( E ) Smyhc1 immunohistochemistry stain of 24 hpf ( hour post fertilization ) zebrafish larvae . Filamentous actin is stained with phalloidin ( red ) . Nuclei are stained with DAPI ( blue ) . Smyhc1 is stained with the F59 antibody ( green ) ( Elworthy et al , 2008 ) . smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae display Smyhc1 in the muscle fibers at 24hpf . smyhc1 - / - larvae at 24 do not stain positively for Smyhc1 . Scale bar represents 50 µm .

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(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.


( A ) Morphologies of smyhc1 + / + , smyhc1 - / + and smyhc1 - / - embryos are grossly normal at 2 days post fertilization ( dpf ) , despite complete paralysis of smyhc1 - / - embryos . In contrast , the smyhc1R673H / + embryos have notochord kinks in larval stages ( arrow head ) , while smyhc1R673H / R673H embryos have much more severe notochord kinks that compress and distort the body axis ( arrow heads ) . The smyhc1R673H / - embryos completely phenocopy smyhc1R673H / R673H embryos . ( B ) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf .

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(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos. (B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf.


( B ) The predicted mendelian ( 1 : 2 : 1 ) and experimental ratio of genotypes of progeny from smyhc1R673H / + in - crosses demonstrates survival at the indicated ages . This ratio quickly skewed toward an overrepresentation of smyhc1 + / + fish with no representation of smyhc1R673H / R673H after 5 dpf . There is also dropout of smyhc1 + / R673H fish into adulthood . Number of fish counted per group is indicated .

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(B) The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1R673H/+ in-crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1+/+ fish with no representation of smyhc1R673H/R673H after 5 dpf. There is also dropout of smyhc1+/R673H fish into adulthood. Number of fish counted per group is indicated.


( A ) Gross morphology of smyhc1 mutant adults . Most smyhc1 - / - adult fish display dorsal tail curvature , while smyhc1R673H / + adults have a shortened body axis and variable spinal curves .

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(A) Gross morphology of smyhc1 mutant adults. Most smyhc1-/- adult fish display dorsal tail curvature, while smyhc1R673H/+ adults have a shortened body axis and variable spinal curves.


( B ) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1 - / - adults , in contrast to the compression and fusion of vertebrae seen in smyhc1R673H / + adults . Distal tail regions are highlighted and enlarged .

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(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.


( C ) Quantification of skeletal phenotype severity in adults . Scale bars represent a length of 5 mm .

(C) Quantification of skeletal phenotype severity in adults. Scale bars represent a length of 5 mm.


( A ) Time course of light - triggered larval movements were manually counted at indicated times between 24 and 48 hpf . The smyhc1 - / - larvae ( n = 36 ) were paralyzed up to 48 hpf . smyhc1R673H / + mutants ( n = 15 ) twitched slightly less than their smyhc1 + / + siblings ( n = 21 ) at 34hpf . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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(A) Time course of light-triggered larval movements were manually counted at indicated times between 24 and 48 hpf. The smyhc1-/- larvae (n = 36) were paralyzed up to 48 hpf. smyhc1R673H/+ mutants (n = 15) twitched slightly less than their smyhc1+/+ siblings (n = 21) at 34hpf. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( B ) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software ( Noldus Ethovision ) . At 6 dpf , both smyhc1 - / - ( n = 24 ) and smyhc1R673H / + ( n = 11 ) larvae travelled significantly less than smyhc1 + / + larvae ( n = 13 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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(B) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1-/- (n = 24) and smyhc1R673H/+ (n = 11) larvae travelled significantly less than smyhc1+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( C ) Diagram of swim tunnel used to quantify adult swimming behavior . Water flow was gradually increased until the fish fatigued and were collected at the grate .

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(C) Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.


( D ) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization ( mpf ) . smyhc1 + / + ( n = 6 ) , smyhc1 - / - ( n = 5 ) , smyhc1R673H / + ( n = 5 ) .

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(D) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization (mpf). smyhc1+/+ (n = 6), smyhc1-/- (n = 5), smyhc1R673H/+ (n = 5).


( A ) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae , stained with phalloidin - rhodamine to detect actin . Puncta of filamentous actin are indicated with white arrowheads , misshapen myofibers are indicated with white arrows and outlined . ( B ) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae , stained with phalloidin - rhodamine . Puncta of filamentous actin are indicated with white arrowheads , distorted myofibers are indicated with arrows and outlined , and frayed myofibers are indicated with yellow arrowhead .

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(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead.


( C ) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . ( D ) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . Bundle of actin ringed with α - actinin indicated with white - bordered black arrowhead .

(C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead.


( E ) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 54 ) , smyhc1 - / - ( n = 31 ) , smyhc1R673H / + ( n = 45 ) , and smyhc1R673H / R673H ( n = 49 ) . Distance between myosepta was measured perpendicular to rostral - caudal body axis at defined mid - body regions in phalloidin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( F ) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 51 ) , smyhc1 - / - ( n = 32 ) , smyhc1R673H / + ( n = 64 ) , and smyhc1R673H / R673H ( n = 38 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 54), smyhc1-/- (n = 31), smyhc1R673H/+ (n = 45), and smyhc1R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral-caudal body axis at defined mid-body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (F) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 51), smyhc1-/- (n = 32), smyhc1R673H/+ (n = 64), and smyhc1R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( G ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 380 ) , smyhc1R673H / + ( n = 293 ) , and smyhc1R673H / R673H ( n = 268 ) . Distance between z - discs was measured in anti - α - actinin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( H ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 249 ) , smyhc1 - / - ( n = 278 ) , smyhc1R673H / + ( n = 246 ) , and smyhc1R673H / R673H ( n = 252 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Z-disc intervals (sarcomere length) of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 380), smyhc1R673H/+ (n = 293), and smyhc1R673H/R673H (n = 268). Distance between z-discs was measured in anti-α-actinin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (H) Z-disc intervals (sarcomere length) of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 249), smyhc1-/- (n = 278), smyhc1R673H/+ (n = 246), and smyhc1R673H/R673H (n = 252). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( A ) Representative morphologies of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 2 dpf after 24 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . All para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis . ( B ) Representative images of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 3dpf after 48 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . Para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis .

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

(A) Representative morphologies of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 2 dpf after 24 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. All para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis. (B) Representative images of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 3dpf after 48 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis.


ATPase staining of GAS and SOL muscles of WT and DKO mice ( left ) . Corresponding percentage of type I fibers were determined ( right ) ( n = 5 ) . Scale bars : 100 μm .

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ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.


Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice ( n = 3 for PGC1α analysis ; n = 5 for PLIN5 analysis ) .

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Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis).


Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR - 183 and miR - 96 agomirs ( Ago - 183 / 96 ) or antagomirs ( Ant - 183 / 96 ) ( n = 3 ) .

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Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).


Mitochondrial respiration profile of C2C12 cells transfected with Ago - 183 / 96 ( J ) or Ant - 183 / 96 ( K ) . Cellular oxygen consumption rate ( OCR ) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR ( n = 3 or 4 per group ) .

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

Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).


Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in GAS muscles of WT and DKO mice . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in GAS muscles of WT and DKO mice. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).


Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice . Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice. Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).


LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR - 183 / 96 ( N ) or antagomir - 183 / 96 ( P ) stained with Nile red ( n = 5 ) . Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR - 183 / 96 ( O ) or antagomir - 183 / 96 ( Q ) . Scale bars : 5 μm .

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LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.


Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 agomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 . Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 antagomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 .

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Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.


Relative mRNA expression ( I , left ) and western blot analysis ( J , top ) of ATGL in C2C12 cells transfected with agomir for miR - 183 ( Ago - 183 ) or miR - 96 ( Ago - 96 ) . Relative mRNA expression ( I , right ) and western blot analysis ( J , bottom ) of ATGL in C2C12 cells transfected with antagomir for miR - 183 ( Ant - 183 ) or miR - 96 ( Ant - 96 ) . ( n = 5 per group for RT - PCR analysis )

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Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis)


Relative mRNA expression of ATGL , PGC1α , PLIN5 , and HSL in C2C12 cells transfected with ATGL ( n = 5 ) .

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Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).


Relative mRNA expression of PGC1α , PLIN5 , and HSL in C2C12 cells transfected with Ant - 183 / 96 and siRNA of PPARδ ( siPPARδ ) .

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Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).


Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice ( n = 3 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice .

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Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice (n=3). Western blot analysis of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice.


Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months ( n = 5 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months .

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Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5). Western blot analysis of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months.


Concentration of serum triglyceride ( TAG ) ( left ) and non - esterified fatty acid ( NEFA ) ( right ) in WT and DKO mice after HFD feeding for 2 months ( n = 5 ) .

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Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).


Western blot analysis of FoxO1 , PDK4 , p - PDHA1 , PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months .

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Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months.


Glucose tolerance test ( GTT ) and area under the curve ( AUC ) data for GTT in WT and DKO mice after HFD feeding ( n = 10 - 11 ) .

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Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).


B Growth curves of the indicated strains shown as population doublings ( pds ) on the y axis and hours in liquid culture on the x axis . Before inoculation in liquid YES medium , single cells underwent approximately 25 pds to produce visible colonies on solid medium . For each strain , averages and SDs ( error bars ) of three biological replicates are shown . Arrows indicate times of harvesting and corresponding pds for experiments shown in ( C ) and ( D ) .

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B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).


C Telo - PCR analysis of telomere length in wt ( ter1 + and est1 + ) and two independent clones ( a and b ) deleted for ter1 + or est1 + grown for ~ 25 + 10 ( ~ 35 ) , ~ 25 + 14 ( ~ 39 ) and ~ 25 + 18 ( ~ 43 ) pds in YES medium . PCRs were performed with the subtelomeric oligonucleotide oF5 , located 620 bp upstream of the first telomeric repeat , and the oligo ( dG ) - containing oligonucleotide odG18 . Marker ( m ) molecular weights are on the left in kilobases . tels : telomere - containing Telo - PCR products .

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C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.


D qRT - PCR analysis of G - rich TERRA , polyA + TERRA and ARRET / αARRET levels in cells as in ( B ) . Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains ( ter1 + and est1 + ) grown in parallel . Bars and error bars are averages and SD from 4 ( ter1Δ clones ) or 3 ( est1Δ clones ) independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to wt ; two - tailed Student ' s t test ) .

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D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).


E ChIP experiments with anti - RNA polymerase II ( RPII ) antibodies and extracts from ter1 + and ter1Δ cells grown for ~ 43 pds . Quantitative PCRs were performed using oligonucleotides oF1 + oR1 ( subtel ) or oligonucleotides amplifying a region from the actively transcribed act1 + locus . Values are expressed as fold increase over ter1 + . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to ter1 + ; two - tailed Student ' s t test ) . The western blot to the right shows no difference in RNPII levels between ter1 + and ter1Δ cells used in ChIP experiments . Ponceau Sstaining serves as loading control .

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E ChIP experiments with anti-RNA polymerase II (RPII) antibodies and extracts from ter1+ and ter1Δ cells grown for ~ 43 pds. Quantitative PCRs were performed using oligonucleotides oF1 + oR1 (subtel) or oligonucleotides amplifying a region from the actively transcribed act1+ locus. Values are expressed as fold increase over ter1+. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to ter1+; two-tailed Student's t test). The western blot to the right shows no difference in RNPII levels between ter1+ and ter1Δ cells used in ChIP experiments. Ponceau Sstaining serves as loading control.


G Western blot analysis of cellular fractions from ter1 + and ter1Δ cells grown for ~ 43 pds . Total proteins ( tot ) and equivalents of soluble ( sol ) and insoluble ( ins ) proteins were loaded . Actin serves as a control for soluble proteins , H3K9ac for chromatin - bound proteins .

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G Western blot analysis of cellular fractions from ter1+ and ter1Δ cells grown for ~ 43 pds. Total proteins (tot) and equivalents of soluble (sol) and insoluble (ins) proteins were loaded. Actin serves as a control for soluble proteins, H3K9ac for chromatin-bound proteins.


H qRT - PCR analysis of total TERRA and polyA + TERRA in cellular fractions as in ( G ) . Values are expressed after normalization to total cellular extracts . Bars and error bars are averages and SD from 3 independent experiments .

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H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.


A RIP experiments were performed using anti - myc antibodies and extracts from Trt1 - myc expressing strains followed by qRT - PCR to detect G - rich TERRA , polyA + TERRA , ARRET , TER1 and ACT1 . Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged ( unt ) wt strain . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - tailed Student ' s t test ) .

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A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


B RIP experiments as in ( A ) where , prior to RNA extraction , washed beads were resuspended in DNaseI buffer ( DNbuff ) and incubated for 1 hour at 30 ° C in presence or absence of DNaseI . Values represent fractions of input polyA + TERRA detected in immunoprecipitated material expressed as fold increase over an untagged ( unt ) wt strain . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - tailed Student ' s t test ) .

O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O

B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


C RIP experiments were performed using anti - myc antibodies and extracts from Trt1 - myc expressing strains ( either ter1 + or ter1Δ ) cultured in YES medium for ~ 43 pds . Values represent polyA + TERRA detected in immunoprecipitated material expressed as a fraction of the input ( graph on the left ) or after normalization to ACT1 RNA in the corresponding input ( on the right ) , and expressed as fold increase over an untagged wt strain ( unt ter1 + ) . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to unt ter1 + ; two - tailed Student ' s t test ) .

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C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).


D Western blot analysis of Trt1 - myc in cells used in ( C ) . Ponceau S staining serves as a loading control .

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D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.


B Genomic DNA from wt cells ( CAF13 ) and cells carrying two tiTELs ( CAF110 ) maintained in YES medium was digested with HindIII and Southern blot - hybridized consecutively with the indicated probes . RET refers to telomeric sequences ( tiTELs or natural telomeres , nTELs ) retained in the upper part of the gel ; REL refers to telomeric sequences released in the lower part of the gel . The black arrow points to the natural nmt1 + sequence on chromosome III . Marker molecular weights are on the left in kilobases .

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B Genomic DNA from wt cells (CAF13) and cells carrying two tiTELs (CAF110) maintained in YES medium was digested with HindIII and Southern blot-hybridized consecutively with the indicated probes. RET refers to telomeric sequences (tiTELs or natural telomeres, nTELs) retained in the upper part of the gel; REL refers to telomeric sequences released in the lower part of the gel. The black arrow points to the natural nmt1+ sequence on chromosome III. Marker molecular weights are on the left in kilobases.


D qRT - PCR analysis of tiTERRA in cells carrying no tiTEL ( CAF13 ) , one tiTEL ( CAF545 ) or two tiTELs ( CAF110 ) cultured in EMM medium in presence or absence of Thiamine ( THI ; repressed and induced condition , respectively ) for 24 h . RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA ( graph on the left ) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA ( on the right ) . Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions ( THI + ) . Bars and error bars are averages and SD from 3 independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to CAF545 THI + ; two - tailed Student ' s t test ) .

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D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student's t test).


E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs ( CAF110 ) treated with combinations of THI and Trichostatin A ( TSA ) for 24 h . Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C - rich ( oC ) or G - rich ( oG ) telomeric repeats . To confirm similar hybridization efficiencies of the two oligonucleotides , northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA ( upper right insets ) . After signal detection , RNAmembranes were stripped and re - hybridized with U6 probes to assure equal loading . Marker molecular weights are on the left in kilobases .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-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 O O O 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 Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.


A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs ( CAF110 ) grown for 24 h in EMM medium containing THI and TSA as indicated . DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe . Marker molecular weights are on the left in kilobases . Numbers at the bottom indicate gel lanes .

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A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


B Genomic DNA from cells carrying two tiTELs and deleted for trt1 + ( CAF113 ) was analyzed as in ( A ) . Marker molecular weights are on the left in kilobases . Numbers at the bottom indicate gel lanes .

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B Genomic DNA from cells carrying two tiTELs and deleted for trt1+ (CAF113) was analyzed as in (A). Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


C ter1 + cells carrying two tiTELs ( CAF110 , left panel ) or trt1Δ cells carrying one tiTEL ( MKSP2104 , central panel ) were cultured in EMM medium in presence of THI for 24 h ( THI + ) or in absence of THI for 24 , 48 or 72 h . In the right panel , cells carrying two tiTELs ( CAF110 ) were treated with TSA for 24 h in the presence or absence of THI . TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform . The statistical analysis for each set of conditions is displayed above each panel . P values for each comparison were derived from the Student ' s t test between each condition and the THI + sample using Welch ' s correction when appropriate . The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red ( center panel ) .

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C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student's t test between each condition and the THI+ sample using Welch's correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).


A qRT - PCR analysis of G - rich and polyA + tiTERRA in cells carrying one tiTEL and expressing Trt1 - myc ( CAF610 ) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h . Values are normalized to ACT1 mRNA and expressed as fold increase over THI + TSA - samples . Bars and error bars are averages and SD from 4 independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ( relative to THI + TSA - ; two - tailed Student ' s t test ) .

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A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).


B Telo - PCR analysis of tiTELs using genomic DNA extracted from cells as in ( A ) . Marker ( m ) molecular weights are on the right in base pairs .

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B Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs.


C RIP experiments performed using anti - myc antibodies and extracts from cells as in ( A ) followed by qRT - PCR analysis of G - rich and polyA + tiTERRA . Values correspond to fraction of input RNA expressed as fold increase over an untagged ( unt ) control tiTEL strain ( CAF545 ) . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to unt ; two - tailed Student ' s t test ) .

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C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).


D ChIP experiments performed using anti - myc antibodies and extracts from cells as in ( A ) followed by qPCR using tiTEL specific oligonucleotides . Values correspond to fraction of input DNA expressed as fold increase over THI + TSA - . Unt : untagged strain ( CAF545 ) . Bars and error bars are averages and SD from at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to THI + TSA - ; two - tailed Student ' s t test ) .

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D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).


mRNA and protein expression of CiTLR5a - HA ( D ) , CiTLR5b - HA ( D ) , CiTLR5a - GFP ( E ) , and CiTLR5b - GFP ( E ) plasmids in FHM cells were examined by qRT - PCR ( upper panel ) with EF1α gene as an internal control and by immunoblotting ( IB ) ( lower panel ) with β - tubulin as a reference ( n = 4 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( D ) and ( E ) . n represents biological replicates . * * * P < 0 . 001 .

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mRNA and protein expression of CiTLR5a-HA (D), CiTLR5b-HA (D), CiTLR5a-GFP (E), and CiTLR5b-GFP (E) plasmids in FHM cells were examined by qRT-PCR (upper panel) with EF1α gene as an internal control and by immunoblotting (IB) (lower panel) with β-tubulin as a reference (n = 4). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D) and (E). n represents biological replicates. ***P < 0.001.


FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h . Co - IP was performed with anti - HA monoclonal antibody . Mouse IgG was used as control . Immunoblotting was performed with anti - HA and anti - GFP antibodies , respectively .

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

FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h. Co-IP was performed with anti-HA monoclonal antibody. Mouse IgG was used as control. Immunoblotting was performed with anti-HA and anti-GFP antibodies, respectively.


Lysates of FHM cells overexpressing CiTLR5a were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies .

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

Lysates of FHM cells overexpressing CiTLR5a were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.


Lysates of FHM cells overexpressing CiTLR5b were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies .

Lysates of FHM cells overexpressing CiTLR5b were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.


Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin - poly ( I : C ) , biotin - ssRNA ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA antibodies .

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

Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin-poly(I:C), biotin-ssRNA (1 μg/mL) at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA antibodies.


N , O CiUNC93B1 - Myc and CiTLR5a - HA ( M ) or CiTLR5b - HA ( N ) were transfected into FHM cells . Co - IP and immunoblotting were conducted to test the interactions between CiUNC93B1 - Myc and CiTLR5a - HA or CiTLR5b - HA .

N, O CiUNC93B1-Myc and CiTLR5a-HA (M) or CiTLR5b-HA (N) were transfected into FHM cells. Co-IP and immunoblotting were conducted to test the interactions between CiUNC93B1-Myc and CiTLR5a-HA or CiTLR5b-HA.


O , P Protein expression of IFN1 ( O ) and NF - κB1 ( P ) in spleen tissue were investigated by immunoblotting ( IB ) after RNAi in vivo for 24 h , and A . hydrophila or GCRV challenge for 24 h ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

O, P Protein expression of IFN1 (O) and NF-κB1 (P) in spleen tissue were investigated by immunoblotting (IB) after RNAi in vivo for 24 h, and A. hydrophila or GCRV challenge for 24 h (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull - down assays and analyzed by immunoblotting ( IB ) . Vector and wild type ( WT ) were used as controls . Relative band intensity ratios were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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

Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull-down assays and analyzed by immunoblotting (IB). Vector and wild type (WT) were used as controls. Relative band intensity ratios were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for n represents biological replicates. *P < 0.05, **P < 0.01.


Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b ( including region deletants ) were incubated with biotin - poly ( I : C ) . Vector was used as a control . Pull - down and immunoblotting ( IB ) were performed .

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

Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b (including region deletants) were incubated with biotin-poly(I:C). Vector was used as a control. Pull-down and immunoblotting (IB) were performed.


H - K Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b site mutants were incubated with biotin - poly ( I : C ) . Vector and wild type were used as controls . Pull - down and immunoblotting were carried out ( H , J ) . ( I , K ) Relative band intensity ratios of ( H ) and ( J ) were analyzed by ImageJ , respectively ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( I ) , and ( K ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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

H-K Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b site mutants were incubated with biotin-poly(I:C). Vector and wild type were used as controls. Pull-down and immunoblotting were carried out (H, J). (I, K) Relative band intensity ratios of (H) and (J) were analyzed by ImageJ, respectively (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (I), and (K). n represents biological replicates. *P < 0.05, **P < 0.01.


Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting ( IB ) with anti - HA antibody .

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

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting (IB) with anti-HA antibody.


Mutants with the predicted N - glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight .

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

Mutants with the predicted N-glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight.


Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin - poly ( I : C ) ( I ) Vector and wild type were used as controls . Pull - down and immunoblotting assays were conducted ( I , ( J , Relative band intensity ratios of ( I ) were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( J ) , n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin-poly(I:C) (I) Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted (I, (J, Relative band intensity ratios of (I) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (J), n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC ( K ) . Vector and wild type were used as controls . Pull - down and immunoblotting assays were conducted K ) . L ) Relative band intensity ratios of ( K ) were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( L ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC (K). Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted K). L) Relative band intensity ratios of (K) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (L). n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Lysates of FHM cells overexpressing LoTLR5 ( A ) , DrTLR5a ( B ) , DrTLR5b ( B ) , IpTLR5 - 1 ( C ) , IpTLR5 - 2 ( C ) , IpTLR5S ( C ) , OmTLR5M ( D ) , OmTLR5S ( D ) , were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads and analyzed by immunoblotting ( IB ) with anti - His antibody .

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

Lysates of FHM cells overexpressing LoTLR5 (A), DrTLR5a (B), DrTLR5b (B), IpTLR5-1 (C), IpTLR5-2 (C), IpTLR5S (C), OmTLR5M (D), OmTLR5S (D), were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.


Lysates of FHM cells overexpressing HsTLR5 ( E ) were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads and analyzed by immunoblotting ( IB ) with anti - His antibody .

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

Lysates of FHM cells overexpressing HsTLR5 (E) were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.


Lysates of FHM cells overexpressing LoTLR5 ( F ) , DrTLR5a ( G ) , DrTLR5b ( G ) , IpTLR5 - 1 ( H ) , IpTLR5 - 2 ( H ) , IpTLR5S ( H ) , were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti - GFP antibody .

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

Lysates of FHM cells overexpressing LoTLR5 (F), DrTLR5a (G), DrTLR5b (G), IpTLR5-1 (H), IpTLR5-2 (H), IpTLR5S (H), were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.


Lysates of FHM cells overexpressing OmTLR5M ( I ) , OmTLR5S ( I ) , and HsTLR5 ( J ) were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti - GFP antibody .

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

Lysates of FHM cells overexpressing OmTLR5M (I), OmTLR5S (I), and HsTLR5 (J) were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.


Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin - poly ( I : C ) and analyzed by immunoblotting ( N ) , and relative band intensity ratios were analyzed by ImageJ ( O ) ( n = 3 ) . Vector and wild type ( WT ) were used as controls . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( O ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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

Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin-poly(I:C) and analyzed by immunoblotting (N), and relative band intensity ratios were analyzed by ImageJ (O) (n = 3). Vector and wild type (WT) were used as controls. Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (O). n represents biological replicates. *P < 0.05, **P < 0.01.


Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type ( WT ) or Nlrc3 - / - mice and treated with LPS ( 100 ng / ml ) for 48 hours .

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

Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type (WT) or Nlrc3-/- mice and treated with LPS (100 ng/ml) for 48 hours.


ELISA of cytokines in culture supernatants of DCs treated


The intracellular production of IFN - γ and IL - 17 by CD4 + T cells among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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

The intracellular production of IFN-γ and IL-17 by CD4+ T cells among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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

cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


thymidine incorporation proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

thymidine incorporation proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


CFSE proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

CFSE proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


WT and Nlrc3 - / - mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized WT ( n = 10 ) and Nlrc3 - / - mice ( n = 10 ) .

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

WT and Nlrc3-/- mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized WT (n= 10) and Nlrc3-/- mice (n= 10).


Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells from the spinal cord and brain of WT or Nlrc3 - / - mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel .

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

Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells from the spinal cord and brain of WT or Nlrc3-/- mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.


Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3 - / - mice 26 d after EAE induction .

Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3-/- mice 26 d after EAE induction.


Expression of Nlrc3 , Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3 - / - mice 26 d after EAE induction , presented relative to that of Gapdh .

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

Expression of Nlrc3, Il12, Il6, Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3-/- mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Recall response to MOG ( 35 - 55 ) by splenocytes isolated from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . CFSE proliferation assay of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. CFSE proliferation assay of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . cytokine secretion of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. cytokine secretion of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for phosphorylated p65 ( p - p65 ) , total p65 , p - AKT , AKT , p - p38 , p38 , p - ERK , ERK , p - JNK , JNK and GAPDH .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Concentrations of IL - 12 , IL - 6 , IL - 23 and IL - 27 in supernatants were detected by ELISA .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


Activity phosphorylation of p38 were detected in DCs in the spleens from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . Pooled data are presented in the right panel .

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

Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel.


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Frequencies of CD4 + T cells that express IFN - γ and IL - 17A in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 and Il23 mRNA in DCs sorted from spleens of DC ( WT ) , DC ( NLRC3 - KO ) , DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for p - p38 , p38 , NLRC3 and GAPDH . Densitometry quantification of band intensity were presented in the right panel .

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

DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Enzyme - linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours .

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

DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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

DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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

DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Mean clinical scores of EAE ( n = 5 mice per group ) . Arrows indicate DC vaccine administration .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Recall proliferative to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . cytokine response ( D ) to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


C Representative plot showing gating strategy for CD4 + T cell phenotypes . Effector Memory RA ( TEMRA ) : CD45RA + CD62L - ; Effector Memory ( EM ) : CD45RA - CD62L - ; Central Memory ( CM ) : CD45RA - CD62L + ; Stem Cell Memory ( TSCM ) : CD45RA + CD62L + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative plot showing gating strategy for CD4+ T cell phenotypes. Effector Memory RA (TEMRA): CD45RA+ CD62L-; Effector Memory (EM): CD45RA-CD62L-; Central Memory (CM): CD45RA-CD62L+; Stem Cell Memory (TSCM): CD45RA+ CD62L+.


D Percentage of GFP + cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor ( HD ; n = 11 ) or patient ( Pt ; n = 1 ) derived CD4 + T cells , measured by FACS analysis . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of GFP+ cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor (HD; n=11) or patient (Pt; n=1) derived CD4+ T cells, measured by FACS analysis. Median ± IQR.


E Percentage of GFP + cells or HDR in T cells from ( D ) . Median ± IQR .

E Percentage of GFP+ cells or HDR in T cells from (D). Median ± IQR.


F Population composition in male HD or patient derived untreated ( n = 9 HD UT , 1 Pt UT ) or bulk edited T cells ( Treat ) from ( D ) . For each subpopulation , the comparisons between HD Treated vs UT were performed by using LME models , with random effects defined to account for the same donor and for different number of replicates per donor within group . All p - values were adjusted with Bonferroni ' s correction to account for multiple testing . In all LME models , the percentages were used in square root scale to meet the assumption of normality of the residuals of the model . Mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Population composition in male HD or patient derived untreated (n=9 HD UT, 1 Pt UT) or bulk edited T cells (Treat) from (D). For each subpopulation, the comparisons between HD Treated vs UT were performed by using LME models, with random effects defined to account for the same donor and for different number of replicates per donor within group. All p-values were adjusted with Bonferroni's correction to account for multiple testing. In all LME models, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. Mean ± SEM.


G Percentage of TCRBV families detected by spectratyping . UT , sorted edited ( GFP + ) , sorted non - edited ( GFP - ) CD4 + T cells derived from male HD ( n = 1 ) or patient ( Pt ; n = 1 ) were analyzed at 17 days after CD40LG editing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of TCRBV families detected by spectratyping. UT, sorted edited (GFP+), sorted non-edited (GFP-) CD4+ T cells derived from male HD (n=1) or patient (Pt; n=1) were analyzed at 17 days after CD40LG editing.


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by Relative Fluorescence Intensity ( RFI , normalized to T0 ; left ) and percentage ( right ) on UT ( n = 3 ) , edited ( GFP + ) or unedited ( GFP - ) HD or Pt CD4 + T cells from ( D ) ( n = 9 HD , 1 Pt ) . Longitudinal comparisons between HD GFP + vs GFP - were performed with an LME model , accounting for multiple donors and separately for RFI and % CD40L + cells ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to 8 hours time - point ( * * * * p < 0 . 0001 and * p = 0 . 0450 , respectively ) . # measured on the small fraction of CD40L + cells . Median ± IQR .

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

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by Relative Fluorescence Intensity (RFI, normalized to T0; left) and percentage (right) on UT (n=3), edited (GFP+) or unedited (GFP-) HD or Pt CD4+ T cells from (D) (n=9 HD, 1 Pt). Longitudinal comparisons between HD GFP+ vs GFP- were performed with an LME model, accounting for multiple donors and separately for RFI and %CD40L+ cells (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to 8 hours time-point (****p<0.0001 and *p=0.0450, respectively). #measured on the small fraction of CD40L+ cells. Median ± IQR.


I Representative plots showing CD40L and GFP expression in UT or bulk edited ( Tretated ) CD4 + T cells derived from male HD or Pt from ( D ) at 8 hr after PMA / Ionomycin stimulation .

I Representative plots showing CD40L and GFP expression in UT or bulk edited (Tretated) CD4+ T cells derived from male HD or Pt from (D) at 8 hr after PMA/Ionomycin stimulation.


K Left : IgG positive spots resulting from B - T cell co - culture . Right : IgG + secreting B cells , evaluated by ELISPOT assay . B cells were isolated from PB of HD and co - cultured with male HD or Pt sorted GFP + , GFP - and UT T cells , resting ( R ) or stimulated with beads ( B ) or PMA / Ionomycin ( PI ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 1 for each group ) .

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

K Left: IgG positive spots resulting from B-T cell co-culture. Right: IgG+ secreting B cells, evaluated by ELISPOT assay. B cells were isolated from PB of HD and co-cultured with male HD or Pt sorted GFP+, GFP- and UT T cells, resting (R) or stimulated with beads (B) or PMA/Ionomycin (PI). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=1 for each group).


L Left : Histograms representing proliferation results from B - T cell co - culture . Right : Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co - cultured with HD or Pt T cells from ( K ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 2 for each group ) . Mean ± SEM .

L Left: Histograms representing proliferation results from B-T cell co-culture. Right: Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co-cultured with HD or Pt T cells from (K). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=2 for each group). Mean ± SEM.


B Percentage of NGFR + cells or HDR at 17 days after CD40LG editing of male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells ( n = 7 for each group ) . Median ± IQR .

B Percentage of NGFR+ cells or HDR at 17 days after CD40LG editing of male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells (n=7 for each group). Median ± IQR.


C Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by RFI ( normalized to T0 ) on UT ( n = 2 ) , edited ( NGFR + ) or unedited ( NGFR - ) HD or Pt derived CD4 + T cells ( n = 4 HD , 1 Pt ) . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by RFI (normalized to T0) on UT (n=2), edited (NGFR+) or unedited (NGFR-) HD or Pt derived CD4+ T cells (n=4 HD, 1 Pt). Median ± IQR.


D Representative plots showing CD40L and NGFR expression in UT or bulk edited ( Treated ) CD4 + T cells derived from male HD or Pt from ( C ) before and 8 hr after Pma / Ionomycin stimulation .

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

D Representative plots showing CD40L and NGFR expression in UT or bulk edited (Treated) CD4+ T cells derived from male HD or Pt from (C) before and 8 hr after Pma/Ionomycin stimulation.


E Population composition in male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells from ( B ) ( n = 7 for each group ) . Friedman test ( to account for the same donor ) with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Population composition in male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells from (B) (n=7 for each group). Friedman test (to account for the same donor) with p-values adjusted with Bonferroni's correction to account for multiple testing. Mean ± SEM.


F Human CD45 + cell engraftment in PB after transplantation of male HD UT ( n = 5 ) , bulk edited ( NGFR bulk ; n = 5 ) or sorted NGFR + ( n = 5 ) CD4 + T cells . Longitudinal comparisons were performed with an LME model , followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Human CD45+ cell engraftment in PB after transplantation of male HD UT (n=5), bulk edited (NGFR bulk; n=5) or sorted NGFR+ (n=5) CD4+ T cells. Longitudinal comparisons were performed with an LME model, followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups. Median ± IQR.


G Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( F ) ( n = 5 for each group , measured in engrafted mice with sufficient blood material ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable ( see Appendix Supplementary Statistical Methods ) . UT group was not included in the analysis . Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection , highlighting a constant ( and significantly different , * * * * p < 0 . 0001 ) behavior of the groups over time . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (F) (n=5 for each group, measured in engrafted mice with sufficient blood material). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable (see Appendix Supplementary Statistical Methods). UT group was not included in the analysis. Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection, highlighting a constant (and significantly different, ****p<0.0001) behavior of the groups over time. Median ± IQR.


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleens of mice from ( F ) ( n = 1 for each group , except for NGFR - unedited group , n = 3 ) . Median ± IQR .

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

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleens of mice from (F) (n=1 for each group, except for NGFR- unedited group, n=3). Median ± IQR.


I Dot plot depicting the ratio between expression of CD40LG edited mRNA ( codon - usage optimized , CO ) and CD40LG wild - type mRNA ( WT ) in cells edited with three different donor templates from Fig EV2H . Three independent experiments ( n = 3 EF1a . GFP , 1 IRES . NGFR , 3 IRES . GFP ) . Median ± IQR .

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

I Dot plot depicting the ratio between expression of CD40LG edited mRNA (codon-usage optimized, CO) and CD40LG wild-type mRNA (WT) in cells edited with three different donor templates from Fig EV2H. Three independent experiments (n=3 EF1a.GFP, 1 IRES.NGFR, 3 IRES.GFP). Median ± IQR.


J Time course of expression of WT - CD40LG mRNA or CO - CD40LG mRNA , measured as fold change ( FC ) on IPO8 housekeeping gene and normalized to T0 . Sorted edited ( + ) and sorted unedited ( - ) CD4 + T cells were analyzed before and 3 , 6 , 12 hr after Actinomycin D treatment ( n = 1 for each group ) .

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

J Time course of expression of WT-CD40LG mRNA or CO-CD40LG mRNA, measured as fold change (FC) on IPO8 housekeeping gene and normalized to T0. Sorted edited (+) and sorted unedited (-) CD4+ T cells were analyzed before and 3, 6, 12 hr after Actinomycin D treatment (n=1 for each group).