( c ) HCT116 cells ( 107 ) or stable FoxO1 - knockdown HCT116 cells ( 107 ) were injected into the left or right hind legs of three nude mice . Six weeks later the mice were killed and the tumours were weighed and analysed ( bottom graph ) . Tumour weights were 0 . 08 ± 0 . 10 g ( without FoxO1 RNAi ) and 0 . 48 ± 0 . 06 g ( with FoxO1 RNAi ) , P = 0 . 020 . Data are means ± sd . n = 3 .

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(c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3.


( d ) Protein was extracted from one paired colon tumour ( T ) or adjacent normal tissue ( N ) from one patient , and western blotting was performed to detect changes in FoxO1 or p62 .

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(d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62.


( e ) Proteins were extracted from 16 paired samples from patients for western blotting with anti - FoxO1 or with anti - p62 antibody . Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β - actin band . FoxO1 of normal tissue , 0 . 37 ; FoxO1 of tumour tissue , 0 . 10 ; P = 0 . 001 , two - sided t - test . p62 of normal tissue , 0 . 32 ; p62 of tumour tissue , 1 . 00 ; P = 0 . 009 , two - sided t - test . Data in e are means ± s . d . ( n = 16 ) .

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(e) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).


Representative dual - channel kymographs showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Time - lapse images ( B ) showing colocalization and comigration ( white arrows ) of Rheb with mitochondria in a retrograde direction along axons during a 148 - s recording period . Cortical neurons were transfected with GFP - Rheb and DsRed - Mito at 5 - 7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10 - 12 DIV .

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Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.


quantitative analysis showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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 B-MEASURED_VAR 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 O O O O O O

quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . The percentages of mitochondria targeted by Rheb or Rheb - SSVM in the presence or absence of CCCP were quantified ,

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Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively


representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . Rheb - SSVM , a farnesylation deficient mutant of Rheb , fails to be recruited to mitochondria upon dissipating ∆ ψm . Mitochondria associated with Rheb were marked by white arrows .

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representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.


F - H Representative dual - channel kymographs ( F ) and quantitative analysis ( G , H ) showing that Rheb - mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation . Note that Rheb , but not Rheb - SSVM , is associated with autophagic vacuoles ( AVs ) within axons upon ∆ ψm dissipation . Rheb - associated AVs move in a predominant retrograde direction along axons . The number of Rheb - associated AVs ( Rheb - AVs ) with or without CCCP treatment and the relative motilities of AVs and Rheb - AVs in CCCP - treated axons were quantified ,

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F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively


Representative dual - channel kymographs showing that Rheb enhances mitophagy in CCCP - treated axons .

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Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.


quantitative analysis showing that Rheb enhances mitophagy in CCCP - treated axons . The numbers of mitochondria ( Mito ) , AVs , and Mito - AVs / mitophagosomes , the percentage of Mito within AVs , were

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quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified


quantitative analysis the percentage of Mito within AVs , and the motilities of AVs and Mito - AVs along axons with and without elevated Rheb expression were

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quantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified


D , E Representative images ( D ) and quantitative analysis ( E ) showing that mitophagy in axons requires Rheb and Nix , but not Parkin . The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP - treated axons expressing scrambled shRNA , Rheb shRNA , Nix shRNA , or Parkin shRNA were quantified , respectively ( E ) .

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D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).


F , G Quantitative analysis ( F ) and representative blots ( G ) showing increased mitochondrial association with Rheb and LC3 - II in mature cortical neurons upon ∆ ψm depolarization . Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post - nuclear supernatant ( P ) , mitochondria - enriched membrane fraction ( M ) , and cytosolic supernatant ( S ) . Equal amounts of protein ( 20 μg ) were sequentially immunoblotted with antibodies against mitophagic / autophagic proteins Rheb , LC3 , and Rab7 , mitochondrial protein VDAC , and cytosolic protein GAPDH on the same membranes after stripping between each antibody application . The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH , compared to post - nuclear supernatant . Protein levels in mitochondrial fractions of CCCP - treated neurons were normalized to those in neurons incubated with DMSO . Data were quantified from four independent repeats .

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F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.


Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP - treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA ( H ) .

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Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).


representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light . The ratio has been false colored with the indicated heat map , with high intensity indicative of ROGFP fluorescence in a more oxidative

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representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level


Aberrant accumulation of mitophagosomes co - labeled by antibodies against LC3 and cytochrome c ( Cyto c ) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains . Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse

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Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brains


The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified from the total number of brain slice sections ( n ) indicated on the top of the

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The average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the bars


C Clustering of mitophagosomes ( white arrows ) co - labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen / dystrophic presynaptic terminals indicated by synaptophysin ( SYP ) surrounding amyloid plaques in AD mouse brains .

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C Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains.


Quantitative analysis showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in

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Quantitative analysis showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses


representative TEM images showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . White arrow - labeled mitophagosomes are not readily detected in WT mouse brains . Black arrows marked amphisomes or degradative autophagic vacuoles ( AVd ) .

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representative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd).


F , G Mitophagic accumulation at Aβ - enriched synapses in AD mouse brains . Note that the levels of mitophagic / autophagic proteins ( red box ) along with Aβ ( green box ) are increased in synaptic fractions isolated from AD mouse brains . Equal amounts ( 10 μg ) of synaptosomal preparations ( Syn ) and post - nuclear supernatants ( PNS ) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP / Aβ , Rheb , LC3 , p62 , and Rab7 , as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH ( F ) . Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls . Data were quantified from five repeats ( G ) .

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F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).


H , I Representative blots ( H ) and quantitative analysis ( I ) showing increased levels of Rheb along with LC3 - II , p62 , and Rab7 ( red box ) in Mito fractions purified from AD mouse brains . The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115 , relative to PNS fractions . Data were quantified from four repeats .

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H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.


Representative images showing increased mitochondrial association with Rheb in AD axons , relative to that of WT littermate controls . Note that CCCP treatment enhances Rheb localization to mitochondria . The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of

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Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP


quantitative analysis The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified ,

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quantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively


C - Representative dual - channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons . mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were

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C- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified


quantitative analysis , the numbers of Mito , AVs , and Mito - AVs , and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified ,

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quantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively


quantitative analysis Relative motility of AVs and Mito - AVs / mitophagosomes in WT or mutant hAPP axons with and without CCCP were

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quantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantified


F , G Quantitative analysis ( F ) and representative blots ( G ) showing that ∆ ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains . Note increased localization of Rheb along with LC3 - II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment ( red box ) . Data were quantified from three independent repeats . P : post - nuclear supernatants ; M : mitochondria - enriched membrane fractions ; S : cytosolic supernatants .

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F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.


Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons , which is exacerbated after dissipating ∆ ψm . Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


A , B Representative images ( A ) and quantitative analysis ( B ) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin - cKO mouse brains . Note that white arrow - labeled mitophagosomes are not readily observed in WT mouse brains . The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified .

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A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified.


Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin - mutant mouse brains . The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Equal amounts ( 10 μg ) of Syn and post - nuclear supernatants (

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Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNS


quantitative analysis The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls WT and snapin - mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH . Data were quantified from five independent repeats .

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quantitative analysis The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats.


E , F Representative blots ( E ) and quantitative analysis ( F ) showing mitophagic accumulation in the brains of snapin conditional KO ( cKO ) mice by crossing with Thy1 - cre Tg mice . Note increased levels of Rheb along with LC3 - II , p62 , and Rab7 in mitochondrial ( Mito ) fractions isolated from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Data were quantified from five independent repeats . PNS : post - nuclear supernatants .

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E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants.


G , H Representative TEM images ( G ) and quantitative analysis ( H ) showing abnormal presynaptic retention of mitophagosome - like structures in the hippocampal regions of snapin - deficient mouse brains . Mitophagosomes , indicated by black arrows , are not readily detected in WT mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in parentheses ( H ) .

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G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H).


I , J Representative images ( I ) and quantitative analysis ( J ) showing that deletion of snapin in mouse brains induces synaptic damage . Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin - mutant mice crossed with CamKII - cre Tg mice at the age of 8 months , relative to that of WT littermate controls .

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I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.


Representative dual - channel kymographs ( A ) and quantitative analysis ( B showing that overexpression of Snapin , but not Snapin - L99K , a Snapin mutant deficient in dynein motor binding , reduces mitophagic accumulation within AD axons incubated with CCCP .

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Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.


Representative quantitative analysis showing that overexpression of Snapin , but not Snapin - L99K , a

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Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a Snapin


D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin - L99K were quantified and normalized to those of mutant hAPP controls . Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment .

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D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.


A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV - mCherry or AAV - mCherry - Snapin . mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus ( DG ) of mutant hAPP mouse brains . NeuN , a neuronal nuclear marker , indicates neurons .

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A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c * : color is converted from blue to red for better

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrast


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression


D , E Quantitative analysis ( D ) and representative images ( E ) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV - mCherry - Snapin . Blue indicates the signal of DAPI staining . The percentage area of SYP - labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV - mCherry ( D ) . Data information : Data were quantified from a total number of 40 - 44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection .

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D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.


B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co - transfected into HEK293 cells with HA - tagged Pellino - 1N plasmid . Cell lysates were immunoprecipitated with anti - HA antibody and immunoblotted with anti - Myc antibody . Data are representative of at least three independent experiments . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates .

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B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.


C , D The SBE - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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C, D The SBE-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


C , D The 5xNF - κB - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with LPS for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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C, D The 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


E CMT - 93 cell lines stably expressing Smad6 amino acids 400 - 441 were treated with LPS for the indicated time and expression of IκBα , IKKα , and phospho - IKKα / β was monitored by immunoblotting . As a positive control , CMT - 93 cells were pre - treated with TGF - β1 for 2 h . CMT - 93 cells stably expressing the empty vector pMSCV - puro were used as a negative control . β - actin was used as a loading control . All data are representative of three independent experiments .

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E CMT-93 cell lines stably expressing Smad6 amino acids 400-441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.


B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) was co - transfected with the HA - tagged full - length Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated ( IP ) with anti - Myc or anti - HA antibody , and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , CS3MTBXA - 6xMyc , was transfected as a negative control . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates . Data are representative of at least three independent experiments .

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B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.


C The Myc - Smad6 ( 422 - 441 ) plasmid was co - transfected with full - length Flag - IRAK1 , Flag - TRAF6 , Flag - MyD88 , or HA - Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti - Myc antibody . IgG was added as a negative control for IP . Data are representative of at least three independent experiments .

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C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild - type FICD . Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species . The elution profile of a monomeric FICDL258D - H363A mutant is also shown for reference , alongside the elution times of molecular weight standards .

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B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild-type FICD. Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species. The elution profile of a monomeric FICDL258D-H363A mutant is also shown for reference, alongside the elution times of molecular weight standards.


C Representative immunoblot comparing the ability of wild - type and Arg371Ser FICD ( at a wide range of enzyme concentrations ) to catalyse the accumulation of BiP - AMP in the presence of physiological ATP concentrations ( 5 mM ) . At low enzyme concentrations ( and early time points ) the AMPylation defect imposed by Arg371Ser is manifest . At higher enzyme concentrations where the deAMPylation activity of the wild - type enzyme begins to dominate , the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP - AMP ( relative to the same concentration of wild - type enzyme ) . D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C ( mean values ± SEM , n = 4 , biological replicates ) .

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C Representative immunoblot comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP in the presence of physiological ATP concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP (relative to the same concentration of wild-type enzyme). D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C (mean values ± SEM, n = 4, biological replicates).


F Representative immunoblot comparing the accumulation of BiP - AMP in reactions constituted with the indicated concentrations of wild - type and Arg371Ser FICD , performed as in A ( upper panel ) . The lower panel is a Coomassie - stained gel of the reaction constituents . An N - terminally extended version of the wild - type enzyme ( residues 45 - 458was used in lanes 2 and 5 - 7 , to distinguish its migration on the gel from the mutant ( residues 104 - 445 ) . The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F ( from n = 3 , biological replicates , mean values ± SEM ) .

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F Representative immunoblot comparing the accumulation of BiP-AMP in reactions constituted with the indicated concentrations of wild-type and Arg371Ser FICD, performed as in A (upper panel). The lower panel is a Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).


A Immunoblots of AMPylated proteins detected with a pan - AMP antibody , BiP , FICD and eIF2α ( a loading control ) in lysates of FICD ∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD . Signals corresponding to AMPylated BiP , auto - AMPylated FICDE234G , total BiP , FICD and eIF2α are indicated . The novel species reactive with the anti - FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S ( * ) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369 .

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A Immunoblots of AMPylated proteins detected with a pan-AMP antibody, BiP, FICD and eIF2α (a loading control) in lysates of FICD∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD and eIF2α are indicated. The novel species reactive with the anti-FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.


C Quantification of the data from repeats of the experiment as shown in B . The upper two graphs show the percent of cells with high CHOP : : GFP or high XBP : : Turq for each transfection . The lower panel provides quantitation of the median fluorescence intensity for each reporter . Mean ± SD , n = 4 , biological replicates .

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C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.


B On the left are immunoblots of AMPylated proteins detected with pan - AMP , BiP and eIF2α ( a loading control ) reactive antibodies in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) for the indicated time . In the panel on the right are immunoblots of samples immunoprecipitated with a BiP - specific antiserum from the lysates of the samples shown on the left .

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B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.


C Immunoblots as in B in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) and / or thapsigargin ( Tg ) for the indicated periods prior to harvest . D Quantification of ratio of the BiP - AMP to total BiP signal ( expressed in arbitrary units ) of repeats of the experiment shown in C . Mean ± SD of the BiP - AMP signal normalized to total BiP signal from the same samples are depicted , ( P values by two tailed t - test , n = 6 , biological replicates ) .

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C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).


A Representative immunoblot of AMPylated proteins in FICD ∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes ( replicated three times ) . Note that the hyperactive E234G mutant bearing the K124E - H131A mutations that compromise its ability to interact with BiP , is nonetheless consistently auto - AMPylated .

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A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.


C Bar diagram of the amount of alkaline phosphatase [ normalised to cytoplasmic luciferase ( a transfection marker ) ] secreted from FICD ∆ CHO cells co - expressing the indicated FICD enzymes . The K124E - H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [ Mean ± SD , P values by ANOVA with Šídák ' s multiple comparisons test , n = 8 - 16 ( biological replicates ) ] .

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C Bar diagram of the amount of alkaline phosphatase [normalised to cytoplasmic luciferase (a transfection marker)] secreted from FICD∆ CHO cells co-expressing the indicated FICD enzymes. The K124E-H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [Mean ± SD, P values by ANOVA with Šídák's multiple comparisons test, n = 8-16 (biological replicates)].


( A ) Oxygen consumption ratio , , in the indicated VSMC types .

(A) Oxygen consumption ratio, , in the indicated VSMC types.


( B ) mitochondrial ATP synthesis in the indicated VSMC types .

(B) mitochondrial ATP synthesis in the indicated VSMC types.


( C ) mitochondrial membrane potential ( MP ) , in the indicated VSMC types .

(C) mitochondrial membrane potential (MP), in the indicated VSMC types.


( A ) Cytosolic ATP synthesis in the indicated VSMC types , measured by incorporation of phosphate - 32 ( 32Pi ) into ADP . [ 32Pi ] - ATP was separated from 32Pi using the molybdate method , as explained in the Methods section .

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(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.


(B) Intracellular lactate concentration


( C ) external acidification in the indicated VSMC types .

(C) external acidification in the indicated VSMC types.


( D ) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi / mL calcium - 45 ( 45Ca2 + ) as a radiotracer .

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(D) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi/mL calcium-45 (45Ca2+) as a radiotracer.


( E ) Magnesium concentration in isolated mitochondria .

(E) Magnesium concentration in isolated mitochondria.


( B ) Representative time - course of 2 mmol / L phosphate on calcification of LmnaG609G / + VSMCs ( up ) . Calcification was visualized with Alizarin red . Representative microscopic images ( 10x ; scale bar : 100 µm ) showing calcification of treated and untreated LmnaG609G / + VSMCs ( down ) .

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(B) Representative time-course of 2 mmol/L phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 µm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down).


( C - F ) Measures of calcium in treated living LmnaG609G / + VSMCs ( C ) , treated fixed LmnaG609G / + VSMCs ( D ) , untreated living LmnaG609G / + VSMCs ( E ) , and untreated fixed LmnaG609G / + VSMCs ( F ) .

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(C-F) Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F).


( G ) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells ( ΔCa2 + ) .

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(G) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+).


( A ) Magnesium intake in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( B ) Plasma magnesium concentration in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( C ) Calcium content of aortas obtained from 34 - week - old wild - type mice and untreated and treated LmnaG609G / + mice ( n = 20 ) .

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(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).


( D ) Body masses of 34 - week - old untreated and treated LmnaG609G / + mice .

(D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice.


( E ) Kaplan - Meier graph for untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16).


( F ) Representative photographs of 40 - wk - old wild - type , untreated and treated LmnaG609G / + mice .

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(F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.


( A ) ATP concentration in liver homogenates obtained from 34 - week - old wild - type , untreated or treated LmnaG609G / + mice .

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(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.


( B ) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups .

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(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.


( C ) Magnesium concentration in isolated mitochondria .

(C) Magnesium concentration in isolated mitochondria.


( D ) Activities of the indicated mitochondrial complexes ( I , II , III , IV , and V ) in the absence or presence of rotenone ( 2 µmol / L ) , 2 - thenoyltrifluoroacetone ( TTFA ; 1 mmol / L ) , antimycin A ( 10 µmol / L ) , potassium cyanide ( KCN ; 1 mmol / L ) , or oligomycin ( 10 µmol / L ) .

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(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).


( B ) Light microscopy images of LGCs , FBGCs and osteoclasts stained with Giemsa staining ( upper panel ) . Representative Hoechst dye images acquired by ImageStream ( lower panel ) of sorted cells showing mononuclear ( mono ) and multinucleated ( multi ) populations for each cell type .

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(B) Light microscopy images of LGCs, FBGCs and osteoclasts stained with Giemsa staining (upper panel). Representative Hoechst dye images acquired by ImageStream (lower panel) of sorted cells showing mononuclear (mono) and multinucleated (multi) populations for each cell type.


( C ) RNA - seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type .

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(C) RNA-seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type.


( C ) MRC1 surface marker expression ( red ) acquired by ImageStream in mononuclear ( mono ) and multinucleated ( multi ) LGCs , FBGCs and osteoclasts stained for Hoechst . Bar graphs ( lower panel ) represent normalized MRC1 mean fluorescence intensity ( MFI ) , measured by ImageStream ; n = 4 donors .

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(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.


( E ) CSF1R , MRC1 , TLR2 and FOS relative expression measured by qRT - PCR for LGCs ( upper ) , FBGCs ( middle ) and osteoclasts ( lower ) , following the cell membrane fusion regulator DC - STAMP knockdown . si - Ctrl , scrambled siRNA ; si - DC - STAMP , DC - STAMP siRNA ; n = 7 donors .

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(E) CSF1R, MRC1, TLR2 and FOS relative expression measured by qRT-PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC-STAMP knockdown. si-Ctrl, scrambled siRNA; si-DC-STAMP, DC-STAMP siRNA; n=7 donors.


( E ) The effect of lysosomotropic agents ( hydroxychloroquine , hydroxy ; ammonium chloride , NH4Cl ) and v - ATPase inhibitors ( Bafilomycin A1 , Baf A1 ; Concanamycin A , Con A ) on fusion and multinucleation in LGCs ( upper panel ) , FBGCs ( middle panel ) and osteoclasts ( lower panel ) . To test the effect of lysosome dysfunction on cellular iron , FeCl3 was supplemented . Error bars are median with interquartile range ; significance tested by one - way Anova followed by Sídák ' s multiple comparisons on log transformed data ; n = 4 donors ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 ; * * * * , P < 0 . 0001 .

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(E) The effect of lysosomotropic agents (hydroxychloroquine, hydroxy; ammonium chloride, NH4Cl) and v-ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl3 was supplemented. Error bars are median with interquartile range; significance tested by one-way Anova followed by Sídák's multiple comparisons on log transformed data; n=4 donors; *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001.


( E ) B7 - H3 surface marker expression ( red ) acquired by ImageStream in mononuclear and multinucleated LGCs , FBGCs and osteoclasts stained for Hoechst . Bar graphs ( lower panel ) show normalized B7 - H3 mean fluorescence intensity ( MFI ) , measured by ImageStream ; n = 4 donors . Error bars are mean ± SD ; significance tested by paired t - test ; * * , P < 0 . 01 ; ns , non - significant .

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(E) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) Phagocytosis quantified by ImageStream analysis in LGCs , FBGCs and osteoclasts . Representative images showing the uptake of S . Aureus bioparticles ( yellow ) in mononuclear ( mono ) and multinucleated ( multi ) cells . Bar graphs ( lower panel ) show normalized S . Aureus bioparticles mean fluorescence intensity ( MFI ) measured by ImageStream ; n = 5 donors . Error bars are mean ± SD ; significance tested by paired t - test ; * * , P < 0 . 01 ; ns , non - significant .

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(C) Phagocytosis quantified by ImageStream analysis in LGCs, FBGCs and osteoclasts. Representative images showing the uptake of S. Aureus bioparticles (yellow) in mononuclear (mono) and multinucleated (multi) cells. Bar graphs (lower panel) show normalized S. Aureus bioparticles mean fluorescence intensity (MFI) measured by ImageStream; n=5 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) TRAP immunohistochemistry ( upper panel ) and hydroxyapatite resorption ( lower panel ) in LGCs , FBGCs and osteoclasts . Note the presence of TRAP + MGCs in all cell types and hydroxyapatite resorption only in osteoclasts .

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(C) TRAP immunohistochemistry (upper panel) and hydroxyapatite resorption (lower panel) in LGCs, FBGCs and osteoclasts. Note the presence of TRAP+ MGCs in all cell types and hydroxyapatite resorption only in osteoclasts.


( D ) Oxygen consumption rate ( OCR , upper panel ) , its related readout ( middle panel ) and extracellular acidification rate ( ECAR , lower panel ) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts ; n = 4 donors . ( E ) Extracellular acidification rate ( ECAR , left ) and glycolysis ( right ) in mononuclear and multinucleated osteoclasts ; n = 4 donors .

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(D) Oxygen consumption rate (OCR, upper panel), its related readout (middle panel) and extracellular acidification rate (ECAR, lower panel) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts; n=4 donors. (E) Extracellular acidification rate (ECAR, left) and glycolysis (right) in mononuclear and multinucleated osteoclasts; n=4 donors.


( F ) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts ; n = 3 donors .

(F) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts; n=3 donors.


( G ) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number ; n = 6 donors .

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(G) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number; n=6 donors.


B Latency to enter dark compartment for Gadd45a - WT ( white bars , n = 11 ) and Gadd45a - KO ( blue bars , n = 11 ) . A significant reduction was found in the Gadd45a - KO at 24 hours and 7 days after the training . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * = p < 0 . 05 .

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B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


C In situ hybridization to detect Gadd45a mRNA in adult wild - type hippocampus ( n = 8 animals ; antisense , left panel ; sense , middle panel ) and in Gadd45a - KO sections ( n = 6 animals ; right panel ) . Scale bars : 500 µm ( black bar ) .

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C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar).


D Experience - dependent changes in mRNA levels were evaluated under control conditions ( home cage ) and exposed to the PA ( context , and context + shock groups ; n = 6 for all groups ) , respectively . Hippocampal Gadd45a mRNA levels remained unchanged , while Gadd45b was induced ( context ; t10 = 2 . 427 , context + shock t10 = 2 . 319 ) . Concomitantly , mRNA levels of activity - regulated cytoskeleton protein ( Arc ) , as a proxy of neuronal activation , were increased in mice exposed to the PA context ( t10 = 3 . 406 ) and context + shock ( t10 = 3 . 141 ) . Values shown are mean ± SEM ; 1 - Way ANOVA and unpaired t - test : * = p < 0 . 05 , * * * = p < 0 . 001 .

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D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.


Gadd45a mRNA level in the hippocampus of Gadd45a - overexpressing mice ( red , n = 7 ) was significantly increased ( t10 = 4 . 189 ) as compared to Gadd45a - WT ( n = 5 ) . Values shown are mean ± SEM ; unpaired t - test : * * = p < 0 . 01 .

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Gadd45a mRNA level in the hippocampus of Gadd45a-overexpressing mice (red, n=7) was significantly increased (t10=4.189) as compared to Gadd45a-WT (n=5). Values shown are mean ± SEM; unpaired t-test: ** = p<0.01.