G proliferation rate of L929 pretreated with different CMs was analyzed using CCK - 8 kit ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , ns : not significant ( P > 0 . 05 ) vs . normal medium group ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison in G ) in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G) in Graph Pad Prism 8.


H Immunofluorescent for α - SMA type I Collagen in L929 after 48 h treatment with different CMs ( scale bar : 10 μm ; n = 6 , biological replicates ) .

O O O O O O O O O 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 Immunofluorescent staining for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (scale bar: 10 μm; n=6, biological replicates).


B Gross view of wound closure process was captured during twenty - one - day in vivo experiments ( scale bar : 500 μm ; n = 8 , biological replicates ) . C of wound closure determined at each time point ( * * * * P < 0 . 0001 ns : not significant ( P > 0 . 05 ) vs . blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison in ( C ) in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gross view of the wound closure process was captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates). C The percentage of wound closure determined at each time point (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (C) in Graph Pad Prism 8.


D Full - thickness skin samples containing entire wound sites were stained with H & E ( day 7 day 14 ) Masson ( day 14 ) ( scale bar : 500 μm , n = 8 , biological replicates ) , under are higher magnification that indicates an in red boxed region in day 7 day 14 ( scale bar : 100 μm ; n = 8 , biological replicates ) ( gt : granulation tissue , he : hyperproliferative epithelium , black arrow : wound edges ) .

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D Full-thickness skin samples containing entire wound sites were stained with H&E (day 7 and day 14) and Masson (day 14) (scale bar: 500 μm, n=8, biological replicates), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).


F Representative multiphoton projection of blood vessels ( lectin ) in healed skin wound tissue after administration of different treatments at day 21 postinjury , uninjured skin was analyzed as positive control ( Up : of entire sampling sites ( yellow circular region ) containing healed wound sites ( green circular region ) ; Down : of higher magnification that indicates an in blue boxed region in upper ; scale bar : 100 μm ; n = 3 , biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 postinjury, uninjured skin was analyzed as positive control (Up: the images of entire sampling sites (the yellow circular region) containing healed wound sites (the green circular region); Down: the images of higher magnification that indicates an area in the blue boxed region in the upper images; scale bar: 100 μm; n=3, biological replicates).


Western blot of total brain lysates of wild type , Tmem106b - / - , Grn - / - Grn - / - / Tmem106b - / - mice using antibody 6F2 to detect TMEM106B . ( 4 . 5 - month - old mice with given genotype ; n = 3 biological replicates per genotype ) .

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

Western blot analysis of total brain lysates of wild type, Tmem106b-/-, Grn-/- and Grn-/-/Tmem106b-/- mice using antibody 6F2 to detect TMEM106B. (4.5-month-old mice with the given genotype; n = 3 biological replicates per genotype).


Hind limb clasping wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - Grn - / - / Tmem106b - / - mice . Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of .

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Hind limb clasping test wild type, Tmem106b-/-, Grn-/-, Grn+/-/Tmem106b-/- and Grn-/-/Tmem106b-/- mice. Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age.


Rotarod performance of wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - double knockout at 3 to 4 months of . Number of used for , 10 wild type , 12 Tmem106b - / - , 5 Grn - / - , 7 Grn + / - / Tmem106b - / - , 3 Grn - / - / Tmem106b - / - mice .

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

Rotarod performance of wild type, Tmem106b-/-, Grn-/-, Grn+/- /Tmem106b-/- and double knockout animals at 3 to 4 months of age. Number of animals used for analysis, 10 wild type, 12 Tmem106b-/-, 5 Grn-/-, 7 Grn+/-/Tmem106b-/-, 3 Grn-/-/Tmem106b-/- mice.


Representative picture of a 4 . 5 - month - old double knockout ( Grn - / - / Tmem106b - / - ) mouse after manual flip .

O O O O O 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 picture of a 4.5-month-old double knockout (Grn-/-/Tmem106b-/- ) mouse after manual flip.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by Neuropathology panel of NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for volcano plots . Gliosis Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed brain mRNAs from Tmem106b - / - wild type mice . From 680 detected genes 21 are significantly upregulated , while 10 are significantly reduced .

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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by Neuropathology panel of NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for volcano plots . Gliosis Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of Grn - / - wild type mice . From 680 detected genes 12 are significantly upregulated , while 6 are significantly reduced .

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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by Neuropathology panel of NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for volcano plots . Gliosis Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot for Grn - / - / Tmem106b - / - wild type mice . From 680 detected genes 79 are significantly upregulated , while 24 are significantly reduced .

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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 are significantly reduced.


D . Display of differently expressed genes overlapping between analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in respective mouse models with more than 20 % change in at least one mouse model . Note that 9 genes are significantly altered in 3 genotypes in comparison to wild type . 7 genes are exclusively overlapping between Grn - / - Grn - / - / Tmem106b - / - mice . Tmem106b - / - Grn - / - / Tmem106b - / - mice share 15 significantly altered genes . Tmem106b - / - Grn - / - mice share one significant altered gene . Data represent mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD.


of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 4 Tmem106b - / - mice ) detected by Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for in volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed mRNA of Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 21 are significantly upregulated , while 12 are significantly reduced .

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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 4 Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed mRNA of Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 21 are significantly upregulated, while 12 are significantly reduced.


of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type , 4 Grn - / mice ) detected by Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for in volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of differentially expressed mRNA in Grn - / - wild type mice . From 622 detected genes 32 are significantly upregulated , while only 1 is significantly reduced .

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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type, 4 Grn-/ mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of differentially expressed mRNA in Grn-/- and wild type mice. From 622 detected genes 32 are significantly upregulated, while only 1 is significantly reduced.


of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 5 Grn - / - / Tmem106b - / - mice ) detected by Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when extent of change was above 20 % as indicated by vertical lines for in volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot of differentially expressed brain mRNAs from Grn - / - / Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 110 are significantly upregulated , while 16 are significantly reduced .

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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 5 Grn-/-/Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot of differentially expressed brain mRNAs from Grn-/-/Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 110 are significantly upregulated, while 16 are significantly reduced.


D . Display of differently expressed genes overlapping between analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in respective mouse models with more than 20 % change in at least one mouse model . Note that 19 genes are significantly altered in 3 genotypes in comparison to wild type . 17 genes are exclusively overlapping between Grn - / - Grn - / - / Tmem106b - / - mice . Tmem106b - / - Grn - / - / Tmem106b - / - mice share 17 significantly altered genes . Data represent mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type. 17 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 17 significantly altered genes. Data represent the mean ± SD.


Western blot of GFAP IBA1 in total brain lysates from 4 . 5 - month - old mice with given genotype . of A . expression was normalized to in wild type . Data represent mean ± SD .

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

Western blot analysis of GFAP and IBA1 in total brain lysates from 4.5-month-old mice with the given genotype. Quantification of A. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


Expression of GFAP in sagittal brain sections . Representative of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Expression of IBA1 in sagittal brain sections . Representative of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Scale bar indicates 100 µm .

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Expression of GFAP in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Expression of IBA1 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Scale bar indicates 100 µm.


In vivo 18F - GE180 TSPO μPET imaging . Two Grn - / - / Tmem106b - / - five wild type mice underwent longitudinal TSPO μPET at 2 . 0 3 . 5 months of . analyses were performed by PMOD ( V3 . 5 , PMOD technologies ) . Normalization of injected activity was performed by previously validated myocardium correction method .

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

In vivo 18F-GE180 TSPO μPET imaging. Two female Grn-/-/Tmem106b-/- and five female wild type mice underwent longitudinal TSPO μPET at 2.0 and 3.5 months of age. All analyses were performed by PMOD (V3.5, PMOD technologies). Normalization of injected activity was performed by the previously validated myocardium correction method.


Western blot of CD68 , CLEC7A , TREM2 ApoE in total brain lysates from 4 . 5 - month - old mice with given genotype . Asterisk indicate unspecific band . ( n = 3 biological replicates per genotype ) . of A . expression was normalized to in wild type .

Western blot analysis of CD68, CLEC7A, TREM2 and ApoE in total brain lysates from 4.5-month-old mice with the given genotype. Asterisk indicate unspecific band. (n = 3 biological replicates per genotype). Quantification of A. Protein expression was normalized to levels in wild type animals.


Expression of CD68 microglial marker IBA1 in sagittal brain sections . Representative of indicated brain regions ( TH = Thalamus ) . Expression of CLEC7A microglial marker IBA1 in sagittal brain sections . Representative of indicated brain regions ( MY = medulla ) . Expression of TREM2 microglial marker IBA1 in sagittal brain sections . Representative of indicated brain regions ( MY = medulla ) . Scale bar indicates 25 µm .

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

Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.


Western blot of cathepsin ( Cat ) D , B , L in total brain lysates from 4 . 5 - month - old mice with indicated genotype . Different maturation variants are indicated ( p = preform , sc = single chain , hc = heavy chain ) ( n = 3 biological replicates per genotype ) . of proteolytically generated variants of cathepsin D , B , L normalized to in wild type from ( A ) .

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

Western blot analysis of cathepsin (Cat) D, B, and L in total brain lysates from 4.5-month-old mice with the indicated genotype. Different maturation variants are indicated (p = preform, sc = single chain, hc = heavy chain) (n = 3 biological replicates per genotype). Quantification of proteolytically generated variants of cathepsin D, B, and L normalized to levels in wild type animals from (A).


Catalytic activity of cathepsin D , B , L . Total brain lysates from 4 . 5 - month - old mice were incubated with quenched fluorogenic substrate for Cat D , Cat B Cat L . linear increase of signal was measured then normalized to wildtype . Data represent mean normalized activity ± SD . ( n = 3 biological replicates per genotype ) .

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

Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).


Immunofluorescence of lipofuscin ( red ) IBA1 ( green ) in sagittal brain sections . Representative of Thalamus ( TH ) . Scale bar indicates 25 µm .

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

Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm.


Western blot of ubiquitin autophagy marker LC3 in RIPA , p62 in RIPA urea total brain lysates from 4 . 5 - month - old mice with indicated genotype . Calnexin used as a loading control . ( n = 3 biological replicates per genotype ) . of B . expression was normalized to in wild type . Data represent mean ± SD .

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 O

Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


Immunofluorescence of p62 in sagittal brain sections . Representative of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; Medulla = MY ) . Scale bar indicates 100 µm .

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

Immunofluorescence analysis of p62 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; Medulla = MY). Scale bar indicates 100 µm.


Localization of p62 mainly in NeuN positive in cortex ( CTX ) of 4 . 5 - month - old mice . Minor overlap of with microglial marker IBA1 . Scale bar indicates 15 µm .

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

Localization of p62 mainly in NeuN positive cells in the cortex (CTX) of 4.5-month-old mice. Minor overlap of aggregates with microglial marker IBA1. Scale bar indicates 15 µm.


Complete overlap of co - of p62 with NeuN in cortex ( CTX ) not with reactive astrocycte marker GFAP . Scale bar indicates 15 µm .

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

Complete overlap of co-staining of p62 with NeuN in the cortex (CTX) and not with the reactive astrocycte marker GFAP. Scale bar indicates 15 µm.


Immunofluorescence of TDP - 43 in sagittal brain sections . Representative of indicated brain regions ( CTX = Cortex ; MB = Midbrain ) . arrow heads indicate TDP - 43 aggregation . Scale bar indicates 100 µm . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of midbrain , scale bar zoom in indicates 50 μm . ( 4 . 5 months of ) .

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

Immunofluorescence analysis of TDP-43 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; MB = Midbrain). White arrow heads indicate TDP-43 aggregation. Scale bar indicates 100 µm. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain, scale bar zoom in indicates 50 μm. (Animals 4.5 months of age).


Immunofluorescence of total TDP - 43 phosphorylated TDP - 43 ( p - TDP - 43 ) in sagittal brain sections by confocal microscopy . Representative of one confocal layer in midbrain . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of midbrain . Scale bar indicates 25 µm . ( 4 . 5 months of ) .

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

Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).


Western blot of TDP - 43 phosphorylated TDP - 43 ( p - TDP - 43 ) in sequential high salt ( HS ) / RIPA / Urea fractionations of total brain lysates from 4 . 5 - month - old mice with indicated genotype . of C - terminal fragments of TDP - 43 ( CTF ) in RIPA fraction . p - TDP - 43 analyzed in Urea fraction . of ( C ) expression was normalized to in wild type . Data represent mean ± SD .

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

Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


, B . survival of Ssrp1fl / fl ; CreERT2 + / + mice of different ages ( ) after start of tamoxifen ( TMX ) treatment . B . Kaplan - Meier survival curves . Log - rank p = 0 . 032 for oldest group versus other groups ; p = 0 . 013 for versus ; for other comparisons p > 0 . 05 . n = 6 - 10 mice .

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, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.


C . Normalized blood in Ssrp1fl / fl ; CreERT2 + / + mice treated with vehicle or tamoxifen on day 7 after start of treatment . Mean ± SD , * indicates p < 0 . 05 ( unpaired t - ) . n = 3 mice .

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C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.


A . H & E of mouse organ sections . Scale bars : spleen , colon 100 μM , bone marrow - 50 μM .

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A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.


A , B . Effect of tamoxifen on replication in spleen ( A ) intestine ( B ) of Ssrp1fl / fl ; CreERT2 + / + mice . EdU was given to mice three days after end of tamoxifen treatment ( 1 hour before tissue collection ) . Organ sections were stained for total ( Hoechst ) , EdU , SSRP1 . Scale bars - 100 μM . C . Quantitation of EdU . Mean number of EdU positive per field of view + / - SD . n > 5 fields of view per mouse . 3 mice were analyzed . D . Quantitation of caspase - 3 . Mean number of positive per fields of view + / - SD , n > 5 fields of view per mouse Two mice were analyzed . Data information : P - values for C D were calculated using unpaired t - .

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A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.


D . Fold - change in proportion of in each cluster shown in panel A in samples from tamoxifen - treated versus vehicle - treated mice . types are identified by color as in panel B .

D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.


D . UMAP plots with color - coded intestinal identified by transcriptional signatures of intestinal or by expression of Lgr5 or Gkn3 genes .

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D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.


A , B . Effect of 4 - OHT administration on colony formation by bone marrow of different genotypes . A . of colonies from bone marrow kept in culture for seven days in presence or absence of 4 - OHT . B . Quantitation of bone marrow colony . Mean colony number per plate ± SD , n = 3 plates per mouse . P values < 0 . 05 are shown ( unpaired t - ) . 2 mice of each genotype were analyzed . C , D . Growth of intestinal organoids in presence or absence of 4 - OHT . C . of small intestine organoids on different days after plating . D . Number of organoids in presence absence of 4 - OHT . Mean fold change between number of organoids in control 4 - OHT treated wells ± SD , n = 4 wells per mouse . P < 0 . 05 are shown ( unpaired t - ) . Organoids were isolated from one mouse of each genotype in two independent experiments .

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A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.


F , G . Effect of 4 - OHT administration on intestinal organoids of different genotypes kept in presence ( undifferentiated ) or absence ( differentiated ) of R - spondin - 1 . F . viability of organoids assessed by resorufin . Mean ± SD , n = 3 wells per mouse . P < 0 . 05 are shown ( unpaired t - ) . Organoids were isolated from two mice of each genotype . G . IF of intestinal organoids from Ssrp1fl / fl ; CreERT2 + / + mice kept in presence or absence of R - Spondin - 1 . organoids were stained with antibodies to LGR5 SSRP1 counterstained with Hoechst ( ) .

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F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).


Effect of 4 - OHT administration on MSCs their differentiated counterparts . H . Methylene blue viability for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days then treated with vehicle or 4 - OHT . Mean ± SD , n = 3 plates per mouse . P < 0 . 05 are shown ( unpaired t - ) . MSCs were isolated from two mice of each genotype .

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Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype.


A . EU incorporation measured by flow cytometry . Histograms of EU signals from two biological replicates for each condition ( except no EU ) . B . SSRP1 of in A .

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A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A.


G . Box plots of mean read density ( y - axis ) at gene ( bins 21 - 100 out of 100 between TSS - TES ) for genes genes expressed at different . * * * - p < 1e - 10 ( paired t - ) . n = 2 biological replicates . Central band is mean , box is interquartile range , whiskers are minimum maximum .

G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum.


H . Comparison of Hoechst 33342 binding to live control 4 - OHT - treated MSCs from different genotypes . Data are presented as median ± SD , n = 4 biological replicates . P < 0 . 05 is shown ( unpaired t - ) .

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H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).


( F ) Representative of GFP - positive HEK293 at day 7 post - transduction using 1x106 TU / of RGD4C . TPA . GFP or RGD4C . AAVP . GFP particles . Non - targeted ( NT ) particles were included as control . Data shown are representative of five independent experiments , performed each in triplicates . Scale bar , 100 μm .

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(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.


( A ) of gene expression in HEK293 after transduction with 0 . 5x106 , 1x106 , 2x106 TU / of RGD4C . TPA , RGD4C . AAVP or non - targeted ( NT ) particles carrying Lucia . Luciferase activity was measured at days 1 to 6 post - transduction . Data shown are representative of three independent experiments ( n = 3 ) , are expressed as mean + . One - way ANOVA with Tukey ' s HSD was used for data .

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(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.


( B ) Representative of FITC - labelled TPA AAVP particle diffusion in Matrigel at 5 30 minutes post - inoculation ( n = 3 ) . Scale bar , 100 μm . ( C ) Particle diffusion using a vertical trans - well system partitioned by Matrigel . Samples were collected from wells below Matrigel at days 1 to 7 , n = 3 , technical replicates . Data are expressed as mean + , independent t - was used for data .

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(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.


( D ) Comparison of AAVP TPA internalisation efficiency in HEK293 as evaluated by of phage capsid , then quantified by FACS at 2 4 hours post - transduction , 0 . 1X106TU / , normalised to particle . Data are expressed as mean + of n = 3 independent experiments , independent t - was used for data .

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(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.


( E ) of ITRs in cytosol nucleus of HEK293 24 hours post - transduction with 0 . 5x106 TU / of RGD4C . TPA RGD4C . AAVP , using qPCR with primers specific to AAV - 2 ITRs . Data are representative of n = 3 independent experiments expressed as mean + , independent t - was used for data .

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(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.


( F ) of Lucia reporter gene expression following transduction of human SEBTA003 glioblastoma G26 glioblastoma - derived neural with 1x106 TU / . Data are expressed as mean + of n = 3 independent experiments . One - way ANOVA with Tukey ' s HSD was used for data .

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(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.


( G ) 3D GBM001 spheres subjected to transduction by increasing doses of TPA AAVP encoding Lucia . Experiments were repeated three times performed in triplicates . Data are shown as mean + are representative of one experiment . Independent t - was used for data .

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(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.


( B ) ELISA of TNFα production in media collected on days 3 to 5 post - transduction of human GBM001 spheres at 1x106 TU / of targeted ( RGD4C ) or non - targeted ( NT ) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences . Data are representative of one experiment expressed as mean + . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD was used for data .

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(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.


( C ) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C . TPA . TNFαIL2 to NOD / SCID mice bearing subcutaneous human GBM001 . Tumors healthy organs were harvested at day 18 post - transduction analysed using RT - qPCR to quantify TNFαIL2 transcripts . Data shown are expressed as mean + , from n = 3 mice . Two - way ANOVA was used for data .

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(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.


( D ) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene , at day 18 post targeted TNFαIL2 cytokine treatment . ( E ) of growth in representative mice , from D , using bioluminescence data ( total flux ) expressed as fold changes of luminescence flux at different time points compared to day 0 .

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(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.


( B ) Investigation of TPA . IL15IgK in comparison to native TPA . IL15 in B16 . F1 CT26 . CL25 . TPA constructs were transfected into , then bioactivity of secreted IL15 was assessed by evaluating effects of media from transfected on proliferation of mouse CTLL - 2 . Data are representative of one experiment expressed as mean + . Experiments were repeated twice performed in triplicates , one - way ANOVA with Tukey ' s HSD was used for data

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(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis


( C ) Targeted ( RGD4C ) non - targeted ( NT ) TPA particles carrying IL15IgK were produced used to transduce B16 . F1 or CT26 . CL25 at 0 . 5x106 or 1x106 TU / . Then secreted IL15 was quantified by ELISA in media collected on day 5 post - transduction . RGD4C . TPA without IL15IgK ( mock ) was also included as control . Experiments were repeated twice shown are results from a representative experiment . Data are expressed as mean + , one - way ANOVA with Tukey ' s HSD was used for data .

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(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.


( D ) Evaluation of CTLL - 2 proliferation in presence of media collected from B16 . F1 or CT26 . CL25 after transduction with TPA particles carrying IL15IgK . treated with a recombinant IL2 cytokine were included as a positive control . Data are representative of one experiment , expressed as mean + shown as change in CTLL - 2 number relative to treatment initiation day . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD was used to analyse data .

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(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.


( A ) - bearing BALB / c mice with established subcutaneous CT26 . CL25 tumors were intravenously injected with a single dose , 5x1010 TU / mouse , of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Gene delivery was evaluated by quantifying IL15IgK mRNA expression in tumors healthy tissues at day 5 post TPA administration . Data are representative of one experiment , n = 3 , expressed as mean + . Two - way ANOVA was used for data .

(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.


( B ) Safety of dose escalation regimens assessed at day 5 following TPA delivery , by evaluating serum of LDH from n = 2 mice / TPA dose of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Serums from untreated mice were also analysed .

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(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.


( C ) Representative - bearing mice showing bioluminescence imaging of luciferase on days 0 5 post administration with 5x1010 TU of RGD4C . TPA . IL15IgK ( RGD4C ) or non - targeted ( NT ) particles . CT26 . CL25 stably express a luciferase Luc reporter gene . Untreated mice were also included as controls ( CTRL ) .

(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).


( D ) Changes in viability in mice ( n = 4 ) from day 0 to day 5 post TPA administration . viability was measured by bioluminescence activity . central band represents median of data . boxes upper lower lines represent quartile 3 quartile 1 of data , respectively . whiskers represent maximum minimum outliers of data . Non - parametric Kruskal - Wallis was used for data .

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(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.


( E ) Kaplan Meier curves showing survival benefit for - bearing mice ( n = 4 ) from experimental groups .

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(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.


( F ) weights ( n = 4 ) were monitored during therapy experiments ad expressed as mean + .

(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.


( A ) immune profile of CT26 . CL25 tumors was investigated using RT - qPCR at day 5 after TPA . IL15IgK administration . A panel of immunological mRNA transcripts were selected for included CD8a CD8b as markers for CD8 + T population ; NKp46 for NK population . Markers for cytotoxic - killing by CD8 + T ( GZMa , GZMb ) by NK ( Prf1 ) were also analysed . Expression of TBX21 was evaluated as a marker for Th1 response mediated by interferon gamma ( IFN - γ ) , FoxP3 for Treg . Data shown are representative of n = 5 mice expressed as mean + . Non - parametric Mann - Whitney was used for GZMb TBX2 data , independent t - to analyse data of other markers .

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(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.


( B ) TPA particles encoding IL12 were used to treat immunocompetent BALB / c mice bearing subcutaneous CT26 . CL25 tumors labelled with a Luc reporter gene . Representative - bearing mice at day 5 post - treatment with targeted RGD4C . TPA . IL12 ( RGD4C ) or non - targeted TPA . IL12 ( NT ) .

(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).


( C ) of total flux change in tumors , n = 4 mice , after IL12 immunotherapy . central band represents median of data . boxes upper lower lines represent quartile 3 quartile 1 of data , respectively . whiskers represent maximum minimum outliers of data . Non - parametric Mann - Whitney was used for data .

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(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.


( D ) Kaplan Meier curves showing survival benefit for - bearing mice ( n = 4 ) from two experimental groups .

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(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.


( E ) Serial of IL12 in media of B16 . F10 treated with RGD4C . TPA . IL12 or controls . Data are representative of one experiment , n = 3 biological repeats , shown as mean + SD Experiments were repeated three times . One - way ANOVA with Tukey ' s HSD was used for data .

(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.


( F ) RGD4C . TPA . IL12 immunotherapy of subcutaneous B16 . F10 tumors in C57BL / 6 mice ( n = 4 ) . Data are shown as mean + independent t - was used for data . volume changes are shown over time .

(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.


B . Genome views of read density profiles on representative loci enriched ( left ) or low ( right ) in FLAG - HP1γ RNAchIP ( IP ) relative to input ( inp . ) in overlaid replicate samples from HP1γ stimulated or not with PMA . Orange arrow , position of locus analyzed in D .

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B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.


C . Genome - wide scatter plot of IP input normalized ( norm . ) read per gene for mean of triplicates ; n = 21 , 754 ; r , Pearson ' s correlation coefficient between IP input .

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C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input.


D . Relative quantities of in RNAchIP samples , detected by RT - qPCR with primers aligning on intron1 of Fosl1 gene as depicted in B ( orange arrow ) . n = 3 independent experiments . Data information : Histograms represent mean s . d . Dots represent data points . P - values indicate significantly higher difference between IP in HP1γ in KO ( * * * P < 0 . 001 ; two - Student ' s t - ) .

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D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments. Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


E . Heat maps of IP input signal centered on summit of RNAchIP peaks of IP versus input detected by MACS2 ( 10 , 013 2609 peaks ) for a representative replicate ( replicate 1 ) . Black arrows represent peak summit .

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E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit.


F . of RNAchIP peaks overlapping with indicated features in MEF samples from ENCODE database ( green bars ) . Overlaps was evaluated by comparison with list of merged peaks whose genomic location was randomized among genes with one hundred permutations ( random , gray bars ) . Data information : Histograms represent mean s . d . Dots represent data points . P - values indicate significantly higher difference between IP in HP1γ in KO ( * * * P < 0 . 001 ; two - Student ' s t - ) .

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F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


B . RNAchIP peaks are enriched in CACACA GAGAGA motifs . Number of peaks containing at least one exact hexameric motif as indicated , compared to average number of motifs in peaks whose genomic location was randomized among genes with ten permutations ( random , bars , represent mean s . d . ) .

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B. RNAchIP peaks are enriched in CACACA and GAGAGA motifs. Number of peaks containing at least one exact hexameric motif as indicated, compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations (random, light bars, represent mean and s.d.).


C , D . Distribution profiles of average RNAchIP signal in both IP ( warm colors ) input ( blue colors ) over ± 2kb centered on intronic ( C ) exonic ( D ) CACACA hexameric motifs oriented in same orientation ( sense ) or in opposite orientation ( antisense ) relative to overlapping annotated transcript . antisense CACACA motifs were obtained by querying transcript sequences with TGTGTG motif .

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C, D. Distribution profiles of average RNAchIP signal in both IP (warm colors) and input (blue colors) over ±2kb centered on intronic (C) and exonic (D) CACACA hexameric motifs oriented in the same orientation (sense) or in opposite orientation (antisense) relative to the overlapping annotated transcript. The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif.


E . Left , representative example of a locus on Ppp3ca gene surrounding an RNAchIP merged peak , showing RNAchIP read density as in Fig 1B . Red orange arrowheads correspond to oriented CACACA GAGAGA motifs , respectively . Right , of surrounding a CACACA motif highlighted in red , as well as a second a neighboring imperfect motif . was used to design a CACACA - containing oligonucleotide probe ( CACACA ) , compared to a control probe ( no - CA ) devoid of any related motif .

E. Left, representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak, showing the RNAchIP read density as in Fig 1B. Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs, respectively. Right, sequence of the RNA surrounding a CACACA motif highlighted in red, as well as a second a neighboring imperfect motif. The sequence was used to design a CACACA-containing RNA oligonucleotide probe (CACACA), compared to a control probe (no-CA) devoid of any related motif.


F , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST , tested for its direct interaction with Cy3 - labeled oligonucleotide probes probes in gels , either free ( arrowheads ) or as shifted / complexes ( red arrowheads ) were detected by their Cy3 . Representative of 3 independent experiments .

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F, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


G , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST , tested for its direct interaction with Cy3 - labeled dsDNA oligonucleotide probes probes in gels , either free ( arrowheads ) or as shifted / complexes ( red arrowheads ) were detected by their Cy3 . Representative of 3 independent experiments .

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G, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled dsDNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


H . Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST , tested for its direct interaction with Cy3 - labeled oligonucleotide probes probes in gels , either free ( arrowheads ) or as shifted / complexes ( red arrowheads ) were detected by their Cy3 . Representative of 3 independent experiments .

H. Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


A . Fold enrichment values on repeat masker classes on Refseq genes , on basis of uniquely aligned reads , shown as mean s . d . , n = 6 biological replicates . Dashed represents an IP / Input of 1 , taken as a reference .

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A. Fold enrichment values on all repeat masker classes and on Refseq genes, on the basis of all uniquely aligned reads, shown as mean and s.d., n=6 biological replicates. Dashed line represents an IP/Input ratio of 1, taken as a reference.


C . Profiles of average RNAchIP signal as in Fig 2B over ± 2kb centered on intragenic , LTR , or SINE repeats annotated in Repeat masker database ( n = 34 , 000 ) . repeats are oriented in same orientation ( sense ) relative to overlapping annotated transcript , unless otherwise specified ( antisense , shuffled ) , as depicted by an oriented box overlapping transcript in 5 ' to 3 ' orientation ( black with arrowhead ) . D . Profiles of average RNAchIP signal , as in ( C ) centered on 5 major families of intragenic SINE repeats .

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C. Profiles of average RNAchIP signal as in Fig 2B over ±2kb centered on intragenic LINE, LTR, or SINE repeats annotated in Repeat masker database (n=34,000). All repeats are oriented in the same orientation (sense) relative to the overlapping annotated transcript, unless otherwise specified (antisense, shuffled), and as depicted by an oriented white box overlapping the transcript in 5' to 3' orientation (black line with arrowhead). D. Profiles of average RNAchIP signal, as in (C) centered on the 5 major families of intragenic SINE repeats.


E . Proportions of families of CACACA - containing SINE repeats within each family , as a of total intragenic sense SINEs .

E. Proportions of the families of CACACA-containing SINE repeats within each family, as a percentage of total intragenic sense SINEs.


F . Profiles of average RNAchIP signal , as in ( D ) centered on subset of intragenic B4 SINEs containing a consensus CACACA motif .

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F. Profiles of average RNAchIP signal, as in (D) centered on the subset of intragenic B4 SINEs containing a consensus CACACA motif.


G . Genomic position of a B4 SINE repeat CACACA consensus motifs at location of RNAchIP

G. Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP peak


H . North - western blot showing direct association between indicated bacterially expressed , purified GST - fusion in vitro transcribed , biotinylated probes on of B4 SINE depicted in ( G ) . Top panels , binding of a 256nt probe corresponding to CACACA - containing B4 SINE , was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3 ' portion ( B4 SINE - Δ3 ' ) . probes hybridized on membranes were detected by their Cy3 . Total GST - fusion loading was visualized by Ponceau S . Representative of two independent experiments .

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H. North-western blot assay showing direct association between the indicated bacterially expressed, purified GST-fusion proteins and in vitro transcribed, biotinylated RNA probes based on the sequence of the B4 SINE depicted in (G). Top panels, binding of a 256nt probe corresponding to the CACACA-containing B4 SINE sequence, was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3' portion (B4 SINE-Δ3'). RNA probes hybridized on the membranes were detected by their Cy3 fluorescence. Total GST-fusion protein loading was visualized by Ponceau S staining. Representative of two independent experiments.


A . Box plot showing distribution of fold enrichment values per genes sorted by exon number , n = 66 , 247 , 389 , 574 genes with 1 , 2 , 3 , 4 exons , respectively . Dashed represents an IP / Input of 1 , taken as a reference . B . Reads from input IP counted in unstimulated PMA stimulated triplicates give significant enrichment values of 1 . 4 0 . 7 on introns exons , respectively , shown as mean s . d . , n = 3 biological replicates . Dots represent data points . C . Box plot of reads belonging to spliced transcripts versus reads in input or RNAchIP samples pooled between + - PMA , calculated as a between split reads total reads , n = 3 biological replicates . Dots represent data points . Data information : box boundaries represent 25th 75th percentiles ; center represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - Student ' s t - .

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

A. Box plot showing the distribution of fold enrichment values per genes sorted by exon number, n= 66, 247, 389, and 574 genes with 1, 2, 3, and 4 exons, respectively. Dashed line represents an IP/Input ratio of 1, taken as a reference. B. Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1.4 and 0.7 on introns and exons, respectively, shown as mean and s.d., n=3 biological replicates. Dots represent individual data points. C. Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and -PMA, calculated as a ratio between split reads and total reads, n=3 biological replicates. Dots represent individual data points. Data information: All box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


D , E . Average distribution profiles of depicted hexameric motifs ( D ) , or SINE repeats ( E ) on RefSeq genes , computed over exons ( black box ) on ± 0 . 7kb of surrounding introns ( left panels ) , or over entire gene bodies including introns ( pre - mRNA ; middle panels ) , or as metagenes over exons only ( Metagene mRNA ; right panels ) .

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D, E. Average distribution profiles of the depicted hexameric motifs (D), or SINE and LINE repeats (E) on RefSeq genes, computed over exons (black box) and on ±0.7kb of surrounding introns (left panels), or over entire gene bodies including introns (pre-mRNA; middle panels), or as metagenes over exons only (Metagene mRNA; right panels).


F . Box plot showing distribution of fold enrichment values on series of introns of comparable ( 4kb in average , ranging from 3 to 5kb ) sorted by number of sense CACACA motifs per intron ( 2 + , two or more motifs ) , n = 5782 , 899 , 960 , 3234 introns for introns , 0 , 1 , 2 + CACACA categories , respectively . R2 , Linear regression coefficient between 0 , 1 , 2 + . Dashed represents an IP / Input of 1 , taken as a reference . Data information : box boundaries in Fig 4 represent 25th 75th percentiles ; center represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - Student ' s t - .

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F. Box plot showing the distribution of fold enrichment values on series of introns of comparable size (4kb in average, ranging from 3 to 5kb) sorted by number of sense CACACA motifs per intron (2+, two or more motifs), n= 5782, 899, 960, and 3234 introns for all introns, 0, 1, and 2+ CACACA categories, respectively. R2, Linear regression coefficient between 0, 1, and 2+. Dashed line represents an IP/Input ratio of 1, taken as a reference. Data information: All box boundaries in Fig 4 represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


G , H . Average read profiles on two groups of RefSeq genes , in - range 4 to 15kb , containing highest ( G ) or lowest ( H ) CACACA motif density .

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G, H. Average read profiles on two groups of RefSeq genes, in the size-range 4 to 15kb, containing the highest (G) or lowest (H) CACACA motif density.


C . Box plot depicting of exon junctions found differentially regulated by MAJIQ between HP1γ ( green ) KO ( orange ) in three categories as in Fig EV5A , classified differential HP1γ / KO junctions , annotated alternative exon junctions , non - annotated de novo cryptic junctions detected in KO only ; n , differential events . Box boundaries represent 25th 75th percentiles ; center represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . Data information : graphes represent mean s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - Student ' s t - ) .

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C. Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ (green) and KO (orange) in three categories as in Fig EV5A, all classified differential HP1γ/KO junctions, annotated alternative exon junctions, non-annotated de novo cryptic junctions detected in KO only; n, differential events. Box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


D . RNAchIP read density per kb on indicated introns for input IP in samples from both untreated PMA stimulated , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Dots represent data points . HP1γ / KO introns are 272 intronic intervals between exon junctions differentially detected in HP1γ KO . Random introns are a matching library of random intronic intervals . E . RNAchIP read density per kb on genes corresponding to introns tested in ( D ) , analyzed as in ( D ) , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Data information : graphes represent mean s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - Student ' s t - ) .

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D. RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells, n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Dots represent individual data points. HP1γ/KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO. Random introns are a matching library of random intronic intervals. E. RNAchIP read density per kb on the genes corresponding to the introns tested in (D), and analyzed as in (D), n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


F . Reads from input IP counted on indicated features in combined unstimulated PMA stimulated triplicate samples , n = 6 biological replicates . Dots represent data points . Data information : graphes represent mean s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; one - Student ' s t - ) .

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F. Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples, n=6 biological replicates. Dots represent individual data points. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; one-tailed Student's t-test).


G . Average distribution profiles of sense SINE repeats on indicated exons on ± 0 . 7kb of surrounding introns .

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G. Average distribution profiles of sense SINE repeats on indicated exons and on ±0.7kb of surrounding introns.


B . RT - PCR of RNAs produced in splicing with Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO lines . top 210bp bottom 120bp amplicons correspond to included form ( beta globin with Prl exon , more included in lane1 , blue arrow , compared to lane 2 ) , skipped form ( beta globin exons only ) of , respectively . Orange arrowheads , position of HBGEX - F HBGEX - R primers used in PCR . C . Histogram showing loss of inclusion of Prl3 exon upon depletion of HP1γ ( in KO ) relative to inclusion in presence of HP1γ with Prl3 wt ( red ) or Prl3 CA mut ( grey ) reporters . on quantitation of 4 experiments as one depicted in B . Values represent fold change relative to HP1γ as a reference ( dashed ) . Graph represents mean s . d . ( * * * P < 0 . 001 ; two - Student ' s t - ; n = 4 ) .

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B. RT-PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines. The top 210bp and bottom 120bp amplicons correspond to the included form (beta globin with Prl exon, more included in lane1, blue arrow, compared to lane 2), and the skipped form (beta globin exons only) of RNA, respectively. Orange arrowheads, position of the HBGEX-F and HBGEX-R primers used in PCR analysis. C. Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ (in KO) relative to inclusion in the presence of HP1γ with the Prl3 wt (red) or Prl3 CA mut (grey) reporters. Based on the quantitation of 4 experiments as the one depicted in B. Values represent the fold change relative to HP1γ as a reference (dashed line). Graph represents mean and s.d. (*** P < 0.001; two-tailed Student's t-test; n=4).


( a ) Electron of IM ( black arrowheads ) associated with ER ( black arrows ) from Atg4BC74A - expressing NIH 3T3 . IM appears to expand between two ER cisternae forms an autophagosome . arrowhead indicates vesicular clusters visible in vicinity of IM . arrow indicates autophagosome .

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(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome.


( b ) of ER - associated autophagic structures ( ER - IM complexes ) to total autophagic structures . Data are expressed as mean ± s . d . of triplicates from representative experiments .

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(b) The ratio of ER-associated autophagic structures (ER-IM complexes) to total autophagic structures. Data are expressed as the mean ± s.d. of triplicates from representative experiments.


( c ) Localization of GFP - DFCP1 with Atg16L1 in both control Atg4BC74A - expressing . Insets show enlarged of boxed regions .

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(c) Localization of GFP-DFCP1 with Atg16L1 in both control and Atg4BC74A-expressing cells. Insets show enlarged images of boxed regions.


( d - g ) Gold - enhanced immunogold electron microscopy revealed that GFP - DFCP1 localized to ER - IM complexes in both control ( d , e ) Atg4BC74A - expressing ( f ) . Immunogold particles are indicated by small arrows . No specific labelling was observed on ER - IM complexes in expressing GFP ( g ) . Large arrows arrowheads indicate ER IM , respectively . Scale bars , 200 nm in a d - g , an d 10 μm in c .

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(d-g) Gold-enhanced immunogold electron microscopy revealed that GFP-DFCP1 localized to ER-IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER-IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d-g, an d 10 μm in c.


Atg4BC74A - expressing ( for Atg16L1 ) or control ( for PDI , COP II β - COP ) were labelled with specific antibodies against indicated markers . left right anti - Atg16L1 anti - PDI are from two different ER - IM complexes . Arrows , arrowheads open arrowheads indicate ER , IMs VTCs , respectively . Small arrows in image from anti - COP II experiment indicate immunogold particles . Scale bars , 200 nm .

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Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER-IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.


( e ) show 1 . 5 - nm tomographic slices of boxed region in c , depict IM ( black arrow ) , whose edge ( red arrowhead ) is connected to associated ER ( black arrowhead ) . Numbers indicate depth of tomographic slices . Scale bars , 200 nm in a , 100 nm in b c , 50 nm in d e .

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(e) Images show 1.5-nm tomographic slices of the boxed region in c, and depict the IM (black arrow), whose edge (red arrowhead) is connected to the associated ER (black arrowhead). Numbers indicate the depth of the tomographic slices. Scale bars, 200 nm in a, 100 nm in b and c, and 50 nm in d and e.


A 2 - nm tomographic slice of each ER - IM , reconstructed from a 200 - nm thick section , their 3D models are depicted in left panels . Red arrows indicate sites at which an edge of IM is connected to ER . Boxed regions are enlarged in right panels . 3D models of boxed regions , shown in a different orientation , depict connection between IM ER ( red arrows ) . Tomographic slices of boxed regions show edge of IM ( red arrowheads ) is connected to ER . Numbers indicate depth of tomographic slices . Scale bars , 100 nm .

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A 2-nm tomographic slice of each ER-IM complex, reconstructed from a 200-nm thick section, and their 3D models are depicted in the left panels. Red arrows indicate sites at which an edge of the IM is connected to the ER. Boxed regions are enlarged in the right panels. 3D models of the boxed regions, shown in a different orientation, depict the connection between the IM and ER (red arrows). Tomographic slices of the boxed regions show the edge of the IM (red arrowheads) is connected to the ER. Numbers indicate the depth of the tomographic slices. Scale bars, 100 nm.


A , B Intracellular NAD NAD / NADH in DC - matched healthy control fibroblasts . values are presented as mean ± SD of four eight replicates in A B , respectively . Student ' s t was performed on DC vs controls .

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A, B Intracellular NAD levels and NAD/NADH ratio in DC and age-matched healthy control fibroblasts. All values are presented as mean ± SD of four and eight replicates in A and B, respectively. Student's t test was performed on DC cells vs controls.


Immunoblots of expression of NAD synthesis consuming , their activities in DC control . are normalized to GAPDH .

Immunoblots of the expression of NAD synthesis and consuming proteins, and their activities in DC and control cells. Protein levels are normalized to GAPDH.


E Immunoblots of expression of NAD synthesis consuming values are presented as mean ± SD of four controls versus five DC samples as shown in D . Student ' s t was performed on DC vs controls . Con : control fibroblasts .

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E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D. Student's t test was performed on DC cells vs controls. Con: control fibroblasts.


F Effects of NR NAM supplementation ( 24 hours ) on NAD in DC fibroblasts . values are presented as mean ± SD of four replicates . One - Way ANOVA was performed on DC in indicated conditions .

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F Effects of NR and NAM supplementation (24 hours) on the NAD levels in DC fibroblasts. All values are presented as mean ± SD of four replicates. One-Way ANOVA was performed on DC cells in indicated conditions.


Quantitative RT - PCR immunoblots of expression of CD38 SARM1 in DC - matched health control fibroblasts . values are presented as mean ± SD of three replicates in quantitative RT - PCR . are normalized to GAPDH , mean ( ± SD ) values of 4 controls vs 5 DC samples are shown . Student ' s t was performed on DC vs controls . Irrelevant intervening lanes have been removed for clarity ( full blots are available online as Source Data ) .

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Quantitative RT-PCR and immunoblots of the expression of CD38 and SARM1 in DC and age-matched health control fibroblasts. All values are presented as mean ± SD of three replicates in quantitative RT-PCR. Protein levels are normalized to GAPDH, and mean (± SD) quantification values of 4 controls vs 5 DC samples are shown. Student's t test was performed on DC cells vs controls. Irrelevant intervening lanes have been removed for clarity (full blots are available online as Source Data).


Immunoblots of indicated in DC fibroblasts treated with DMSO with 2 μM ATM inhibitor , KU - 55933 for 48 hours . Mean ( ± SD ) values of three DC lines with without ATMi treatment are shown . P values on basis of Student ' s t .

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Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor, KU-55933 for 48 hours. Mean (± SD) quantification values of three DC lines with and without ATMi treatment are shown. P values on the basis of Student's t test.


efficacy of CD38 shRNA ( sh - 1 sh - 2 ) was verified by quantitative RT - PCR in DC fibroblasts by RT - PCR immunoblots in A549 . relative CD38 mRNA values in CD38 knockdown were normalized to scrambled shRNA . values are presented as mean ± SD of three replicates . Student ' s t was performed on DC A549 in indicated conditions .

The efficacy of CD38 shRNA (sh-1 and sh-2) was verified by quantitative RT-PCR in DC fibroblasts and by RT-PCR and immunoblots in A549 cells. The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA. All values are presented as mean ± SD of three replicates. Student's t test was performed on DC and A549 cells in indicated conditions.


Intracellular NAD in scramble CD38 knockdown DC fibroblasts . CD38 knockdown DC fibroblasts were treated with PARP1 inhibitor Olaparib ( 400nM ) SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC in indicated conditions .

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Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


immunoblots of expression of indicated in scramble CD38 knockdown DC fibroblasts . CD38 knockdown DC fibroblasts were treated with PARP1 inhibitor Olaparib ( 400nM ) SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC in indicated conditions .

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immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


Intracellular NAD in brain tissues from 6 - month - old G1 G3 Tert - / - mice .

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Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice.


immunoblots of expression of NAD consuming related their activities in brain tissues from 6 - month - old G1 G3 Tert - / - mice . CD38 antibody was validated using CD38 - / - mouse brain tissues .

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immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.


C of immunoblots from indicated in B . Data information : values are presented as mean ± SD of G1 Tert - / - mouse brain tissues versus G3 Tert - / - mouse brain tissues . n = 4 in each group . P values on basis of Student ' s t .

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C Quantification of immunoblots from the indicated proteins in B. Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test.


D NADase activity in G1 G3 Tert - / - mouse brain lysate was determined at various time points during reaction . NADase activity was not detectable in extracts treated with CD38 inhibitor , 78c , at a of 50nM .

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D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.


A Representative immunoblots of expression of PGC1 - 1α in DC treated with vehicle or 3 mM NR . of indicated is from three immunoblots .

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A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.


B - D mitochondrial ROS in DC - matched healthy control supplemented with vehicle NR were measured by flow cytometry from three replicates .

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B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates.


mitochondrial ROS in DC - matched healthy control in scramble CD38 knockdown DC ( D ) were measured by flow cytometry from three replicates .

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Cellular and mitochondrial ROS in DC and age-matched healthy control cells in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.


E Representative captured by transmission electron microscopy of DC1 treated with vehicle or NR . arrows : mitochondria . Yellow arrow : autophagosome - like structures with engulfed mitochondria . Enlarged image within frame is shown . F - H Quantifications of of damaged mitochondria per ( F ) , mitochondrial ( G ) , mitochondrial diameter ( H ) . A minimum of 200 mitochondria counted per group .

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E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown. F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.