Figure 8A . Mean protein concentration in the CSF of Mpdz + / + and Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) . B . Volcano plot showing that the protein contents in sera of P15 - P21 Mpdz + / + and Mpdz - / - mice are similar to each other . C . A volcano plot showing that 23 proteins are at least two - fold significantly more abundant in the CSF of P15 - 21 Mpdz - / - mice ( red circles , protein names are indicated ) whereas only two proteins were more abundant in the CSF of Mpdz + / + mice ( green circles ) . The CSF composition was analyzed in 3 Mpdz - / - and 3 Mpdz + / + mice . D . Images of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + and Mpdz - / - mice , immunolabeled for Nkcc1 . The areas in the marked squares are magnified below . Scale bars , top , 50 μm , bottom 20 μm . E . Immunoblots of LDLR and Mpdz , with β - actin as loading control . The MPDZ immunoblot shows the efficiency of the shRNA - mediated knockdown . Bands from three immunoblots were quantified by normalization to β - actin bands ( mean ± SD ) .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).B. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other.C. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice.D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm.E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).


Figure 1B . Western blots of MDI protein in wild type ( wt ) and mdi1 flies , showing that mdi1 is a protein - null mutation . Tubulin was used as loading control . C . Ovarioles expressing MDI - GFP stained with a mitochondrial marker , ATP synthase ( ATP - S ) . Overlapping signals ( merge ) indicate that MDI localizes to mitochondria . Note that MDI is highly expressed in germ cells at late germarium stages ( left panels ) and in egg chambers ( right panels ) . Bars , 10 m .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.


Figure 2A - C . mtDNA replication in wt ( A ) and mdi1 ovarioles ( B , C ) as illustrated by EdU incorporation . Arrows point to mitochondrial DNA , arrowheads to nuclei . In wt ovaries ( A ) , mtDNA replication starts at germarium stage 2B and lasts into egg - chamber stage 2 ( S2 ) . In most mdi1 ovarioles ( B ) , mtDNA replication is undetectable at these stages , whereas in some ( C ) it appears delayed until germarium stage 3 . Bars , 10 m . D . Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls . Each data point represents the mean of 3 independent replicates . Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . The relative mtDNA level was determined as the average of 3 biological repeats . P values of comparing mdi1 ; nos > mdi to mdi1 : hatching rate , P = 1 . 1995E - 05 ; mtDNA , P = 0 . 0078 . P values of comparing mdi1 ; nos > hAKAP1 to mdi1 : hatching rate , P = 1 . 6998E - 05 ; mtDNA level , P = 0 . 0009 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-C. mtDNA replication in wt (A) and mdi1 ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m.D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.


Figure 3B . Representative images of S2 cells expressing GFP fusions ( green ) of the MDI proteins diagramed in ( A ) . Staining with MitoTracker Red ( red ) to label mitochondria shows an overlapping signal ( yellow ) , indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS . C . Submitochondrial localization of MDI . Mitochondria from cultured cells expressing an MDI - myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes , and digested or not with Protease K ( PK ) . Protein extracts were subjected to western blot analysis . Tom20 , cytochrome C ( cyt . C ) and SOD2 were used as markers of the outer membrane , inter - membrane space and matrix , respectively . MDI behaved like an outer - membrane protein . D . Western blots of MDI protein in wt and mdi knockout ( mdi - ko ) cells confirming the mdi - ko cells completely lacked MDI protein . Actin was used as loading control . E . Representative images of wt or mdi - ko cells expressing a Larp - GFP fusion protein and stained with MitoTracker Red to label mitochondria . The majority of Larp localized to mitochondria in wt cells ( overlapping red and green signal ) , but diffused into the cytoplasm of mdi - ko cells . F . Representative images of mdi - ko cells co - expressing Larp - GFP with MDI or the deletion mutants diagramed in ( A ) fused to the mCherry . Of the 4 MDI deletions that localized to mitochondria , 2 ( MDIΔR and MDIΔTudor ) did not allow Larp - GFP to localize to mitochondria . The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants , and the hatching rate and mtDNA level in their eggs were determined . The result was summarized in ( A ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS.C. Submitochondrial localization of MDI. Mitochondria from cultured cells expressing an MDI-myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes, and digested or not with Protease K (PK). Protein extracts were subjected to western blot analysis. Tom20, cytochrome C (cyt.C) and SOD2 were used as markers of the outer membrane, inter-membrane space and matrix, respectively. MDI behaved like an outer-membrane protein.D. Western blots of MDI protein in wt and mdi knockout (mdi-ko) cells confirming the mdi-ko cells completely lacked MDI protein. Actin was used as loading control.E. Representative images of wt or mdi-ko cells expressing a Larp-GFP fusion protein and stained with MitoTracker Red to label mitochondria. The majority of Larp localized to mitochondria in wt cells (overlapping red and green signal), but diffused into the cytoplasm of mdi-ko cells.F. Representative images of mdi-ko cells co-expressing Larp-GFP with MDI or the deletion mutants diagramed in (A) fused to the mCherry. Of the 4 MDI deletions that localized to mitochondria, 2 (MDIΔR and MDIΔTudor) did not allow Larp-GFP to localize to mitochondria. The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants, and the hatching rate and mtDNA level in their eggs were determined. The result was summarized in (A).


Figure 4A . Co - immunoprecipitation of Larp with MDI - myc in transfected S2 cells . Tubulin was used as loading control . B . mtDNA replication , as illustrated by EdU incorporation , in wt and larp mutant ( larp ) ovarioles . mtDNA replication is dramatically reduced in the larp ovariole . Arrows : mtDNA ; arrowheads : nuclei . C . wt and mdi1 egg chambers stained for Larp ( green ) and ATP - S ( red ) to reveal mitochondria . Larp closely associates with mitochondria in wt egg chambers . Mitochondria in mdi1 flies are clumped together , and completely lack Larp staining . Bars in ( B ) ( C ) : 10 m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Co-immunoprecipitation of Larp with MDI-myc in transfected S2 cells. Tubulin was used as loading control.B. mtDNA replication, as illustrated by EdU incorporation, in wt and larp mutant (larp) ovarioles. mtDNA replication is dramatically reduced in the larp ovariole. Arrows: mtDNA; arrowheads: nuclei.C. wt and mdi1 egg chambers stained for Larp (green) and ATP-S (red) to reveal mitochondria. Larp closely associates with mitochondria in wt egg chambers. Mitochondria in mdi1 flies are clumped together, and completely lack Larp staining. Bars in (B) (C): 10 m.


Figure 5A . Nascent protein synthesis revealed by HpG incorporation ( green ) in wt and mdi1 egg chambers . Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery . Arrows point to HpG signal associated with mitochondria . Mitochondria are marked by a Tom20 - mCherry ( red ) created by inserting mCherry at the endogenous Tom20 locus . B . ER location is unaltered in mdi1 egg chambers . An ER marker ( ER - GFP ) was expressed in wt and mdi1 egg chambers that were co - stained with ATP - S to mark mitochondria . ER localizes to the perinuclear region , cytoplasm and cell periphery in both wt and mdi1 egg chambers .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Nascent protein synthesis revealed by HpG incorporation (green) in wt and mdi1 egg chambers. Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery. Arrows point to HpG signal associated with mitochondria. Mitochondria are marked by a Tom20-mCherry (red) created by inserting mCherry at the endogenous Tom20 locus.B. ER location is unaltered in mdi1 egg chambers. An ER marker (ER-GFP) was expressed in wt and mdi1 egg chambers that were co-stained with ATP-S to mark mitochondria. ER localizes to the perinuclear region, cytoplasm and cell periphery in both wt and mdi1 egg chambers.


Figure 6Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . A . Nascent protein synthesis was monitored by AHA incorporation , and detected by anti - biotin antibody . Tom20 served as a loading control . Note that the synthesis of nuclear - encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20 - Larp ( mdi1 / TL ) . Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . B . Western blots of mitochondrial proteins in ovarioles of wt , mdi1 and mdi1 flies expressing Tom20 - Larp ( mdi1 / TL ) fusion protein . Note that levels of Tamas , TFAM , mRpL19 and COX4 were reduced in mdi1 flies , but restored in mdi1 / TL flies . Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . C . Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary , whereas mtSSB was not affected . Tfamgfp , Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background . Nascent proteins were labeled by AHA incorporation and then the GFP - tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti - biotin antibody . Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . D . Representative profile of 254 nm absorbance of wt , mdi1 and mdi1 / TL ovary extracts . Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . E . Polysome mRNA profiling for tamas , tfam , mtSSB , cox4 and mRPL19 in wt , mdi1 and mdi1 / TL ovary . The percentage of mRNA for each gene in non - polysomal fractions ( N . P , including ribosomal subunits and monosome - associated ) and polysomal fractions ( poly ) was calculated and plotted . The fractions of tamas , tfam , cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt , but were restored in mdi1 / TL flies . N = 4 for all samples . P values of comparing wt to mdi1 : tamas , P = 0 . 0055 ; tfam , P = 0 . 001 ; cox4 , P = 0 . 0066 ; mRPL19 , P = 0 . 0097 . P values of comparing mdi1 / TL to mdi1 : tamas , P = 0 . 0206 ; tfam , P = 0 . 0346 ; cox4 , P = 0 . 0036 ; mRPL19 , P = 0 . 0016 . Detection of nascent protein synthesis in the mitochondrial fraction of the ovary . F . The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1 / TL flies . The proteins were immunopurified with antibodies against the endogenous proteins .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL).Detection of nascent protein synthesis in the mitochondrial fraction of the ovary.B. Western blots of mitochondrial proteins in ovarioles of wt, mdi1 and mdi1 flies expressing Tom20-Larp (mdi1/TL) fusion protein. Note that levels of Tamas, TFAM, mRpL19 and COX4 were reduced in mdi1 flies, but restored in mdi1/TL flies.Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody.Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts.Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. E. Polysome mRNA profiling for tamas, tfam, mtSSB, cox4 and mRPL19 in wt, mdi1 and mdi1/TL ovary. The percentage of mRNA for each gene in non-polysomal fractions (N.P, including ribosomal subunits and monosome-associated) and polysomal fractions (poly) was calculated and plotted. The fractions of tamas, tfam, cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt, but were restored in mdi1/TL flies. N=4 for all samples. P values of comparing wt to mdi1: tamas, P= 0.0055; tfam, P= 0.001; cox4, P=0.0066; mRPL19, P=0.0097. P values of comparing mdi1/TL to mdi1: tamas, P= 0.0206; tfam, P= 0.0346; cox4, P=0.0036; mRPL19, P=0.0016.Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.


Figure 7A . Mitochondrial localization of a Tom20 - LarpGFP ( Tom20 - Larp ) fusion protein in an mdi1 egg chamber stained with ATP - S to reveal mitochondria . Bars , 20 m . B . Hatching rates and mtDNA contents of eggs produced by flies with different genotypes relative to wt control ; each data point represents the mean of 3 independent replicates . Bars , std . Expression of Tom20 - Larp in mdi1 ( mdi1 ; Tom20 - Larp ) significantly restored the mtDNA level and hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . The relative mtDNA level was the average of 3 biological repeats . P values of comparing mdi1 ; Tom20 - Larp to mdi1 : for mtDNA level , P = 0 . 0209 ; for hatching rate , P = 3 . 4758E - 05 .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Mitochondrial localization of a Tom20-LarpGFP (Tom20-Larp) fusion protein in an mdi1 egg chamber stained with ATP-S to reveal mitochondria. Bars, 20 m.B. Hatching rates and mtDNA contents of eggs produced by flies with different genotypes relative to wt control; each data point represents the mean of 3 independent replicates. Bars, std. Expression of Tom20-Larp in mdi1 (mdi1;Tom20-Larp) significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats. P values of comparing mdi1;Tom20-Larp to mdi1: for mtDNA level, P=0.0209; for hatching rate, P=3.4758E-05.


Figure 1 ( A ) Intracellular detection of NOD2 ( bold line in the histogram plot ) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells ( R1 , dot plot ) . One representative experiment out of three experiments is presented . ( B ) Cells were stimulated with 10 μg / mL of MDP for 24 h , and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis . The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported . ( C ) The upregulation of NOD2 gene expression was assessed by quantitative PCR . The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells ( n = 11 ) . ( D ) Subcellular localization of NOD2 was ascertained in unstimulated ( left , × 80 , 000 ) and MDP ‐ stimulated cells ( right , × 63 , 000 ) using an anti ‐ NOD2 antibody and detected with a secondary antibody coupled to 5 ‐ nm gold particles , indicated with arrowheads . Bar represents 150 nm . Micrographs were obtained from one experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Intracellular detection of NOD2 (bold line in the histogram plot) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells (R1, dot plot). One representative experiment out of three experiments is presented.(B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported.(C) The upregulation of NOD2 gene expression was assessed by quantitative PCR. The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells (n = 11).(D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP‐stimulated cells (right, ×63,000) using an anti‐NOD2 antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.


Figure 2NOD2 ligation in alveolar macrophages induces pro ‐ inflammatory cytokine release . Cells were incubated for 24 h in the presence of 10 μg / mL of MDP or 100 ng / mL of LPS . Production of IL ‐ 1β , IL ‐ 6 , TNF ‐ α , and IL ‐ 10 was measured in culture supernatants using Milliplex technology . Depicted are box plots with median values and quartiles for each cytokine . The data are representative of two independent experiments ( n = 11 ) ; * p 0 . 05 and * * p 0 . 01 using the two ‐ tailed Wilcoxon signed ‐ rank test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2NOD2 ligation in alveolar macrophages induces pro‐inflammatory cytokine release. Cells were incubated for 24 h in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS. Production of IL‐1β, IL‐6, TNF‐α, and IL‐10 was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.


Figure 3LL37 and IRGM gene expression is upregulated following NOD2 activation ( A ‐ D ) . Alveolar macrophages were incubated in the presence of 10 μg / mL of MDP or 100 ng / mL of LPS for 24 h . Upregulation of ( A ) LL37 and ( D ) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification . The fold change in gene expression relative to unstimulated cells is depicted . Data are representative of two independent experiments ( n = 7 - 12 ) . Bold lines indicate median values . ( B , E ) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis . ( C , F ) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM . One representative experiment out of three experiments is presented .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values.(B, E) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis.(C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.


Figure 4 ( A ) Intracellular bacterial burden was measured by quantifying CFU , and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0 . Data are shown as medians and quartiles and are representative of n = 7 experiments . The production of ( B ) TNF ‐ α , ( C ) IL ‐ 6 and ( D ) IL ‐ 1β was measured in culture supernatants using Milliplex technology . Box plots depicting median values and quartiles are representative of n = 9 experiments . ( E ) LL37 and ( F ) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification . Fold changes in gene expression relative to unstimulated cells are reported . Bold lines indicate median values of n = 9 . * p 0 . 05 and * * p 0 . 01 using the two ‐ tailed Wilcoxon signed ‐ rank test . ( G ) LL37 and IRGM protein was assessed by western blot . One representative experiment out of three experiments is presented . ( H ) The amount of protein normalized to tubulin relative to infected ‐ only cells was calculated by densitometry . Data are depicted as the means ± SEM and are representative of n = 3 experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Intracellular bacterial burden was measured by quantifying CFU, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments.The production of (B) TNF‐α, (C) IL‐6 and (D) IL‐1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments.(E) LL37 and (F) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.(G) LL37 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented.(H) The amount of protein normalized to tubulin relative to infected‐only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.


Figure 5 ( A , B , D , E , G , H ) The subcellular localization of autophagy proteins was ascertained in untreated and MDP ‐ treated cells using ( A , B ) anti ‐ IRGM , ( D , E ) anti ‐ LC3 , and ( G , H ) anti ‐ ATG16L1 antibodies and detected with a secondary antibody coupled to 5 ‐ nm gold particles indicated with arrowheads ; bar represents 150 nm . ( C , F , I ) Gold particles co ‐ localizing with bacteria were manually counted in ten macrophages of each condition . Means ± SEM are depicted and differences between treatments are indicated , * p 0 . 05 using a two ‐ tailed paired t ‐ test . ( B ) Transmission electron microscopy ( TEM ) magnification × 50 , 000 ; the rest of the micrographs are TEM magnification × 80 , 000 . Data were generated from one experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP‐treated cells using (A, B) anti‐IRGM, (D, E) anti‐LC3, and (G, H) anti‐ATG16L1 antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm.(C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.


Figure 6Macrophages treated with MDP after M . tuberculosis infection develop autophagy vacuoles . Alveolar macrophages were infected with M . tuberculosis H37Rv at an MOI of 5 for 1 h . Nonphagocytosed bacteria were washed away , and the macrophages were incubated for an additional hour . The cells were then treated with 10 μg / mL of MDP for 24 h . Autophagy indicators ( arrowheads ) such as ( A ) vesicles containing organelles ( TEM magnification × 20 , 000 ) and ( B ) onion skin ‐ like lamellar multivesicular bodies ( TEM magnification × 16 , 000 ) were identified by ultrastructural analysis . . ( C , D ) Autophagosomes containing mycobacteria ( Mtb ) are indicated with arrows ( C , TEM magnification × 40 , 000 ; D , TEM magnification × 12 , 000 ) . Micrographs were obtained from one experiment . Bar represents 300 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Macrophages treated with MDP after M. tuberculosis infection develop autophagy vacuoles. Alveolar macrophages were infected with M. tuberculosis H37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h. Autophagy indicators (arrowheads) such as (A) vesicles containing organelles (TEM magnification ×20,000) and (B) onion skin‐like lamellar multivesicular bodies (TEM magnification ×16,000) were identified by ultrastructural analysis..(C, D) Autophagosomes containing mycobacteria (Mtb) are indicated with arrows (C, TEM magnification ×40,000; D, TEM magnification ×12,000). Micrographs were obtained from one experiment. Bar represents 300 nm.


Figure 1Coomassie - stained SDS - PAGE gel showing purified protein from the predicted bacterial OTU constructs . Ub - PA activity - based probe assay for wild - type ( WT ) and catalytic Cys - to - Ala mutants ( CA ) . Strong , Cys - dependent reactivity is indicated with asterisks . Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations . Note that BurkOTU displays an increase in fluorescence polarization , indicative of noncovalent binding . Heatmap representation of DUB activity against the Ub - KG ( TAMRA ) substrate shown in G . , including the WT enzyme and Ala substitutions at the predicted catalytic Cys , general base His , or acidic position . Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Coomassie-stained SDS-PAGE gel showing purified protein from the predicted bacterial OTU constructs.Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.


Figure 2Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala wMelOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala BurkOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Note that the rise in fluorescence polarization signal , indicative of a noncovalent interaction , is specific to the Ub substrate . Heatmap representation of corrected OTU activities toward the Ub and Ub - like fluorescent substrates . In the reactions marked by an asterisk , an unusually high level of noise in fluorescence polarization signal was observed , likely a result of high OTU concentration . Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining . Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining . Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O

Figure 2Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.


Figure 3Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided wMelOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for reference .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.


Figure 4Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided EschOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for clarity .

Figure 4Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.


Figure 5Ub - PA activity - based probe assay for WT vOTU and sequence - permutated vOTUP . Strong reactivity is indicated with asterisks . Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence - permutated vOTUP .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 5Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.


Figure 1A . Enrichment of lamin B1 in the SGZ of DG . B . Immunohistochemical analyses of lamin B1 - high cells . The lamin B1 - high cells express Sox2 or PSA - NCAM , but not Prox1 . Arrowheads indicate Prox1 negative lamin B1 - high cells in the SGZ . C . Immunohistochemical analyses of lamin B1 in pNestin - GFP mice . An open arrowhead indicates RGL - ANSC and a closed arrowhead indicates a non - RGL ( NR ) - NPC . E . Immunocytochemistry of lamin B1 in NPCs and Tuj1 + differentiating neurons 4 days after the induction of differentiation . F . Relative expression of Lmnb1 and Tubb3 revealed by qRT - PCR from NPCs and differentiating neurons at the indicated days . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one sample t - test ( n = 4 ) . G . Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types . F ( 3 , 82 ) = 173 . 1 , P = 1 . 2 e - 34 , n = 12 - 30 cells per cell type , one - way ANOVA followed by Tukey - Kramer test ( * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Enrichment of lamin B1 in the SGZ of DG.B. Immunohistochemical analyses of lamin B1-high cells. The lamin B1-high cells express Sox2 or PSA-NCAM, but not Prox1. Arrowheads indicate Prox1 negative lamin B1-high cells in the SGZ.C. Immunohistochemical analyses of lamin B1 in pNestin-GFP mice. An open arrowhead indicates RGL-ANSC and a closed arrowhead indicates a non-RGL(NR)-NPC.E. Immunocytochemistry of lamin B1 in NPCs and Tuj1+ differentiating neurons 4 days after the induction of differentiation.F. Relative expression of Lmnb1 and Tubb3 revealed by qRT-PCR from NPCs and differentiating neurons at the indicated days. ***P < 0.001, **P < 0.01, *P < 0.05, one sample t-test (n = 4).G. Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types. F (3,82) = 173.1, P = 1.2 e-34, n = 12-30 cells per cell type, one-way ANOVA followed by Tukey-Kramer test (**P < 0.01).


Figure 2A . Age - dependent reduction of lamin B1 in the SGZ . As early as 5 . 5 months of age , the levels of lamin B1 in the SGZ were markedly reduced . Insets show higher magnification images in the SGZ . B - D . Quantification of lamin B1 levels using immunofluorescent signals . Lamin B1 levels in Sox2 - positive , PSA - NCAM - negative - ANSPCs were selectively downregulated at 5 . 5 months of age ( ANPCs , P = 1 . 02129e - 19 ; Neuroblasts , P = 0 . 09 , DGCs , P = 6 . 4011e - 15 , ANOVA ) . * * P < 0 . 001 , ANOVA followed by Tukey - Kramer test . ( 3 animals , 30 cells per conditions ) E . Age - dependent reduction of lamin B1 in RGL - ANSCs ( upper ) and NR - ANPC ( lower ) from 2 to 5 . 5 months of age . Arrowheads indicate RGL - ANSCs or NR - ANPCs . F . Quantification of lamin B1 levels in RGL - ANSCs and ANPCs . * P < 0 . 05 , * * * P < 0 . 001 , ( 4 animals , 18 - 32 cells per conditions , Mann - Whitney test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Age-dependent reduction of lamin B1 in the SGZ. As early as 5.5 months of age, the levels of lamin B1 in the SGZ were markedly reduced. Insets show higher magnification images in the SGZ.B-D. Quantification of lamin B1 levels using immunofluorescent signals. Lamin B1 levels in Sox2-positive, PSA-NCAM-negative-ANSPCs were selectively downregulated at 5.5 months of age (ANPCs, P = 1.02129e-19; Neuroblasts, P = 0.09, DGCs, P = 6.4011e-15, ANOVA). ** P < 0.001, ANOVA followed by Tukey-Kramer test. (3 animals, 30 cells per conditions)E. Age-dependent reduction of lamin B1 in RGL-ANSCs (upper) and NR-ANPC (lower) from 2 to 5.5 months of age. Arrowheads indicate RGL-ANSCs or NR-ANPCs. F. Quantification of lamin B1 levels in RGL -ANSCs and ANPCs. * P < 0.05, *** P < 0.001, (4 animals, 18-32 cells per conditions, Mann-Whitney test).


Figure 3Open field ( OF ) test . ( B ) Color - coded distribution of the exploratory path in the open field test . X - Y axes of squares correspond to the OF arena . Old WT mice as well as Lmnb1 - cKO mice spend more time in the periphery of the OF arena . Open field ( OF ) test . ( C ) Total distance traveled during the OF test . No significant difference among groups . F ( 2 , 25 ) = 2 . 23 , P = 0 . 13 , ANOVA . ( D ) Velocity of travel during the OF test . F ( 2 , 25 ) = 1 . 60 , P = 0 . 22 , ANOVA . N . S . ( not significant ) . ( E ) Fraction of time spent in the periphery . F ( 2 , 25 ) = 10 . 71 , P = 0 . 00044 , ANOVA followed by Tukey Kramer , * * P < 0 . 01 . ( F ) Fraction of traveled distance in the periphery , F ( 2 , 25 ) = 3 . 84 , P = 0 . 035 , ANOVA followed by Tukey Kramer , * P < 0 . 05 ( n : WT = 10 , WT - old = 7 , Lmnb1 - cKO = 11 for the OF test ) . H - J . Novelty suppressed feeding . ( H ) Total feeding time during the NSF test , P = 0 . 0068 , Kruskal - Wallis test followed by Dunn ' s test . * * P < 0 . 01 . ( n : WT = 6 , WT - old = 7 , Lmnb1 - cKO = 6 for novelty suppressed feeding ) . ( I ) Food consumption during the NSF test , P = 0 . 014 , Kruskal - Wallis test followed by Dunn ' s test . * P < 0 . 05 . ( J ) Total number of feeding instances during NSF test . N . S . ( not significant ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Open field (OF) test. (B) Color-coded distribution of the exploratory path in the open field test. X-Y axes of squares correspond to the OF arena. Old WT mice as well as Lmnb1-cKO mice spend more time in the periphery of the OF arena.Open field (OF) test. (C) Total distance traveled during the OF test. No significant difference among groups. F(2, 25) = 2.23, P = 0.13, ANOVA. (D) Velocity of travel during the OF test. F(2, 25) = 1.60, P = 0.22, ANOVA. N.S. (not significant). (E) Fraction of time spent in the periphery. F(2, 25) = 10.71, P = 0.00044, ANOVA followed by Tukey Kramer, ** P < 0.01. (F) Fraction of traveled distance in the periphery, F(2, 25) = 3.84, P = 0.035, ANOVA followed by Tukey Kramer, * P < 0.05 (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test).H-J. Novelty suppressed feeding. (H) Total feeding time during the NSF test, P = 0.0068, Kruskal-Wallis test followed by Dunn's test. ** P < 0.01. (n: WT = 6, WT-old =7, Lmnb1-cKO = 6 for novelty suppressed feeding). (I) Food consumption during the NSF test, P = 0.014, Kruskal-Wallis test followed by Dunn's test. * P < 0.05. (J) Total number of feeding instances during NSF test. N.S. (not significant).


Figure 4B , C . Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment . Note that the numbers of DCX - positive cells are increased . Boxes indicate magnified regions in C . D , E , F . Quantification three weeks after TAM treatment . Among EYFP - positive cells , the density of RGL - ANSCs is not different ( P = 0 . 65 , t - test ) ( D ) . The density of ANPCs is significantly decreased ( P = 0 . 0037 , t - test ) ( E ) whereas the density of DCX - positive cells is markedly increased ( P = 0 . 014 , t - test ) ( F ) . ( n = 4 for Control , n = 3 for cKO ) . Open arrowheads indicate respective cells . G , H , I . Quantification two months after the induction of KO ( n = 3 for Control , n = 4 for cKO ) . The density of RGL - ANSCs is not significantly different ( P = 0 . 29 , t - test ) ( G ) . The density of ANPCs is significantly decreased . ( P = 0 . 0092 , t - test ) ( H ) . The density of neuroblasts is not significantly different ( P = 0 . 76 , t - test ) ( I ) . J , K , L . Quantification of proliferating cells three weeks after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 027 , Mann Whitney test ) ( J ) , and that of Ki67 + ANPCs is also significantly decreased . ( P = 0 . 034 , t - test ) ( K ) . The density of Ki67 + neuroblasts is significantly increased ( P = 0 . 0011 , t - test ) ( N ) ( n = 4 for Control , n = 4 for cKO ) ( L ) . M , N , O . Quantification of proliferating cells two months after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 0086 , t - test ) ( M ) , and densities of Ki67 + ANPCs and Ki67 + neuroblasts are also significantly decreased . ( P = 0 . 023 , P = 0 . 025 , t - test ) ( n = 3 for Control , n = 3 for cKO ) ( N , O ) . P . Declined return to RGL - ANSCs . Left , experimental scheme . Analysis was performed 3 weeks after BrdU injections . Middle , a representative image of BrdU + GFAP + RGL - ANSCs . Right , Quantification of BrdU + GFAP + RGL - ANSCs ( P = 0 . 019 , n = 4 for Control , n = 4 for cKO ) .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B, C. Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment. Note that the numbers of DCX-positive cells are increased. Boxes indicate magnified regions in C.D, E, F. Quantification three weeks after TAM treatment. Among EYFP-positive cells, the density of RGL-ANSCs is not different (P = 0.65, t-test) (D). The density of ANPCs is significantly decreased (P = 0.0037, t-test) (E) whereas the density of DCX-positive cells is markedly increased (P = 0.014, t-test) (F). (n = 4 for Control, n =3 for cKO). Open arrowheads indicate respective cells. G, H, I. Quantification two months after the induction of KO (n = 3 for Control, n = 4 for cKO). The density of RGL-ANSCs is not significantly different (P = 0.29, t-test) (G). The density of ANPCs is significantly decreased. (P = 0.0092, t-test) (H). The density of neuroblasts is not significantly different (P = 0.76, t-test) (I). J, K, L. Quantification of proliferating cells three weeks after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.027, Mann Whitney test) (J), and that of Ki67+ANPCs is also significantly decreased. (P = 0.034, t-test) (K). The density of Ki67+ neuroblasts is significantly increased (P = 0.0011, t-test) (N) (n = 4 for Control, n = 4 for cKO) (L). M, N, O. Quantification of proliferating cells two months after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.0086, t-test) (M), and densities of Ki67+ANPCs and Ki67+neuroblasts are also significantly decreased. (P = 0.023, P = 0.025, t-test) (n = 3 for Control, n = 3 for cKO) (N, O). P. Declined return to RGL-ANSCs. Left, experimental scheme. Analysis was performed 3 weeks after BrdU injections. Middle, a representative image of BrdU+GFAP+ RGL-ANSCs. Right, Quantification of BrdU+GFAP+RGL-ANSCs (P = 0.019, n = 4 for Control, n = 4 for cKO).


Figure 5Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM . C . Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM . Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( D ) The density of RGL - ANSCs tends to be reduced in cKO . F ( 2 , 8 ) = 3 . 98 , P = 0 . 063 , One - way ANOVA . ( E ) The density of ANPCs was reduced in cKO - 8M and Cont - 15M mice . F ( 2 , 8 ) = 11 . 49 , P = 0 . 0044 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 01 ) . Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( F ) The density of DCX - positive cells was reduced in WT - old and cKO mice . p = 0 . 0091 , Kruskal - Wallis test followed by Dunn ' s test ( * P < 0 . 05 ) . ( G ) The density of Ki67 + RGL - ANSCs was reduced in cKO - 8M mice ( * P = 0 . 029 , Kruskal - Wallis test followed by Dunn ' s test ) . ( H ) The density of Ki67 + ANPC was not significantly different among three groups ( P = 0 . 49 , Kruskal - Wallis test ) . ( I ) The density of NeuN + EYFP + neurons was reduced in cKO - 8M mice . F ( 2 , 8 ) = 34 . 85 , P = 0 . 00011 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 001 ) . ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M ) .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Representative confocal images of the SGZ 6.5 months after the treatment with TAM.C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM.Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01).Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (F) The density of DCX-positive cells was reduced in WT-old and cKO mice. p = 0.0091, Kruskal-Wallis test followed by Dunn's test (* P < 0.05). (G) The density of Ki67+RGL-ANSCs was reduced in cKO-8M mice (* P = 0.029, Kruskal-Wallis test followed by Dunn's test). (H) The density of Ki67+ANPC was not significantly different among three groups (P = 0.49, Kruskal-Wallis test). (I) The density of NeuN+EYFP+ neurons was reduced in cKO-8M mice. F (2, 8) = 34.85, P = 0.00011, One-way ANOVA followed by Tukey's HSD test (** P < 0.001). (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M).


Figure 6MA plot of differentially expressed genes between control and Lmnb1 - KO NPCs . E . Fraction of direction of gene regulation by LaminB1 . Genes were allocated dependent on where LaminB1 interacts with genes . Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout .

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6MA plot of differentially expressed genes between control and Lmnb1-KO NPCs.E. Fraction of direction of gene regulation by LaminB1. Genes were allocated dependent on where LaminB1 interacts with genes. Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout.


Figure 7Confirmation of Lamin B1 overexpression by qRT - PCR ( * P = 0 . 020 , n = 3 , one - sample t - test ) C . Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs . Scale bar = 10 µm . D , Relative induced expression of differentiation markers upon the withdrawal of FGF2 . 0 % indicates the same expression levels with control . Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation ( Tuj1 , * P = 0 . 023 ; Prox1 , * * * P = 0 . 00012 ; NeuroD1 , * * P = 0 . 0046 ; Ascl1 , * * * P = 0 . 00064 ; S100β , * * * P = 0 . 000097 ; n = 3 , one - sample t - test ) . F , Confocal images after exogenous expression of lamin B1 in EGFP + cells with RV ; LaminB1 - IRES - GFP ( laminB1 - OE . ) Scale bar = 20 µm . G , H , Exogenous expression of laminB1 repressed neuronal differentiation in vivo . An arrowhead indicate DCX + GFP + cell in control ( G , left ) and open arrowheads indicate Sox2 + GFP + cells in lamin B1 - OE cells . The fraction of EGFP + DCX + cells was significantly reduced by lamin B1 overexpression ( * * * P < 0 . 0002 , n = 3 ) , whereas the fractions of EGFP + DCX + Sox2 + cells and the EGFP + Sox2 + were increased ( EGFP + DCX + Sox2 + , * * P = 0 . 0024 ; EGFP + Sox2 + , P = 0 . 058 ; others , * P = 0 . 02 ) . Scale bar = 20 µm .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Confirmation of Lamin B1 overexpression by qRT-PCR (*P = 0.020, n = 3, one-sample t-test)C. Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs. Scale bar = 10 µm.D, Relative induced expression of differentiation markers upon the withdrawal of FGF2. 0 % indicates the same expression levels with control. Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation (Tuj1, *P = 0.023; Prox1, ***P = 0.00012; NeuroD1, ** P = 0.0046; Ascl1, ***P = 0.00064; S100β, ***P = 0.000097; n = 3, one-sample t-test).F, Confocal images after exogenous expression of lamin B1 in EGFP+ cells with RV;LaminB1-IRES-GFP (laminB1-OE.) Scale bar = 20 µm.G, H, Exogenous expression of laminB1 repressed neuronal differentiation in vivo. An arrowhead indicate DCX+GFP+ cell in control (G, left) and open arrowheads indicate Sox2+GFP+ cells in lamin B1-OE cells. The fraction of EGFP+ DCX+ cells was significantly reduced by lamin B1 overexpression (***P < 0.0002, n = 3), whereas the fractions of EGFP+DCX+Sox2+ cells and the EGFP+Sox2+ were increased (EGFP+DCX+Sox2+, **P = 0.0024; EGFP+ Sox2+, P = 0.058; others, *P = 0.02). Scale bar = 20 µm.


Figure 1C . Confocal images of MDA - MB - 231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D - collagen matrix . Cells were imaged and stained with Phalloidin - 568 ( red ) , pMLCII / Alexa ‑ 633 ( false ‑ coloured green ) or imaged for ZsGreen ( false - coloured white ) . Scale 20 μm . D . Single cell morphology assay of MDA - MB - 231 cells cultured in 3D - collagen and analysed for morphology 24 hours after transient knock - down using either a control sequence ( NT , non - targeting ) or a sequence targeting the RASSF1 locus . RASSF1 knock - down marks a reduction in rounded cells . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm.D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


Figure 2C . Western blot analysis of proteins in MDA - MB - 231 cells transiently transfected with siRNA targeting a control sequence ( NT ) or targeting RASSF1 . GAPDH was used as a loading control . D . Immunoprecipitation from MDA - MB - 231 transiently transfected with control ( pcDNA3 ) or HA ‑ RASSF1C plasmid . Pull ‑ down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies . H . Quantification of rounded versus elongated cells in single cell morphology assay in 3D ‑ collagen indicating the degree of mesenchymal - amoeboid transition of MDA - MB - 231 cells when RASSF1C or the described mutants are expressed . I . Representative confocal images show elongated or rounded cells per field of view of MDA ‑ MB - 231 cells transfected with DsRed , DsRASSF1C , DsRASSF1C - R197W or DsRASSF1C - R199F , grown in 3D ‑ collagen and stained with Phalloidin - 568 ( red ) for F ‑ actin and pMLCII / Alexa 633 . Scale bars represent 10 μm .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control.D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.


Figure 3A . Left : Cartoon showing the principle behind the Cre - carrying EVs and how recipient cells switch from DsRed + to eGFP + expression upon Cre - mediated excision of the DsRed locus . Centre : representative images , and right : quantification of T47DDsRed cells that converted to eGFP + in the bottom wells of a transwell system in the presence of MDA - MB - 231CFP ; Cre ; Control or MDA ‑ MB ‑ 231CFP ; Cre ; HA - RASSF1C seeded in the upper wells . Scale bars represent 100 μm . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C . Western blot analysis of EVs isolated from MDA - MB - 231 cells transfected with Control ( pcDNA3 ) or FLAG - RASSF1C with indicated antibodies . Equal amounts of protein were loaded after normalization via microBCA assay . F . Left : Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells , treated with 20 ng / μl EVs derived from MDA - MB - 231 cells , transfected the same constructs used in ( e ) . Right : Quantification of Boyden chamber invasion 24 hours after EV treatment . Scale bars represent 200µm . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Left: Cartoon showing the principle behind the Cre-carrying EVs and how recipient cells switch from DsRed+ to eGFP+ expression upon Cre-mediated excision of the DsRed locus. Centre: representative images, and right: quantification of T47DDsRed cells that converted to eGFP+ in the bottom wells of a transwell system in the presence of MDA-MB-231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C seeded in the upper wells. Scale bars represent 100 μm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.C. Western blot analysis of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3) or FLAG-RASSF1C with indicated antibodies. Equal amounts of protein were loaded after normalization via microBCA assay.F. Left: Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells, treated with 20 ng/μl EVs derived from MDA-MB-231 cells, transfected the same constructs used in (e). Right: Quantification of Boyden chamber invasion 24 hours after EV treatment. Scale bars represent 200µm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.


Figure 4D . Representative confocal images ( upper row ) and quantification of fluorescent intensity of ALDH1 expression in MDA - MB - 231 cells expressing RASSF1C ( black bar in the graph ) or after siRNA treatment against RASSF1 ( grey bar ) . Lower row shows merged images with DAPI ( blue ) , ALDH1 ( green ) and RASSF1C ( red ) . Scale bars represent 20 μm . Data Information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . J . qRT ‑ PCR expression of NANOG , OCT4 , SOX2 in MCF7 cells 24 h after exposure to 50 ng / μl EVs derived from MDA ‑ MB ‑ 231 cells ( treated as indicated ) . Data Information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D. Representative confocal images (upper row) and quantification of fluorescent intensity of ALDH1 expression in MDA-MB-231 cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1 (grey bar). Lower row shows merged images with DAPI (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm. Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.J. qRT‑PCR expression of NANOG, OCT4, SOX2 in MCF7 cells 24 h after exposure to 50 ng/μl EVs derived from MDA‑MB‑231 cells (treated as indicated). Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


Figure 5B . Left : representative confocal images of mammary tumor formed by the co - injection of T47DDsRed reporter cells with either MDA - MB - 231CFP ; Cre ; Control or MDA - MB - 231CFP ; Cre ; HA - RASSF1C . Scale 200 μm . Right : averaged values for all mice for the percent of the eGFP + or CFP + cells as part of the whole tumor . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. Left: representative confocal images of mammary tumor formed by the co-injection of T47DDsRed reporter cells with either MDA-MB-231CFP;Cre;Control or MDA-MB-231CFP;Cre;HA-RASSF1C. Scale 200 μm. Right: averaged values for all mice for the percent of the eGFP+ or CFP+ cells as part of the whole tumor. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.


Figure 6B . Kaplan - Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter / CpG island methylation of RASSF1A , previously reported to be associated with outcome in breast cancer patients . P values were obtained from a log - rank test . C . Left : heatmap depicting methylation patterns across the RASSF1 gene . In light green , RASSF1 - 2γ CpG island is depicted ; in dark green , RASSF1 - 1α CpG island is depicted . Right : Kaplan ‑ Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients . P values were calculated from a log - rank test . Color legend : yellow , low RASSF1A and low RASSF1C ; green , low RASSF1A and high RASSF1C ; red , high RASSF1A and low RASSF1C ; grey , high RASSF1A and high RASSF1C .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B. Kaplan-Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter/CpG island methylation of RASSF1A, previously reported to be associated with outcome in breast cancer patients. P values were obtained from a log-rank test.C. Left: heatmap depicting methylation patterns across the RASSF1 gene. In light green, RASSF1-2γ CpG island is depicted; in dark green, RASSF1-1α CpG island is depicted. Right: Kaplan‑Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients. P values were calculated from a log-rank test. Color legend: yellow, low RASSF1A and low RASSF1C; green, low RASSF1A and high RASSF1C; red, high RASSF1A and low RASSF1C; grey, high RASSF1A and high RASSF1C.


Figure 1A . Exogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 . Data information : Data are representative of three biological replicates with similar results . B . Endogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 in mouse peritoneal macrophages . Data information : Data are representative of three biological replicates with similar results . C . TRIM50 and NLRP3 proteins were expressed and obtained from the in vitro translation system and their interaction were further analyzed . Data information : Data are representative of three biological replicates with similar results . D . Co - IP assay of the interaction between NLRP3 and TRIM50 after stimulation with nigericin for 0 , 15 , 30 and 60 min . Data information : Data are representative of three biological replicates with similar results . E . Co - IP assay of the interaction between NLRP3 and TRIM50 in mouse peritoneal macrophages primed with LPS for 6h and treated with ATP for 30min . Data information : Data are representative of three biological replicates with similar results . F . Confocal microscopy assay of the interaction between TRIM50 and NLRP3 in THP1 cells with stimulation by LPS and ATP . Scale bars : 10μm . Data information : Data are representative of three biological replicates with similar results . G . Schematic diagram of human TRIM50 ( WT ) and its truncation mutants was presented ( top ) , and the interaction of TRIM50 ( TRIM50 truncation mutants ) and NLRP3 was detected by IP assay . Data information : Data are representative of three biological replicates with similar results . H , I . Schematic diagram of human NLRP3 ( WT ) and its truncation mutants ( top ) , and immunoprecipitation assay of the interaction of NLRP3 ( NLRP3 truncation mutants ) with TRIM50 in HEK293T cells . Data information : Data are representative of three biological replicates with similar results .

O B-PANEL_START O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Exogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3. Data information: Data are representative of three biological replicates with similar results.B. Endogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 in mouse peritoneal macrophages. Data information: Data are representative of three biological replicates with similar results.C. TRIM50 and NLRP3 proteins were expressed and obtained from the in vitro translation system and their interaction were further analyzed. Data information: Data are representative of three biological replicates with similar results.D. Co-IP assay of the interaction between NLRP3 and TRIM50 after stimulation with nigericin for 0, 15, 30 and 60 min. Data information: Data are representative of three biological replicates with similar results.E. Co-IP assay of the interaction between NLRP3 and TRIM50 in mouse peritoneal macrophages primed with LPS for 6h and treated with ATP for 30min. Data information: Data are representative of three biological replicates with similar results.F. Confocal microscopy assay of the interaction between TRIM50 and NLRP3 in THP1 cells with stimulation by LPS and ATP. Scale bars: 10μm. Data information: Data are representative of three biological replicates with similar results.G. Schematic diagram of human TRIM50 (WT) and its truncation mutants was presented(top), and the interaction of TRIM50 (TRIM50 truncation mutants) and NLRP3 was detected by IP assay. Data information: Data are representative of three biological replicates with similar results.H, I. Schematic diagram of human NLRP3 (WT) and its truncation mutants (top), and immunoprecipitation assay of the interaction of NLRP3 (NLRP3 truncation mutants) with TRIM50 in HEK293T cells. Data information: Data are representative of three biological replicates with similar results.


Figure 2A . Immunoblot analysis of the NLRP3 protein level in the mouse peritoneal macrophages transfected with Si - Trim50 . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test ( A , Data are representative of three biological replicates with similar results . ( mean ± SDB . qRT - PCR analysis of Nlrp3 and IL - 1β mRNA levels in LPS - primed Si - Trim50 transfected mouse peritoneal macrophages . Data information : P - values are shown , two - way analysis of variance ( ANOVA ) ( B , Data are representative of three biological replicates with similar results . ( mean ± SDC , D . Immunoblot analysis of Caspase1 - P20 ( C ) and Gasdermin D ( D ) of WT or Trim50 - / - mouse peritoneal macrophages after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDE , Immunoblot analysis of Caspase1 - P20 ( E ) of WT or Trim50 - / - BMDM cells after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDF . Immunoblot analysis of Gasdermin D ( F ) of WT or Trim50 - / - BMDM cells after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDG , ELISA analysis of IL - 1β , TNF - α and IL - 6 in the supernatant of WT or Trim50 - / - peritoneal macrophages with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) G , Data are representative of three biological replicates with similar results . ( mean ± SDH . ELISA analysis of IL - 1β , TNF - α and IL - 6 in the supernatant of WT or Trim50 - / - BMDM cells with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) Data are representative of three biological replicates with similar results . ( mean ± SDI . LDH analysis of WT or Trim50 - / - mouse peritoneal macrophages with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) I ) . Data are representative of three biological replicates with similar results . ( mean ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblot analysis of the NLRP3 protein level in the mouse peritoneal macrophages transfected with Si-Trim50. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test (A, Data are representative of three biological replicates with similar results. (mean ± SDB. qRT-PCR analysis of Nlrp3 and IL-1β mRNA levels in LPS-primed Si-Trim50 transfected mouse peritoneal macrophages. Data information: P-values are shown, two-way analysis of variance (ANOVA) (B, Data are representative of three biological replicates with similar results. (mean ± SDC, D. Immunoblot analysis of Caspase1-P20 (C) and Gasdermin D (D) of WT or Trim50-/- mouse peritoneal macrophages after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDE, Immunoblot analysis of Caspase1-P20 (E) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDF. Immunoblot analysis of Gasdermin D (F) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDG, ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) G, Data are representative of three biological replicates with similar results. (mean ± SDH. ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- BMDM cells with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) Data are representative of three biological replicates with similar results. (mean ± SDI. LDH analysis of WT or Trim50-/- mouse peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) I). Data are representative of three biological replicates with similar results. (mean ± SD


Figure 3A . Immunoprecipitation analysis of the NLRP3 ubiquitination in HEK293T cells transfected with HA - ubiquitin , His - NLRP3 and Flag - TRIM50 plasmids . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . B . Immunoprecipitation analysis of the endogenous NLRP3 ubiquitination in WT or Trim50 - / - BMDM cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . C . Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3 , HA - Ubiquitin , TRIM50 or TRIM50 ( ΔRING ) mutant plasmids . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . D . Immunoblot analysis of the ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and the whole cell lysate ( soluble ) in HEK293T cells with nigericin treatment for 30 min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . E . Immunoblot analysis of NLRP3 protein levels in cycloheximide ( CHX ) treated HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ± SDF . Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 or TRIM50 ( ΔRING ) mutant , followed with the further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ± SDG . Immunoblot analysis of NLRP3 in mouse peritoneal macrophages , followed with the further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Immunoprecipitation analysis of the NLRP3 ubiquitination in HEK293T cells transfected with HA-ubiquitin, His-NLRP3 and Flag-TRIM50 plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.B. Immunoprecipitation analysis of the endogenous NLRP3 ubiquitination in WT or Trim50-/- BMDM cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.C. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or TRIM50 (ΔRING) mutant plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.D. Immunoblot analysis of the ASC oligomerization in cross-linked cytosolic pellets (insoluble) and the whole cell lysate (soluble) in HEK293T cells with nigericin treatment for 30 min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.E. Immunoblot analysis of NLRP3 protein levels in cycloheximide (CHX) treated HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SDF. Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 or TRIM50 (ΔRING) mutant, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SDG. Immunoblot analysis of NLRP3 in mouse peritoneal macrophages, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


Figure 4A . SDD - AGE analysis of NLRP3 oligomerization in HEK293T cells transfected with NLRP3 and TRIM50 plasmids . Data information : Data are representative of three biological replicates with similar results . B . SDD - AGE analysis of NLRP3 oligomerization in NLRP3 and TRIM50 transfected HEK293T cells followed with nigericin treatment for 30min . Data information : Data are representative of three biological replicates with similar results . C . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in HEK293T cells transfected with NLRP3 , ASC and TRIM50 plasmids followed with nigericin treatment for 30 min . Data information : Data are representative of three biological replicates with similar results . D . SDD - AGE analysis of NLRP3 oligomerization in WT or Trim50 - / - peritoneal macrophages with LPS treatment for 6h and ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results . E . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in WT or Trim50 - / - peritoneal macrophages by LPS stimulation for 6h and ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results . F . SDD - AGE analysis of NLRP3 oligomerization in WT or Trim50 - / - BMDM cells with LPS treatment for 6h and ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results . G . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in WT or Trim50 - / - BMDM cells by LPS stimulation for 6h and ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results . H . SDD - AGE analysis of the oligomerization of NLRP3 in HEK293T cells transfected with His - NLRP3 and TRIM50 or TRIM50 ( ΔRING ) mutant . Data information : Data are representative of three biological replicates with similar results . I . In vitro NLRP3 oligomerization assay with in - vitro translated TRIM50 and NLRP3 proteins . Data information : Data are representative of three biological replicates with similar results . J . Immunoprecipitation analysis of NLRP3 ubiquitination in HA - ubiquitin , His - NLRP3 and Myc - TRIM50 transfected HEK293T cells with further treatment with tranilast or MCC950 for 24h . Data information : Data are representative of three biological replicates with similar results .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. SDD-AGE analysis of NLRP3 oligomerization in HEK293T cells transfected with NLRP3 and TRIM50 plasmids. Data information: Data are representative of three biological replicates with similar results.B. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 and TRIM50 transfected HEK293T cells followed with nigericin treatment for 30min. Data information: Data are representative of three biological replicates with similar results.C. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in HEK293T cells transfected with NLRP3, ASC and TRIM50 plasmids followed with nigericin treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.D. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- peritoneal macrophages with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.E. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- peritoneal macrophages by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.F. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- BMDM cells with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.G. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- BMDM cells by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.H. SDD-AGE analysis of the oligomerization of NLRP3 in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50(ΔRING) mutant. Data information: Data are representative of three biological replicates with similar results.I. In vitro NLRP3 oligomerization assay with in-vitro translated TRIM50 and NLRP3 proteins. Data information: Data are representative of three biological replicates with similar results.J. Immunoprecipitation analysis of NLRP3 ubiquitination in HA-ubiquitin, His-NLRP3 and Myc-TRIM50 transfected HEK293T cells with further treatment with tranilast or MCC950 for 24h. Data information: Data are representative of three biological replicates with similar results.