D , E The SBE - Luc or 5xNF - κB - Luc reporter plasmids were co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 6 h or LPS for 2 h , and luciferase activities were measured and normalized .

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D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.


F The BRE - Luc reporter plasmid was co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid or full - length Smad6 into RAW264 . 7 cells , respectively . After 24 h , cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized .

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F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized.


G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) or full - length Smad6 ( Myc - Smad6 ) was co - transfected with HA - tagged full - length Smad4 or HA - tagged full - length Pellino - 1 plasmid into HEK293 cells , respectively . Cell lysates were immunoprecipitated ( IP ) with anti - Myc and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , pCS3MTBXA - 6xMyc , was co - transfected with HA - Smad4 or HA - Pellino - 1 as a negative control . Data are representative of at least three independent experiments .

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G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.


A Pre - treatment of Smaducin - 6 reduces LPS - induced interleukin - 6 ( Il6 ) gene expression in a dose - dependent manner in RAW264 . 7 cells . Il6 gene expression was analyzed by qRT - PCR .

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A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by qRT-PCR.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( B ) NF - κB - mediated luciferase gene expression when RAW264 . 7 cells are treated with LPS for 2 h . Luciferase activity in ( B ) was normalized to β - galactosidase activity .

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B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( C ) IκBα degradation and IKKα / β phosphorylation when RAW264 . 7 cells are treated with LPS for 2 h .

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B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h.


D Peli1 knockdown or wild - type humanTHP1 cells were pre - treated with 100 nM Pal - Scram peptide and Smaducin - 6 for 30 min and subsequently treated with LPS for 2 h . Il6 and Peli1 gene expression were analyzed by qRT - PCR . Data show the mean ± SD of three independent experiments .

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D Peli1 knockdown or wild-type humanTHP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by qRT-PCR. Data show the mean ± SD of three independent experiments.


E RAW264 . 7 cells were treated with 100 nM FITC - conjugated Smaducin - 6 and TAT - S6 ( 422 - 441 ) , and localization was observed by confocal microscopy . Scale bar , 10 μm ( magnification , 200 × ) . Nuclei were stained with DAPI .

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E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.


F After pre - treating RAW264 . 7 cells with biotin - conjugated Smaducin - 6 ( 100 nM ) for 30 min , cells were treated with LPS for 2 h . Subsequent precipitation by streptavidin - agarose showed that endogenous Pellino - 1 binds to the Smaducin - 6 peptide . A biotin - conjugated scrambled peptide was used as a negative control .

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F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin-agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.


G - I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin - 6 inhibits the formation of endogenous ( G ) IRAK1 - , ( H ) RIP1 - , or ( I ) IKKε - mediated signaling complexes induced by 2 h LPS treatment , compared to a scrambled peptide . IB , immunoblot ; TCL , total cell lysates . Data shown are representative of three independent experiments .

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G-I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.


Subcutaneous injection of Smaducin - 6 increases the survival rate of severe CLP - induced sepsis mice ( BALB / c mice ) . Different amounts of Smaducin - 6 were subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group per experiment .

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Subcutaneous injection of Smaducin-6 increases the survival rate of severe CLP-induced sepsis mice (BALB/c mice). Different amounts of Smaducin-6 were subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group per experiment.


Smaducin - 6 treatment reduces abnormal nuclei and cell morphology observed in tissues of CLP - induced BALB / c mice , as observed by hematoxylin and eosin ( H / E ) staining . Data shown are representative of five independent experiments . Scale bar , 40 μm ( magnification , 400 × ) .

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Smaducin-6 treatment reduces abnormal nuclei and cell morphology observed in tissues of CLP-induced BALB/c mice, as observed by hematoxylin and eosin (H/E) staining. Data shown are representative of five independent experiments. Scale bar, 40 μm (magnification, 400×).


The survival rates of severe CLP - induced sepsis mice by subcutaneously injected Smaducin - 6 were compared with those by intraperitoneally injected antibiotics ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) . Antibiotics were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . n = 10 mice per group per experiment .

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The survival rates of severe CLP-induced sepsis mice by subcutaneously injected Smaducin-6 were compared with those by intraperitoneally injected antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin). Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. n = 10 mice per group per experiment.


Therapeutic effects of Smaducin - 6 alone , or antibiotics ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) , or different amounts of Smaducin - 6 plus antibiotics were observed in CLP mice . Antibiotics were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . Smaducin - 6 was subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group .

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Therapeutic effects of Smaducin-6 alone, or antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin), or different amounts of Smaducin-6 plus antibiotics were observed in CLP mice. Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. Smaducin-6 was subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group.


Comparison of mortality of severe CLP - induced sepsis mice upon subcutaneous injections of a total of 16 mg / kg Smaducin - 6 or TAT - S6 ( 422 - 441 ) peptide . n = 10 mice per group .

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Comparison of mortality of severe CLP-induced sepsis mice upon subcutaneous injections of a total of 16 mg/kg Smaducin-6 or TAT-S6(422-441) peptide. n = 10 mice per group.


A - I Smaducin - 6 treatment decreased the concentration of IL - 6 , TNF - α , IFN - γ , and IL - 1β in the blood of CLP mice ( A - D ) , but not IL - 4 , IL - 10 , TGF - β1 , IL - 12 , or IL - 17A ( E - I ) . Cytokine concentrations were analyzed by ELISA . n = 10 mice per group per experiment . Data were statistically analyzed by the Dunnett ' s multiple comparison test ( one - way ANOVA ) . * * * P < 0 . 001 , * * P < 0 . 005 , * P < 0 . 05 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A-I Smaducin-6 treatment decreased the concentration of IL-6, TNF-α, IFN-γ, and IL-1β in the blood of CLP mice (A-D), but not IL-4, IL-10, TGF-β1, IL-12, or IL-17A (E-I). Cytokine concentrations were analyzed by ELISA. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001, **P < 0.005, *P < 0.05 compared to sham or vehicle control (CLP + Pal-Scram #1).


J Expression of the IL - 6 protein is reduced in the spleen and liver of Smaducin - 6 - treated CLP mice . Each lane represents independent mice .

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J Expression of the IL-6 protein is reduced in the spleen and liver of Smaducin-6-treated CLP mice. Each lane represents independent mice.


Subcutaneous injection of Smaducin - 6 into CLP mice reduced bacterial loads in peritoneal fluid . n = 10 mice per group per experiment . Data were statistically analyzed by the Mann - Whitney U - test . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Subcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann-Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


Smaducin - 6 increases neutrophil numbers in peritoneal lavage fluids from LPS - induced endotoxemia BALB / c mice . n = 5 mice per group per experiment . Data were statistically analyzed by a t - test . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


CXCR2 expression in human neutrophils , measured by FACS , was decreased by LPS treatment and restored by Smaducin - 6 treatment .

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CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.


Treatment with Smaducin - 6 downregulates LPS - induced GRK2 expression in RAW264 . 7 cells and human neutrophils .

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Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.


CXCR2 - expressing neutrophils isolated from CLP mice in the presence of Smaducin - 6 or Pal - Scram peptide were analyzed by FACS . n = 5 mice per group were used . Data were statistically analyzed by a t - test and show the mean ± SD of at least three independent experiments . * * P < 0 . 005 compared to sham or vehicle controls ( CLP + PBS and CLP + Pal - Scram # 1 ) .

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CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).


GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin - 6 or Pal - Scram # 1 peptide .

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GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.


A , B Quantitative analysis of ( A ) TUNEL - positive cells and ( B ) caspase - 3 - positive cells in IHC in the spleens of CLP + PBS , CLP + Pal - Scram # 1 , and CLP + Smaducin - 6 mice . Three independent experiments ( n = 3 mice per group per experiments ) were performed . At least five hot spots in a section of TUNEL and IHC per experiment were selected , and average count was determined . The data were expressed as a mean percentage of total cell numbers and statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 005 , * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A, B Quantitative analysis of (A) TUNEL-positive cells and (B) caspase-3-positive cells in IHC in the spleens of CLP + PBS, CLP + Pal-Scram #1, and CLP + Smaducin-6 mice. Three independent experiments (n = 3 mice per group per experiments) were performed. At least five hot spots in a section of TUNEL and IHC per experiment were selected, and average count was determined. The data were expressed as a mean percentage of total cell numbers and statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.005, ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


C ELISA showing that Smaducin - 6 treatment reduced IFN - β1 levels in peritoneal lavage fluids compared to Pal - Scram # 1 - treated CLP mice . n = 10 mice per group per experiment . Data were statistically analyzed by the Dunnett ' s multiple comparison test ( one - way ANOVA ) . * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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C ELISA showing that Smaducin-6 treatment reduced IFN-β1 levels in peritoneal lavage fluids compared to Pal-Scram #1-treated CLP mice. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


D , E IFN - β1 ( D ) and TRAIL ( E ) expression detected by FACS in splenocytes is decreased in Smaducin - 6 - treated CLP mice compared to scrambled peptide - treated CLP mice . Data are representative of three independent experiments .

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D, E IFN-β1 (D) and TRAIL (E) expression detected by FACS in splenocytes is decreased in Smaducin-6-treated CLP mice compared to scrambled peptide-treated CLP mice. Data are representative of three independent experiments.


F Decreased expression of TRAIL was detected by IHC in the spleen of CLP - induced mice treated with Smaducin - 6 compared to mice treated with Pal - Scram . Scale bar , 100 μm ( magnification , 200 × ) . Data are representative of three independent experiments .

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F Decreased expression of TRAIL was detected by IHC in the spleen of CLP-induced mice treated with Smaducin-6 compared to mice treated with Pal-Scram. Scale bar, 100 μm (magnification, 200×). Data are representative of three independent experiments.


B . The truncated receptor encoded by RNA1 is endogenously expressed . HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C ( RNA1 ) . SDS - lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 ( Figure EV1 ) . Mousehypothalamus was exposed 10 times longer . In brain , a band around 75kDa of unknown origin is detected by the antiserum ( indicated by a star ) .

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B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).


D . RT - PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition . Below is a quantification of three independent experiments . A statistical evaluation showed significant differences between oligo # 5 ( p = 0 . 00008 ) and oligo # 5 - 3 ( p = 0 . 0019 ) and control .

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D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.


A . Titration of oligo # 5 . An increasing amount of oligo # 5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT - PCR . - RT : negative control without reverse transcriptase . B . Quantification of three independent experiments . The half - maximal effect was seen at 13 . 1 nM concentration .

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A. Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT-PCR. -RT: negative control without reverse transcriptase.B. Quantification of three independent experiments. The half-maximal effect was seen at 13.1 nM concentration.


C . Influence of oligo # 5 shortening on alternative splicing of the serotonin 2C receptor . Oligo # 5 : 5 ' AGU AUU GAG CAU AGC CGC 3 ' ( 18mer ) ; Oligo # 5 - 3 : 5 ' GAG CAU AGC CGC 3 ' ( 12mer ) ; Oligo # 5 - 10 : 5 ' G CAU AGC CGC 3 ' ( 10mer ) were used in transfection assays analyzed by RT - PCR . D . Statistical analysis of the effect of oligonucleotide shortening . The effect of all oligos testes is significant , oligo # 5 : p = 0 . 0015 ; oligo # 5 - 3 : p = 0 . 031 ; oligo # 5 - 10 : p = 0 . 048 , n = 6 .

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C. Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5' AGU AUU GAG CAU AGC CGC 3' (18mer); Oligo#5-3: 5' GAG CAU AGC CGC 3' (12mer); Oligo#5-10: 5' G CAU AGC CGC 3' (10mer) were used in transfection assays analyzed by RT-PCR.D. Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: p= 0.0015; oligo#5-3: p=0.031; oligo#5-10: p= 0.048, n=6.


E . Influence of cy3 label on the 5 ' and 3 ' end of oligo # 5 . Using a phosphate linker , Cy3 was added to the 5 ' or 3 ' phosphate , respectively and the oligonucleotides assayed by RT - PCR . Oligo # 5 with cy3 at the 5 ' end showed 86 % inclusion , with Cy3 at the 3 ' end 17 % exon inclusion . F . Statistical analysis of the effect of oligonucleotide modification . The effect is significant , p = 0 . 009 , n = 4 .

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E. Influence of cy3 label on the 5' and 3' end of oligo#5. Using a phosphate linker, Cy3 was added to the 5' or 3' phosphate, respectively and the oligonucleotides assayed by RT-PCR. Oligo#5 with cy3 at the 5' end showed 86% inclusion, with Cy3 at the 3' end 17% exon inclusion.F. Statistical analysis of the effect of oligonucleotide modification. The effect is significant, p=0.009, n=4.


D . In vitro splicing assay : Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract ( NE ) under splicing conditions for two hours at 30 ºC . The structures of the in vitro splicing products are shown on the right . LI and LII are two lariats formed . Numbers indicate the expected product lengths . E . Quantification of selected bands from four independent experiments . The percentage of each form in the total products is shown . The differences of splice products ( Va + b - MINX ) is significant , p = 0 . 0055 ; n = 3 .

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D. In vitro splicing assay: Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract (NE) under splicing conditions for two hours at 30 ºC. The structures of the in vitro splicing products are shown on the right. LI and LII are two lariats formed. Numbers indicate the expected product lengths.E. Quantification of selected bands from four independent experiments. The percentage of each form in the total products is shown. The differences of splice products (Va+b-MINX) is significant, p=0.0055; n=3.


B . Effect of oligo # 5 on pre - mRNA splicing of the reporter gene , stably transfected into HeLa cells .

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B. Effect of oligo#5 on pre-mRNA splicing of the reporter gene, stably transfected into HeLa cells.


C . Effect of oligo # 5 on the truncated 5HT2C protein . Stable cell lines expressing the construct shown in panel ( A ) were treated with oligo # 3 and oligo # 5 . After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant . The fractions were analyzed by Western Blot using the anti - RNA1 antiserum ( Figure EV1 ) . The protein samples were on the same membrane , but the supernatant fractions were exposed about ten times longer . D . Effect of oligo # 5 on the full - length 5HT2C protein . The protein was prepared as in panel ( C ) and analyzed using a polyclonal anti - GFP antiserum . E . Quantification of panels C and D from three independent experiments . The ratio of protein signal after oligo # 3 and oligo # 5 treatment is shown for RNA1 ( p = 0 . 002 ) and RNA2 ( p = 0 . 0003 ) , n = 3 , respectively .

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C. Effect of oligo#5 on the truncated 5HT2C protein. Stable cell lines expressing the construct shown in panel (A) were treated with oligo#3 and oligo#5. After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant. The fractions were analyzed by Western Blot using the anti-RNA1 antiserum (Figure EV1). The protein samples were on the same membrane, but the supernatant fractions were exposed about ten times longer.D. Effect of oligo#5 on the full-length 5HT2C protein. The protein was prepared as in panel (C) and analyzed using a polyclonal anti-GFP antiserum.E. Quantification of panels C and D from three independent experiments. The ratio of protein signal after oligo#3 and oligo#5 treatment is shown for RNA1 (p=0.002) and RNA2 (p=0.0003), n=3, respectively.


F , G . Effect of oligo # 5 on localization of the EGFP - tagged serotonin 2C receptor . Confocal microscopy images of three representative pictures are shown . H . Quantitification of GFP signal found on the membrane after oligo # 5 addition . The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image . 200 cells from three individual experiments were analyzed , p < 0 . 008 , n = 200 .

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F, G. Effect of oligo#5 on localization of the EGFP-tagged serotonin 2C receptor. Confocal microscopy images of three representative pictures are shown.H. Quantitification of GFP signal found on the membrane after oligo#5 addition. The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image. 200 cells from three individual experiments were analyzed, p<0.008, n=200.


A . Accumulation of Cy3 - labeled oligo # 5 in the area around the 3rd ventricle one hour post injection , shown is the ventral part of a coronal section - 1 . 34 mm from Bregma . The injection coordinates were AP : - 1 . 82 mm , DV : - 4 . 5 mm , ML : 0 . B . Uptake of oligo # 5 in the arcuate nucleus region ( arc ) , enlarged from panel A .

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A. Accumulation of Cy3-labeled oligo#5 in the area around the 3rd ventricle one hour post injection, shown is the ventral part of a coronal section -1.34 mm from Bregma. The injection coordinates were AP: -1.82 mm, DV: -4.5 mm, ML: 0.B. Uptake of oligo#5 in the arcuate nucleus region (arc), enlarged from panel A.


C . Oligo # 5 enters neurons . Enlargement of hypothalamus region taking up oligo # 5 ( red ) , stained for the neuronal marker NeuN ( green ) . Most of the oligo # 5 signal is in neurons , but some is also in NeuN negative cells ( arrow ) , likely glia . D . Oligo # 5 enters some glia cells . Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo # 5 injection . The oligo ( red ) can be found in some GFAP - positive glia cells ( arrow ) , but numerous GFAP - positive cells ( green ) show no oligo # 5 uptake .

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C. Oligo#5 enters neurons. Enlargement of hypothalamus region taking up oligo#5 (red), stained for the neuronal marker NeuN (green). Most of the oligo#5 signal is in neurons, but some is also in NeuN negative cells (arrow), likely glia.D. Oligo#5 enters some glia cells. Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo#5 injection. The oligo (red) can be found in some GFAP-positive glia cells (arrow), but numerous GFAP-positive cells (green) show no oligo#5 uptake.


E . Effect of oligo # 5 on RNA1 / RNA2 ratios in the injected area . The area marked by Cy3 staining in the dorsal hypothalamus , consisting mainly of the arcuate nucleus was microdissected , RNA isolated and analyzed by qPCR . NS : non - significant changes ( 3 hrs : p = 0 . 01 ; 6 hrs : p = 0 . 000001 ; 9 hrs : p = 0 . 02 ; 12 hrs : p = 0 . 06 ; n = 3 )

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E. Effect of oligo#5 on RNA1/RNA2 ratios in the injected area. The area marked by Cy3 staining in the dorsal hypothalamus, consisting mainly of the arcuate nucleus was microdissected, RNA isolated and analyzed by qPCR. NS: non-significant changes (3 hrs: p=0.01; 6 hrs: p=0.000001; 9 hrs: p=0.02; 12 hrs: p=0.06; n=3)


F . Oligo # 5 does not change the RNA2 - editing pattern . The hypothalamic area showing oligo # 5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing . The percent of non - edited reads in total reads is shown ( at least 20 , 000 reads per lane ) .

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F. Oligo#5 does not change the RNA2-editing pattern. The hypothalamic area showing oligo#5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing. The percent of non-edited reads in total reads is shown (at least 20,000 reads per lane).


G . Effect of oligo # 5 on POMC expression . The RNA from panel D was analyzed using qPCR with primers against POMC , normalized to GAPDH ( 3 hrs : p = 0 . 0009 ; 6 hrs : p = 0 . 02 ; 9 hrs : p = 0 . 1 ; 12 hrs : p = 0 . 3 , n = 3 ) .

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G. Effect of oligo#5 on POMC expression. The RNA from panel D was analyzed using qPCR with primers against POMC, normalized to GAPDH (3 hrs: p=0.0009; 6 hrs: p=0.02; 9 hrs: p=0.1; 12 hrs: p=0.3, n=3).


H . Effect of oligo # 5 on food uptake in wild - type mice . C57BL / 6 wild - type mice received a guide canulae and did not have access to food for 16 hours . After injection of 2 µg oligonucleotides , they could freely access food . The food consumption was measured by weighing the food used . A total of 52 animals were used , p = 0 . 001 for the difference between oligo # 5 and control oligonucleotides . Each of the six individual experiments showed statistically significant differences between oligo # 5 and control ( p < 0 . 01 - 0 . 001 ) . Control oligo : an oligonucleotide against human SMN2 .

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H. Effect of oligo#5 on food uptake in wild-type mice. C57BL/6 wild-type mice received a guide canulae and did not have access to food for 16 hours. After injection of 2 µg oligonucleotides, they could freely access food. The food consumption was measured by weighing the food used. A total of 52 animals were used, p=0.001 for the difference between oligo#5 and control oligonucleotides. Each of the six individual experiments showed statistically significant differences between oligo#5 and control (p<0.01 - 0.001). Control oligo: an oligonucleotide against human SMN2.


I . Effect of oligo # 5 on food uptake in ob / ob mice . Ob / obmice received oligo # 5 through ICV injection , similar to panel F . However , the mice could freely access food at all times ( p = 0 . 04 , n = 3 ) .

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I. Effect of oligo#5 on food uptake in ob/ob mice. Ob/obmice received oligo#5 through ICV injection, similar to panel F. However, the mice could freely access food at all times (p=0.04, n=3).


A . 50 µg of cy3 - labelled - oligo # 5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected . ChP : choroid plexus , RSGc : retrosplenial granular cx , c , 3V : 3rd ventricle ; D3V : dorsal 3rd ventricle .

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A. 50 µg of cy3-labelled-oligo#5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected. ChP: choroid plexus, RSGc: retrosplenial granular cx,c, 3V: 3rd ventricle; D3V: dorsal 3rd ventricle.


B . Change of RNA2 / RNA1 ratios after carotid artery injection . The area around the arcuate nucleus was microdissected at the times indicated post injection . The ration of RNA1 / RNA2 was determined using real - time PCR . Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs : p = 0 . 3 ; 4 hrs : p = 0 . 003 , 8 hrs : p = 0 . 0006 , n = 5 )

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B. Change of RNA2/RNA1 ratios after carotid artery injection.The area around the arcuate nucleus was microdissected at the times indicated post injection. The ration of RNA1/RNA2 was determined using real-time PCR. Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs: p=0.3; 4 hrs: p=0.003, 8 hrs: p=0.0006, n=5)


C . Effect of oligo # 5 delivered through a carotid artery catheter on food uptake in wild - type mice . C57BL / 6 wild - type mice with a pre - implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated .

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C. Effect of oligo#5 delivered through a carotid artery catheter on food uptake in wild-type mice. C57BL/6 wild-type mice with a pre-implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated.


D . Long term effect of oligo # 5 action . Food intake was measured for 70 hrs after one injection of 100 µg oligo # 5 through a carotid catheter at time zero .

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D. Long term effect of oligo#5 action. Food intake was measured for 70 hrs after one injection of 100 µg oligo#5 through a carotid catheter at time zero.


a , Survival ratio after TAC ( n = 7 - 14 per group ) .

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a, Survival ratio after TAC (n = 7-14 per group).


b - g , Ten days after TAC . b , Gross appearance of hearts . Scale bar , 2 mm .

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b-g, Ten days after TAC. b, Gross appearance of hearts. Scale bar, 2 mm.


b - g , Ten days after TAC . c , Echocardiography . Scale bars , 0 . 2 s and 5 mm . Echocardiographic parameters ( d ) ( n = 7 - 13 per group ) . LVIDd and LVIDs , end - diastolic and end - systolic left ventricular internal dimension , respectively ; LVFS , left venticular fractional shortening ;

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b-g, Ten days after TAC. c, Echocardiography. Scale bars, 0.2 s and 5 mm. Echocardiographic parameters (d) (n = 7-13 per group). LVIDd and LVIDs, end-diastolic and end-systolic left ventricular internal dimension, respectively; LVFS, left venticular fractional shortening;


b - g , ( e ) Ten days after TAC . physiological parameters ( n = 7 - 13 per group ) .

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b-g, (e) Ten days after TAC.physiological parameters (n = 7-13 per group).


Haematoxylin and eosin - stained ( f ) and azocarmine and aniline blue ( AZAN ) - Mallory - stained ( g ) heart sections . Scale bar , 100 µm . Data are mean ± s . e . m . * P 0 . 05 versus all other groups , * * P 0 . 05 versus sham - operated controls .

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Haematoxylin and eosin-stained (f) and azocarmine and aniline blue (AZAN)-Mallory-stained (g) heart sections. Scale bar, 100 µm. Data are mean ± s.e.m. *P  0.05 versus all other groups, **P  0.05 versus sham-operated controls.


Mice were analysed 2 days after TAC ( a - c ) . a , Haematoxylin and eosin - stained heart sections . Scale bar , 100 µm . b , AZAN - Mallory - stained sections . Scale bar , 100 µm .

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Mice were analysed 2 days after TAC (a-c). a, Haematoxylin and eosin-stained heart sections. Scale bar, 100 µm. b, AZAN-Mallory-stained sections. Scale bar, 100 µm.


Mice were analysed 2 days after TAC ( a - c ) . c , Immunohistochemical analysis using antibodies to CD45 , CD68 , Ly6G and CD3 . Scale bar , 100 µm .

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Mice were analysed 2 days after TAC (a-c). c, Immunohistochemical analysis using antibodies to CD45, CD68, Ly6G and CD3. Scale bar, 100 µm.


Mice were analysed 2 days after TAC ( a - e ) . a , Electron microscopic analysis . Images of mitochondria at higher magnification are shown in subsets . Scale bar , 1 µm . b , Autolysosome after incubation with anti - DNA antibody and 10 nm gold staining . Scale bar , 200 nm . Arrows indicate labelled DNA .

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Mice were analysed 2 days after TAC (a-e). a, Electron microscopic analysis. Images of mitochondria at higher magnification are shown in subsets. Scale bar, 1 µm. b, Autolysosome after incubation with anti-DNA antibody and 10 nm gold staining. Scale bar, 200 nm. Arrows indicate labelled DNA.


Mice were analysed 2 days after TAC Double staining of heart sections with EdU ( green ) and anti - LAMP2a antibody ( red ) ( c ) , EdU ( green ) and anti - LC3 antibody ( red ) ( d ) or EdU and anti - TLR9 antibody ( red ) ( e ) . Arrows indicate EdU - positive and LAMP2a - , LC3 - or TLR9 - positive structures . Scale bar , 10 µm .

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Mice were analysed 2 days after TAC Double staining of heart sections with EdU (green) and anti-LAMP2a antibody (red) (c), EdU (green) and anti-LC3 antibody (red) (d) or EdU and anti-TLR9 antibody (red) (e). Arrows indicate EdU-positive and LAMP2a-, LC3- or TLR9-positive structures. Scale bar, 10 µm.


a , Survival ratio of TAC - operated ODN - treated mice ( n = 6 - 10 per group ) .

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a, Survival ratio of TAC-operated ODN-treated mice (n = 6-10 per group).


b - d , Four days after TAC . b , Echocardiography . Scale bars , 0 . 2 s and 5 mm .

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b-d, Four days after TAC. b, Echocardiography. Scale bars, 0.2 s and 5 mm.


b - d , Four days after TAC . c , Echocardiographic parameters . Open and closed bars represent ODN2088 control - and ODN2088 - treated groups , respectively ( n = 5 - 8 per group ) .

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b-d, Four days after TAC. c, Echocardiographic parameters. Open and closed bars represent ODN2088 control- and ODN2088-treated groups, respectively (n = 5-8 per group).


b - d , Four days after TAC . d , Immunohistochemical analysis . Scale bar , 100 µm .

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b-d, Four days after TAC. d, Immunohistochemical analysis. Scale bar, 100 µm.


TLR9 - deficient mice were analysed 10 weeks after TAC ( e , f ) . e , Scale bars , 0 . 2 s and 5 mm . f , Echocardiographic parameters ( n = 6 - 10 per group ) . Data are mean ± s . e . m . * P 0 . 05 versus all other groups .

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TLR9-deficient mice were analysed 10 weeks after TAC (e, f). e, Scale bars, 0.2 s and 5 mm. f, Echocardiographic parameters (n = 6-10 per group). Data are mean ± s.e.m. *P  0.05 versus all other groups.


Western blots for exercise - associated phosphorylated proteins in ( top ) contracted ( + ) or sham ( - ) rat muscle and ( bottom ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total AMPK and 14 - 3 - 3 were used as loading controls .

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Western blots for exercise-associated phosphorylated proteins in (top) contracted (+) or sham (-) rat muscle and (bottom) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total AMPK and 14-3-3 were used as loading controls.


Volcano plots of phosphopeptide median Log2 fold - change ( contraction / sham surgery ) plotted against the - Log10 p - value in rat contraction ( left ) or mouse running ( right ) . Colored dots represent significantly - regulated phosphopeptides ( p < 0 . 05 , n = 5 , moderated t - test ) . Gray dots represent phosphopeptides that were detected but not significantly altered in exercise .

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Volcano plots of phosphopeptide median Log2 fold-change (contraction/sham surgery) plotted against the -Log10 p-value in rat contraction (left) or mouse running (right). Colored dots represent significantly-regulated phosphopeptides (p < 0.05, n=5, moderated t-test). Gray dots represent phosphopeptides that were detected but not significantly altered in exercise.


Western blots for STIM1 phosphorylated at S257 or S521 in ( left ) contracted ( + ) or sham ( - ) rat muscle and ( right ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total STIM1 and 14 - 3 - 3 were used as loading controls . 14 - 3 - 3 data for mouse running was re - used from Figure 1B .

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Western blots for STIM1 phosphorylated at S257 or S521 in (left) contracted (+) or sham (-) rat muscle and (right) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total STIM1 and 14-3-3 were used as loading controls. 14-3-3 data for mouse running was re-used from Figure 1B.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in L6 rat myoblasts with siRNA - mediated knockdown of endogenous STIM1 ( siSTIM1 ) or transfected with scramble control siRNA ( Control ) . Cells were treated with the AMPK activator A - 769662 ( + ) or vehicle ( - ) for 30 min . Total STIM1 was used to demonstrate STIM1 knockdown efficiency . Total ACC and 14 - 3 - 3 were used as loading controls .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK β1 / β2 knockout ( AMPKβ KO ) mouse endothelial fibroblasts were isolated and treated with A - 769662 ( + ) or vehicle ( - ) for 30 min . AMPKβ was used to demonstrate knockout , and AMPKɑ was used to demonstrate specificity for AMPKβ . Total ACC and STIM1 were used as loading controls .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.


Western blots for total ACC and STIM1 and phosphorylated STIM1 ( S257 and S521 ) and ACC ( S79 ) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP - STIM1 .

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Western blots for total ACC and STIM1 and phosphorylated STIM1 (S257 and S521) and ACC (S79) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP-STIM1.


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.


( A ) Coimmunoprecipitation of SMO - HA and Myc - Smaug proteins . Extracts of Cl8 cells expressing combinations of SMOWT - HA or SMOPKA - SD - HA with Myc - SmaugWT in the absence or presence of HH were immunoprecipitated ( IP ) with anti - HA . The input ( lower panel ) and the IP complexes ( upper panel ) were analyzed by western blot with anti - Myc ( αMyc ) or anti - HA ( αHA ) antibodies . Here and in the other figures , the names of the proteins detected are indicated on the left and the molecular weights on the right , in kDa ; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs . * corresponds to higher molecular weight forms of SMO of unknown origin . Here and in panel F , the black arrow indicates unphosphorylated SMOWT - HA and the bracket indicates the phosphorylated forms of SMOWT - HA or SMOPKA - SD .

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(A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD.


( D , E ) Mapping of the Smaug interaction domain in SMO . Extracts from Cl8 cells transfected with Myc - SmaugWT and various regions of SMO fused to either HA ( D ) or to GFP ( E ) were immunoprecipitated with αMyc ( D ) or αGFP ( E ) and analyzed by western blotting with αMyc ( lower panel in D and upper panel in E ) , αHA ( D , upper panel ) or αGFP ( E , lower panel ) . In : Input ; Sup : supernatant . See also Fig EV1D - F .

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(D, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F.


( F ) Hyperphosphorylated forms of SMO do not interact with Smaug . Left panel : Extracts of Cl8 cells expressing SMOWT - HA with or without Myc - SmaugWT , in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA ( upper panel ) or αMyc antibodies ( lower panel ) . Right panel : Cl8 cells expressing SNAP - SMOWT with Myc - SmaugWT in the presence of HH were extracellularly labelled with a membrane - nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation ( IP ) with anti - Myc . Labelled SNAP - SMO was directly visualized after electrophoresis . Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms . See also Fig EV1G - J .

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(F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J.


( G ) SMO and Smaug colocalize . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT alone ( 1 - 2 ) or together with SMOWT - mCherry ( 3 - 3 ' ' and 4 - 4 ' ' ) either without ( 1 , 3 - 3 " ) or with HH ( 2 , 4 - 4 " ) . The merge images in 3 " and 4 " show GFP - SmaugWT in green and SMOWT - mCh in red . The same results were seen with different fluorescent tags as well ( Fig EV2B ) . The lack of effect of HH on GFP - SmaugWT in absence of SMOWT - mCh is likely due to limiting amounts of endogeneous SMO . A least 20 cells were assayed for each condition . In absence of HH , all co - transfected cells exhibited greater than 90 % colabelled SMOWT - mCherry . Scale bar ( shown in G1 , identical for all panels ) : 10μm . See also Fig EV2 .

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(G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.


( B , C ) Smaug downregulates reporter expression . Mean values of the relative levels of SNAP - GUS reporter ( SNAP - GUS / GFP - SNAP ratio , in arbitrary units ) in extracts of transfected Cl8 cells in the absence ( black ) or presence of λN - SNAP - Smaug ( red ) , λN - SNAP ( pale grey ) or SNAP - Smaug ( dark grey ) . See Fig EV3A for the representative gel and Fig EV3B which shows that λN - SNAP - Smaug has no effect on a SNAP - GUS construct that lacks the 5BoxB . N = 3 ( biological replicates ) . See also Fig EV3A - C . Here and in D - F : SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug . The error bars correspond to the standard deviation of the mean ( SD ) . Statistical analysis was done by a Kruskal - Wallis test by ranks followed by a Dunn test and p values are : black bracket < 0 . 05 , red bracket < 0 . 01 , green bracket < 0 . 0001 . Here and in D - E : 15 ng of pAct . λN - SNAP - smaug plasmid were used per transfection .

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(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection.


, E ) HH / SMO reduces Smaug levels and its overall negative effect on reporter expression . Mean values of the relative levels of reporter expression ( determined as above ) ( D ) or of λN - SNAP - Smaug ( ratio λN - SNAP - Smaug / GFP - SNAP ) ( E ) in the absence of SMO constructs ( red ) or in the presence of SMOWT - HA ( green ) , SMOPKA - SD - HA ( blue ) or SMO ∆ 958 - HA ( yellow ) , without HH ( plain boxes ) or in presence of HH ( striped boxes ) . N = 6 ( independent biological replicates ) , except for " no SMO , no HH " and " SMOWT with HH " conditions where n = 10 ( independent biological replicates ) . A representative gel is shown in Fig EV3D . Note that SMOWT - HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant . See also Fig EV3E - F .

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

, E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.


( F ) HH / SMO reduces Smaug ' s intrinsic repressive ability . A similar experiment was conducted with different amounts of λN - SNAP - smaug expressing plasmid ( ranging from 0 to 50 ng ) as indicated ) . The mean values of the relative levels of reporter ( y - axis ) are plotted against the relative levels of λN - SNAP - Smaug ( x - axis ) . SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug in absence of SMOPKA - SD / HH . The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with ( blue ) and without ( red ) SMOPKA - SD / HH , respectively . N = 3 ( biological triplicates ) . See Fig EV3H and also a representative gel in Fig EV3G .

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

(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.


( A ) HH / SMO promotes slow - migrating forms of Smaug . Western blot analysis of Cl8 cells that transiently express HA - SmaugWT alone , or together with either SMOWT - GFP or SMOPKA - SD - GFP , in the presence or in absence of HH . GMAP serves as a loading control . Here and in the other panels , the antibodies used are indicated on the left . Here and in ( B , C ) , the black arrows indicate the unphosphorylated form of HA - SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA - SmaugWT . NT : not transfected . See a phosphatase assay in Fig EV4A .

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

(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.


( B ) Smaug phosphorylation requires its interaction with SMO . Western blot analysis of Cl8 cells expressing HA - SmaugWT , in presence of HH , in combination with SMO ∆ 958 - GFP , SMOPKA - SD - GFP or SMOPKA - SD , ∆ 978 - GFP , as indicated .

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(B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.


( C ) FU controls Smaug phosphorylation . Extracts of Cl8 cells expressing SNAP - Smaug with SMOPKA - SD FU - SD - GFP ( with HH ) in combination with GFP - FUWT , GAP - GFP - FUWT , Myc - FUEE or GAP - null - FUDANA ( as indicated ) were labeled for the SNAP tag before electrophoresis . After quantification of the gels , the percentage ( % ) of phosphorylated Smaug ( % of phosphorylated forms of SNAP - Smaug total amounts of SNAP - Smaug ) was estimated . In C to E , the mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom , and the statistical analysis was done by one tailed bivariate Wilcoxon rank test . See also fig EV4C , D .

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

(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.


( D ) Phosphorylation of Smaug by activated FU requires the co - expression of SMO . Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN - SNAP - Smaug with GAP - GFP - FUWT and SMOWT alone or together ( as indicated ) N = 3 ( billogical triplicates ) . Note that the effect of GAP - GFP - FUWT and SMOWT together is lower than the one observed with GAP - GFP - FUWT and SMOPKA - SD FU - SD - GFP together ( in panel C ) . This could have multiple nonexclusive causes , including the fact that SMOWT is present at much lower levels than SMOPKA - SD FU - SD [ 38 ] and / or the involvement of another kinase . See also Fig EV4B . Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2 .

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

(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.


( E ) Forcing the interaction of Smaug with activated FU leads to a SMO - independent phosphorylation of Smaug . Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN - SNAP - Smaug with GFP - FU , FU - SBR , FUEE - SBR or FUDANA - SBR , with or without HH , as indicated . N = 3 ( biological triplicates ) . See also Fig EV4C and D . Note that the levels of Smaug phosphorylation observed in presence of FUDANA - SBR are not lower than those seen with GFP - FU . This suggests an incomplete inhibition of endogenous FU , which may be due to the trapping of FUDANA - SBR by Smaug . See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D .

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

(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.


( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The % of phosphorylated SNAP - Smaug protein ( determined as in Fig 3 ) ( A ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control .

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

(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.


( B ) Effect of FUEE - SBR on Smaug activity . The relative levels of reporter expression obtained in the same experiment as in ( A - C ) are represented as a function of the % of phosphorylated Smaug . Color of data points correspond to ng of transfected FUEE - SBR in A - C .

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

(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.


( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The relative levels of of λN - SNAP - Smaug protein ( C ) ( determined as in Fig 2 ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control .

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

(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.


( D ) Effect of FUEE - SBR on Smaug subcellular localization . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT ( 1 ) , FUEE - SBR - mCherry ( 2 ) , FUDANA - SBR - mCh ( 3 ) or expressing GFP - SmaugWT together with FUEE - SBR - mCherry ( 4 - 4 ' ' ) or GFP - SmaugWT together with FUDANA - SBR - mCh ( 5 - 5 ' ' ) . The merge images in 4 " and 5 " show GFP - SmaugWT in green and FUEE - SBR - mCh or FUDANA - SBR - mCh in red . ( 6 ) In presence of FUEE - SBR , 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci . In presence of FUDANA - SBR , 100 % of the cotransfected cells ( n = 241 ) show Smaug and FU distributed in bodies and colocalized . 250 ng of plasmid were used for each construct . Scale bar ( shown in D1 , identical for all panels ) :

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

(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ


( A ) Effect of FUEE - SBR and HH / SMO on endogeneous Smaug target mRNAs in Cl8 cells . Relative mRNA levels estimated using semi - quantitative RT - PCR for various Smaug ' s targets in Cl8 cells expressing FUEE - SBR ( black ) , FUEE ( dark grey ) , or FUDANA - SBR ( light grey ) were reported with respect to levels of a GFP control . mRNA levels were set to 1 for the GFP control . The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3 . See also Fig EV5E . Here and in Fig EV5D : RNA encoding mitochondrial proteins are written in blue . N = 3 ( biological triplicates ) and the quantifications were repeated 3 times on each sample . The results are presented as the means ± standard error . The differences between groups were assessed using Student ' s t - tests with Prism V8 . 2 . 1 software . A p value < 0 . 05 was considered to indicate a statistically significant

O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference


( B ) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc . Relative levels of Smaug ' s targets mRNAs in MS1096 ; UAS - hh or hhts and their respective controls ( as in Fig EV5X ) . N = 3 , with xx disc for each phenotype .

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(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.


( C ) Effect of smaug mutation on [ fu1 ] wing class distribution . Percent distribution of phenotypic classes ( defined in Appendix Figure S4 ) in fu1 males in presence of zero ( fu1 ) , one ( fu1 ; ; smaug47 / + ) or two copies ( fu1 ; ; smaug47 / smaug47 ) of the smaug47 allele . The wings were double - blindly classified in " weak " and " strong " according to the LV3 - V4 defects ( see Fig EV6B ) . * Chi2 statistical analysis gave a p of 0 , 015 for fu1 and fu1 ; ; smaug47 / + distributions and of 0 , 0024 when comparing fu1 ; ; smaug47 / + to fu1 ; ; smaug47 / smaug47 .

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

(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.


C ) MiNA analysis of HeLa cells stained with the membrane potential - independent dye MitoTracker ™ Green ( MTG , 100 nM ) to visualize and determine the mitochondrial area ( = mitochondrial footprint ) . Left : HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Co - localization shown in yellow . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . D ) Left : HeLa IF1 - KO cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . E ) Left : HeLa IF1 - H49K cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . Co - localization shown in yellow . Scale bars = 10 µm . F )

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C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)