

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B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.


C ) Hippocampal neurons in primary culture were grown in the presence of Lactacystin ( 10 μM ) , fixed at two hour intervals and immunolabeled against Shank3 / ProSAP2 . Two representative fields of view at t = 0 and t = 24h . Scale bar , 10μm . D ) Average synaptic Shank3 / ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin . Fluorescence was normalized to mean fluorescence at t = 0 . Numbers under data points reflect the numbers of fields of view analyzed at each time point . Error bars , SEM .

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C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.


B ) Comparisons of H / L ratios obtained for 1409 proteins in lactacystin and untreated cells . H / L ratios are averages of 2 experiments .

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B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.


A ) Fold changes in the H / L ratios ( ) measured for each protein in the experiments described in Fig . 3 ( expressed as log2 ( H / L ) Lactacystin - log2 ( H / L ) control ) . H / L ratios used here are the average of ratios obtained in two experiments for each protein and each condition . Proteins were sorted according to the fold change in H / L ratios - from the proteins whose ratios were most strongly reduced ( ~ 8 - fold reduction ) to proteins whose H / L ratios increased to the greatest extent ( > 8 - fold increase ) . Red circles indicate the location of synaptic proteins along this sorted list . The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance . B ) The apparent fold - change in protein half - lives under certain assumptions described in Results . See Materials and Methods for further details on the calculation of the apparent fold change in half - lives .

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A) Fold changes in the H/L ratios ( ) measured for each protein in the experiments described in Fig. 3 (expressed as log2(H/L)Lactacystin - log2(H/L)control). H/L ratios used here are the average of ratios obtained in two experiments for each protein and each condition. Proteins were sorted according to the fold change in H/L ratios - from the proteins whose ratios were most strongly reduced (~8-fold reduction) to proteins whose H/L ratios increased to the greatest extent (>8-fold increase). Red circles indicate the location of synaptic proteins along this sorted list. The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance. B) The apparent fold-change in protein half-lives under certain assumptions described in Results. See Materials and Methods for further details on the calculation of the apparent fold change in half-lives.


A ) Average log2 ( H / M ) ratios for 1 , 750 proteins at four time points in three separate experiments . Each time point shows the average and standard deviation of three mean log2 ( H / M ) values obtained in three separate experiments . Each mean , in turn , represents the mean log2 ( H / M ) value measured in the population of 1 , 750 proteins in that experiment and time point . B ) Distribution of log2 ( H / M ) values for 1 , 750 proteins at t = 0 . The log2 ( H / M ) value of each protein represents the average value for that protein in the three experiments . In principle , log2 ( H / M ) for all proteins at t = 0 should be 0 , but in practice they are distributed around 0 as shown here . The H / M ratios for 3 standard deviation limits are shown beneath the X axis . C , D ) The same as in A and B but for two experiments in which Epoxomicin was used ( 1 , 823 proteins ) . E , F ) The same as in A and B but for the combined data from all experiments ( 3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used ; 1 , 469 proteins ) .

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A) Average log2(H/M) ratios for 1,750 proteins at four time points in three separate experiments. Each time point shows the average and standard deviation of three mean log2(H/M) values obtained in three separate experiments. Each mean, in turn, represents the mean log2(H/M) value measured in the population of 1,750 proteins in that experiment and time point. B) Distribution of log2(H/M) values for 1,750 proteins at t=0. The log2(H/M) value of each protein represents the average value for that protein in the three experiments. In principle, log2(H/M) for all proteins at t=0 should be 0, but in practice they are distributed around 0 as shown here. The H/M ratios for 3 standard deviation limits are shown beneath the X axis. C, D) The same as in A and B but for two experiments in which Epoxomicin was used (1,823 proteins). E, F) The same as in A and B but for the combined data from all experiments (3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used; 1,469 proteins).


A ) log2 ( H / M ) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin . Each point represents average log2 ( H / M ) from 3 experiments . B ) log2 ( H / M ) values for the same 5 synaptic proteins in experiments in which epoxomicin was used . Each point represents average log2 ( H / M ) from 2 experiments . C ) Average ( ± SEM ) log2 ( H / M ) ratios for the same 5 synaptic proteins , based on all five experiments ( 3 lactacystin , 2 epoxomicin ) . P values - two - tailed Welch ' s t - test , comparing the five log2 ( H / M ) measurements at t = 24 to those measured for the same proteins at t = 0 .

O 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 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 B-CONTROLLED_VAR 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

A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.


D , E , F ) Average ( ± SEM ) Log2 ( H / M ) values for three groups of synaptic proteins ( synaptic vesicle , presynaptic , postsynaptic ) , based on all five experiments . None of the changes observed for these proteins were statistically significant . The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H / M normalization process ( Compare with Fig . 6E ; see Results for further details ) .

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D,E,F) Average (± SEM) Log2(H/M) values for three groups of synaptic proteins (synaptic vesicle, presynaptic, postsynaptic), based on all five experiments. None of the changes observed for these proteins were statistically significant. The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H/M normalization process (Compare with Fig. 6E; see Results for further details).


G ) log2 ( H / M ) values at t = 24 hours for a group of well - studied synaptic proteins , sorted from highest to lowest . P values ( two - tailed Welch ' s t - test ) of comparisons with log2 ( H / M ) values for the same proteins at t = 0 are shown in parentheses . Proteins that exhibited statistically significant changes ( P ≤ 0 . 05 ) at t = 24h are shown in red . Proteins for which ( 0 . 05 < P ≤ 0 . 10 ) at t = 24h are shown in blue / red . The gray region depicts three standard deviations of log2 ( H / M ) values at t = 0 ( Fig . 6F ) , to provide a measure of the method ' s sensitivity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) log2(H/M) values at t=24 hours for a group of well-studied synaptic proteins, sorted from highest to lowest. P values (two-tailed Welch's t-test) of comparisons with log2(H/M) values for the same proteins at t=0 are shown in parentheses. Proteins that exhibited statistically significant changes (P≤0.05) at t=24h are shown in red. Proteins for which (0.05 < P ≤ 0.10) at t=24h are shown in blue/red. The gray region depicts three standard deviations of log2(H/M) values at t=0 (Fig. 6F), to provide a measure of the method's sensitivity.


Measured log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 1 , 416 proteins for which half - life estimates were available .

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

Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.


Measured Log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 174 synaptic proteins for which half - life estimates were available .

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

Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.


( A ) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag - HA - tagged C9ORF72 and captured through consecutive anti - Flag and anti - HA affinity purification steps .

O O O O O O O O O O O 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) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps.


( B ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and / or Flag - tagged SMCR8 and / or Flag - tagged WDR41 .

O O O O O O O O O O O O O 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) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.


( C ) Coomassie blue staining of Nickel - NTA affinity purification of HIS - C9ORF72 , SMCR8 and WDR41 co - expressed in baculovirus infected insect cells .

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(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.


( D ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with various Flag - tagged Rab GTPases .

(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.


( E ) Immunoblot against endogenous C9orf72 , Wdr41 , Rab8a , Rab39b , Rab5 and Rab7 of control ( IgG alone ) or endogenous Smcr8 immunoprecipitated from adult mouse brain .

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(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.


( F ) αP32 - radiolabelled GDP release from GST - tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41 .

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

(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.


( G ) Identical GDP release assay as in ( F ) but using recombinant purified GST - tagged RAB39b instead of RAB8a . Error bars indicate s . e . m . Experiments were repeated 3 times ( n = 3 ) .

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

(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).


( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP - RFP - LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Right panel , quantification of LC3B puncta .

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

(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.


( B ) Upper panel , immunoblot analysis of endogenous LC3B ( Map1lc3b ) , C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Lower panel , real time RT - qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA .

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(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.


( C ) Left panel , representative images of immunofluorescence labeling of endogenous P62 ( Sqstm1 ) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of P62 aggregates .

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(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.


( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD .

O O O O O O O O O O O O O O O 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) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.


( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 and HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32 - radiolabelled ATP . Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) .

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(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).


( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS - tagged C9ORF72 : SMCR8 : WDR41 complex purified from baculovirus infected insect cells .

O O O O O O O O O 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) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.


( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting the 3 ' UTR of Smcr8 mRNA and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A and T796A ; TD , S402D and T796D ; UD , S400D , S492D , S562D and T666D ) . Right panel , quantification of P62 aggregates .

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

(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.


( E ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of endogenous Tdp - 43 , C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA .

(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.


( B ) Left panel , representative images of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of cytoplasmic Tdp - 43 aggregates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.


( C ) Left panel , representative images of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on primary cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , quantification of cells with cytoplasmic aggregates of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

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(C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , quantification of ATXN2 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.


( B ) Cell viability ( tetrazolium assay ) of GT1 - 7 neuronal cells co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

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(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( C ) Tracing of the swimming trajectories of 48 hours post - fertilization zebrafish larvae following light touch . ( D - F ) Quantification of the touch - evoked swimming distance ( D ) , average velocity ( E ) , and maximum velocity attained ( F ) shows significant functional impairment of the zebrafish injected with HA - tagged ATXN2 with intermediate length of polyQ ( Q30x ) and antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to the sole injection of AMO against C9orf72 .

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(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.


( G ) Representative images of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . n = 3 .

(G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


E and F . TLC analysis of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total RNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . The FL - hcGAS concentration was 0 . 265 mg / mL . The tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 85 mg / mL . The extracted total RNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 5 mg / mL . NC indicates the negative control that has only the FL - hcGAS . dsDNA ISD at a concentration of 0 . 05 mg / mL was used as a positive control . All the samples were prepared in 20 mM HEPES at pH 7 . 5 and 150 mM NaCl .

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E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


F . Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments .

F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.


G . The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . H . The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) analysis of the fluorescence signals of cGAS vs . RNA ( n = 13 , biological replicates ) , and mCherry vs . RNA ( n = 9 , biological replicates ) . The median values were calculated and used for evaluation of significance . * * * P < 0 . 001 ( two - tailed Mann - Whitney test ) .

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G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


D . Fluorescence microscopy images showing the replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . E . Fluorescence microscopy images showing the replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . F . Quantification of the replacement in " D " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of Cy5 - ISD over FTSC - tRNA in the four parts were calculated , averaged and plotted . G . Quantification of the replacement in " E " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in the four parts were calculated , averaged and plotted .

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D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.


The 3 ' - UTR sequence of mUcp1 RNA . Canonical CPEs ( UUUUA1 - 2U in bold ) , the reported BRF1 - binding site ( UAUUUAU , gray box ) and predicted IMP2 - binding motifs ( AYAYA and YA2YA , underlined ) are denoted . Ucp1L and Ucp1S carrying long and short 3 ' - UTRs , respectively , are resulted from alternative use of two polyadenylation signals ( Hex : AAUAAA , open box ) . ORF , open reading frame . The arrowhead marks the end of Ucp1S sequence . Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes

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The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes


Schematic comparison of mUcp1L and hUcp1 . Poly ( A ) signal ( Hex , open box ) and CPE ( black box ) are denoted . The RT - PCR results from human , WT ( Ucp1 + / + ) and UCP1 - KO ( Ucp1 - / - ) BAT RNA samples

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Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples


Dual luciferase reporter assay and RT - qPCR . The reporter plasmids , Fluc - mUcp1S , - mUcp1L or - hUcp1L 3 ' - UTR , and Rluc were co - transfected into differentiated HIB1B adipocytes . The transfected cells were used for luciferase activity assay and RT - qPCR . Data are mean ± SD from 3 independent experiments . Translation efficiency was defined as normalized activity ( Fluc / Rluc ) divided by normalized RNA level ( Fluc / Rluc ) . * * * p < 0 . 001 compared with Fluc - mUcp1S , one - way ANOVA

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Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA


Northern blotting of Ucp1 and 18S rRNA and RT - PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples ( 2 mice per genotype , 2 - 3 mo old ) . The short PCR fragments were isolated for sequencing

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Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing


Western blot analysis using WT and Ucp1 ∆ L BAT lysates . The protein level of UCP1 was normalized to GAPDH ( 4 mo old , n = 4 per group ) . * * p < 0 . 01 , Student ' s t test

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Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test


Dual luciferase reporter assay . The reporter plasmids , Fluc - mUcp1L 3 ' - UTR , and Rluc were co - transfected with the plasmid expressing EGFP , or myc - tagged CPEB ( immunoblots shown at the left ) into differentiated HIB1B adipocytes ( Data from 3 independent experiments ) . * p < 0 . 05 compared with EGFP , one - way ANOVA

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Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA


Western blot analysis using BAT lysates prepared from various CPEB - WT and KO male littermates ( 4 mo old , n = 4 per group ) . * p < 0 . 05 , Student ' s t test

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Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test


Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2 - WT and - KO male mice at room temperature or after 5 - h cold exposure ( CE ) at 4oC ( 5 mice per genotype , 4 - 7 mo old )

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Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)


Western blot analysis and RT - qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT ( 13 mo old , n = 3 - 4 per group )

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Western blot analysis and RT-qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT (13 mo old, n = 3-4 per group)


Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old ( n = 3 per group ) . Protein levels were normalized to that of GAPDH . RT - qPCR analysis of Ucp1 mRNA level in BAT collected from ( B ) relative to Gapdh mRNA

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Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old (n = 3 per group). Protein levels were normalized to that of GAPDH. RT-qPCR analysis of Ucp1 mRNA level in BAT collected from (B) relative to Gapdh mRNA level


Hematoxylin and eosin sections of BAT from WT and CPEB2 - KO female mice at 4 and 12 mo old . Scale bars , 1

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Hematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mm


Lipid droplet size and cell number ( i . e . , number of nuclei ) in WT and KO BAT ( representative images shown in 4D ) were quantified and displayed as arbitrary units . Three tissue sections from each BAT and 3 mice per group were

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Lipid droplet size and cell number (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzed


Weight and representative images of WT and KO BAT . Three mice per group were

Weight and representative images of WT and KO BAT. Three mice per group were analyzed


CPEB2 - WT and - KO mice were transduced with adeno - associated virus ( AAV ) expressing full - length ( AAV _ CP2 ) or RNA - binding domain ( AAV _ RBD ) of CPEB2 . Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV _ CP2 for 2 weeks , with Hoechst staining of nuclei . Scale bar , 10 µm . See more images in Fig EV4D

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CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D


Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction ( n = 6 mice per group ) . * p < 0 . 05 , Student ' s t test

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Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction (n = 6 mice per group). *p < 0.05, Student's t test


BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12 - h light time ( n = 7 mice per group ) . * p < 0 . 05 , Student ' s t test

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BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12-h light time (n = 7 mice per group). *p < 0.05, Student's t test


BAT temperature in WT and KO male mice , and of AAV _ CP2 - transduced WT and KO male mice ( n = 7 mice per group ) before and after i . p . injection of CL316243 ( CL ) . * p < 0 . 05 , repeated - measures two - way ANOVA

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BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA


Normalized RPF count profile averaged for all E . coli transcripts . Profiles generated for cells grown in the absence and presence of IPTG ( 1 mM ) . Start and stop codons are shaded .

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Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.


Bar chart of all measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for cells grown in the absence and presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG ( 1 mM ) .

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

Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).